Use of bhlhe22 in immunotherapy of prostate cancer bone metastasis

Abstract

The application discloses application of BHLHE22 in immunotherapy of prostate cancer bone metastasis, and relates to the technical field of new drug use. The application proves that BHLHE22 expression is up-regulated in bone metastatic prostate cancer patients, BHLHE22 drives the formation of an immunosuppressive bone microenvironment and promotes prostate cancer bone metastasis; it is revealed that the BHLHE22 / PRMT5 / CSF2 pathway drives the formation of an immunosuppressive bone microenvironment and promotes the molecular mechanism of prostate cancer bone metastasis. The application proposes a new method for screening BHLHE22-positive prostate cancer patient subgroups for immunotherapy based on the expression of BHLHE22 in prostate cancer patients. At the same time, BHLHE22 is proposed as a new biomarker to guide ICT treatment and a new strategy for combined PRMT5 / CSF2 treatment. The application develops a new use of drugs for preventing and treating prostate cancer bone metastasis, improves the therapeutic effect of ICT, and discovers the new medical value of BHLHE22.

Description

Technical Field

[0001] This invention relates to the technical field of novel uses of pharmaceuticals, specifically to the application of BHLHE22 in the immunotherapy of bone metastases from prostate cancer. Background Technology

[0002] Prostate cancer (PCa) is a common malignant tumor in men. In the United States, it is the leading cause of cancer death among men, and in my country, its incidence is increasing year by year. As of 2020, globally, prostate cancer is the second most common and fifth leading cause of cancer death in men. While the overall incidence of prostate cancer has remained stable, the detection rate of advanced prostate cancer has increased from 3.9% to 8.2% over the past decade. Bone is the most common site of metastasis in advanced prostate cancer, affecting over 80% of patients. Bone-related complications caused by metastasis severely reduce the quality of life and survival time of patients with advanced prostate cancer. Current treatments for prostate cancer bone metastases primarily aim to prevent disease progression and alleviate clinical symptoms; a cure is difficult to achieve. Therefore, inhibiting the occurrence of prostate cancer bone metastases, exploring the endogenous and exogenous factors influencing the development and metastasis of PCa cells, revealing their underlying molecular mechanisms, and developing targeted therapies are of great significance.

[0003] Novel treatment options for patients with advanced prostate cancer bone metastases include: androgen deprivation therapy (ADT) represented by AR antagonists, targeted therapies such as PARP inhibitors, radioactive isotope therapy such as radium-223 and lutetium-177, bone-targeted drug therapy such as bisphosphonates and denosumab, and immunotherapy. Currently, immunotherapy, represented by immune checkpoint inhibitors (ICT), has been proven effective against various solid tumors. However, the endogenous molecular characteristics of tumor cells and the immunosuppressive state specific to the tumor microenvironment in patients with prostate cancer bone metastases prevent ICT from effectively improving the prognosis of these patients. Therefore, elucidating the endogenous treatment resistance mechanisms of PCa cells, alleviating the tumor immunosuppressive microenvironment in patients with prostate cancer bone metastases, and improving the efficacy of immunotherapy are of great clinical significance.

[0004] Transcription factors (TFs) are a class of genes that regulate gene expression. Their expression at different times and spaces affects various cellular physiological processes and determines cell fate. Previous studies have shown that BHLHE22 regulates gene expression through the formation of transcriptional complexes in neurodevelopmental research. However, in the field of tumor research, there are currently no reports on the function of BHLHE22. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of BHLHE22 in the immunotherapy of bone metastases of prostate cancer.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: the application of a molecular marker in predicting or detecting bone metastasis of prostate cancer, wherein the molecular marker is BHLHE22; the application is not for disease diagnosis and treatment.

[0007] BHLHE22 is a member of the helical-loop-helical (bHLH) transcription factor class B superfamily, located on chromosome 8 q12.3. BHLHE22 is primarily expressed in pancreatic, telencephalon, and cerebellar tissues, and is a typical tissue-specific gene. MDSCs are a heterogeneous population of immature, undifferentiated myeloid progenitor cells that have not differentiated into macrophages, neutrophils, or dendritic cells. MDSCs suppress antitumor immune responses through multiple mechanisms. The inventors of this application have discovered that the transcription factor BHLHE22 is upregulated in bone metastases of prostate cancer (PCa), inducing the accumulation of MDSCs within the bone microenvironment, driving the formation of an immunosuppressive microenvironment, and promoting PCa bone metastasis. Therefore, detecting BHLHE22 expression in tissues can predict or detect prostate cancer bone metastasis.

[0008] This invention also provides the use of BHLHE22 in the preparation of products for predicting or detecting bone metastases in prostate cancer.

[0009] As a preferred embodiment of the application described in this invention, the product includes a reagent for detecting the expression level of BHLHE22 in a sample.

[0010] In a preferred embodiment of the application described in this invention, the expression level of BHLHE22 in the sample is upregulated.

[0011] The inventors of this application found that BHLHE22 expression is upregulated in primary prostate cancer lesions with bone metastases, and further upregulated in bone metastatic tissues. Furthermore, upregulated BHLHE22 expression is significantly positively correlated with poor clinical prognosis in prostate cancer patients, including overall survival and survival without bone metastases. Therefore, detecting BHLHE22 expression in primary prostate cancer lesions or bone metastatic tissues can predict or detect bone metastases in prostate cancer.

[0012] As a preferred embodiment of the application described in this invention, the reagents include those for detecting BHLHE22 expression levels by RT-PCR, real-time quantitative PCR, immunoassay, or microarray.

[0013] In a preferred embodiment of the application described in this invention, the sample being tested is a human tissue sample.

[0014] In a preferred embodiment of the application described in this invention, the human tissue sample is a primary prostate cancer lesion or bone metastasis tissue.

[0015] The present invention also provides a kit for predicting or detecting bone metastases in prostate cancer, the kit containing reagents capable of detecting the expression level of BHLHE22.

[0016] The bone microenvironment is conducive to the colonization and growth of metastatic tumor cells. The bone marrow microenvironment promotes immune escape of disseminated tumor cells through multiple mechanisms, leading to drug resistance and recurrence. Metastatic PCa (proliferative proliferative cytokine) is a "cold" tumor, rich in immunosuppressive cytokines; it also shows extensive infiltration of Treg cells and MDSCs, and lacks APCs, NK cells, Th1 cells, and CD8+. + T cells. Therefore, therapeutic strategies that alleviate immunosuppression mediated by immunosuppressive cell populations and activate the function of effector immune cells have promising application prospects. The inventors of this application found that PCa cells with high BHLHE22 expression induced the expansion and infiltration of MDSCs, and MDSCs depleted CD4+. + T and CD8 + T cells create an immunosuppressive bone microenvironment, promoting bone metastasis in prostate cancer. Therefore, downregulating BHLHE22 expression reduces MDSC infiltration and inhibits prostate cancer bone metastasis.

[0017] CSF2 is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts, which function as cytokines. The inventors of this application discovered that BHLHE22 transcribes and activates CSF2 expression by binding to the CSF2 promoter. Experiments showed that anti-CSF2 combined with ICT therapy significantly improved the therapeutic efficacy of ICT and reduced BHLHE22 expression. + PCa cell-induced MDSCs expansion and infiltration, reduced CD4 + T and CD8 + T cell depletion, activation of CD8 + T cells help to relieve the bone immunosuppressive microenvironment.

[0018] This invention also provides the use of GSK3326595 in combination with a PD-1 inhibitor in the preparation of a medicament for the prevention and treatment of bone metastases from prostate cancer.

[0019] The present invention also provides a pharmaceutical composition for the prevention and treatment of bone metastases of prostate cancer, the pharmaceutical composition comprising the following substances: (1) PRMT5 antagonist GSK3326595; (2) PD-1 inhibitor pembrolizumab.

[0020] PRMT5, short for protein arginine methyltransferase 5, is an epigenetic modifier that catalyzes monomethylation and symmetrical dimethylation of arginine residues on histones and non-histone proteins, regulating various cellular processes and participating in cancer formation and progression. The inventors of this application discovered that BHLHE22 binds to PRMT5, and PRMT5 catalyzes methylation at histone H4R3me2a and H3R2me2s sites, transcriptionally activating CSF2 expression. Experiments showed that the PRMT5 antagonist GSK3326595, combined with ICT treatment, significantly improved the therapeutic efficacy of ICT and reduced BHLHE22 expression. + PCa cell-induced MDSCs expansion and infiltration, reduced CD4 + T and CD8 + T cell depletion, activation of CD8 + T cells help to relieve the bone immunosuppressive microenvironment.

[0021] Beneficial effects of this invention: This invention provides the application of BHLHE22 in the immunotherapy of prostate cancer bone metastases. This invention confirms that upregulation of BHLHE22 expression promotes prostate cancer bone metastasis and that BHLHE22-driven immunosuppressive microenvironment for prostate cancer bone metastases is formed. This invention reveals the important role of the BHLHE22 / PRMT5 / CSF2 pathway in driving the formation of an immunosuppressive bone microenvironment and promoting prostate cancer bone metastasis. This invention applies BHLHE22 to the immunotherapy of prostate cancer bone metastases, not only providing a new source for the preparation of drugs for the prevention and treatment of prostate cancer bone metastases, but also uncovering new pharmaceutical value of BHLHE22. Attached Figure Description

[0022] Figure 1A: Representative IHC staining results and quantitative analysis of BHLHE22 expression in PCa / nBM (n=132), PCa / BM (n=60), and BM (n=30). Scale bar, 100µm and 50µm. *P<0.05, ***P<0.001. One-way ANOVA; B: Expression level of BHLHE22 in metastatic bone tissue, primary prostate, and other organ metastatic sites based on GSE77930 sequencing results. ***P<0.001. One-way ANOVA; C: Expression level of BHLHE22 in PCa / nBM and PCa / BM based on TCGA-PRAD database analysis. **P<0.01. Mann-Whitney test; D shows the disease-free survival curves for PCa patients with low BHLHE22 expression (0–33% quantile) and high BHLHE22 expression (66–100% quantile) based on TCGA-PRAD database data, analyzed by Kaplan-Meier. P = 0.012, log-rank test; E shows the overall survival curves for PCa patients with low BHLHE22 expression (0–50% quantile) and high BHLHE22 expression (50–100% quantile) based on Kaplan-Meier. P = 0.007, log-rank test; F shows the bone metastasis-free survival curves for PCa patients with low BHLHE22 expression (0–50% quantile) and high BHLHE22 expression (50–100% quantile) based on Kaplan-Meier. P = 0.005, log-rank test.

[0023] Figure 2AC represents BLI signal monitoring and H&E stained section images and quantitative results of the BALB / c nude mouse bone metastasis model after left ventricular injection of RM-1 cells. ns, not significant. t-test; DF represents BLI signal monitoring and H&E stained section images and quantitative results of the BALB / c nude mouse bone metastasis model after left ventricular injection of PC-3 cells. ns, not significant. t-test; G represents representative BLI signal monitoring results of the C57BL / 6J mouse bone metastasis model after left ventricular injection of RM-1 cells; H represents representative microCT images of bone metastasis lesions in C57BL / 6J mice (arrows indicate osteolytic lesions). Scale bar, 1 mm; I represents representative H&E stained section images of the femur / tibia of C57BL / 6J mice (T, tumor; N, adjacent non-tumor tissue). Scale bar, 200 μm and 50 μm; J represents the quantitative results of microCT osteolytic area. ***P<0.001. t-test. (K) Quantitative results of H&E stained tumor lesion extent. ***P<0.001. t-test; L is the Kaplan-Meier survival curve for the overall survival rate of C57BL / 6J mice in each group; M is the Kaplan-Meier survival curve for the survival rate without bone metastasis of C57BL / 6J mice in each group. P=0.001(L), P=0.004(M). Log-Rank test.

[0024] Figure 3 A shows that BHLHE22 is located on chromosome 8, as indicated by the UCSC database. The PhyloP algorithm was used to calculate the sequence conservation score of BHLHE22 among different species during evolution (black bars indicate sequence consistency, gray bars indicate sequence inconsistency, and double lines indicate sequence deletion). BC shows the Transwell migration / invasion experiment results and quantitative statistics for RM-1 and PC-3 cells. ns, not significant. One-way ANOVA. DE shows the gene set enrichment analysis (GSEA) based on the TCGA-PRAD database and RNA-seq results, revealing the enrichment of immune negative regulatory pathways in high BHLHE22 expression. F shows the Gene Ontology analysis based on RNA-seq results, showing that the immune response gene set in high BHLHE22 expression is enriched in the Top 10.

[0025] Figure 4 : AB represents BHLHE22, DAPI staining and CD33 staining in human prostate cancer tissue samples PCa / BM(A) and BM(B). + MDSCs, CD8 +Representative multilabeled tissue immunofluorescence staining images of T cell infiltration. Samples were stratified according to low (0–50th percentile) and high (50–100th percentile) BHLHE22 expression. Scale bar, 50 μm; C is a bone marrow section of C57BL / 6J mouse bone metastases injected with RM-1 cells in the left ventricle. + T, CD8 + T, Gr-1 + Representative multilabeled immunofluorescence staining images of MDSC cell infiltration. Scale bar, 50 μm; DE represents CD33 concentration per high-power field (200×) in human prostate cancer tissue samples PCa / BM (D) and BM (E). + MDSCs and CD8 + Quantitative results of T cell count. *P<0.05,**P<0.01,***P<0.001. t-test; F represents the number of CD33 cells per high-power field (200×) in bone marrow sections from C57BL / 6J mice injected with RM-1 cells in the left ventricle. + MDSCs and CD4 + T, CD8 + Quantitative results of T cell count. **P<0.01,***P<0.001. t-test; GH was determined by multicolor flow cytometry. Results showed that C57BL / 6J mice had bone metastases infiltrating CD45 cells. + 7AAD - Cells (G) and CD3 + CD11b - Cell (H) proportion and quantification results. ns, not significant. t-test; I represents multicolor flow cytometry results showing that C57BL / 6J mice had bone metastases and bone marrow tumors infiltrating CD11b. + Gr-1 + (MDSCs), Ly6C + Ly6G lo (M-MDSCs), Ly6C - Ly6G + (PMN-MDSCs) and Gr-1 + Arg-1 + The proportion of cells; J represents tumor-infiltrating MDSCs, M-MDSCs, PMN-MDSCs, and Arg-1. + Quantitative results of MDSCs. *P<0.05, ***P<0.001. t-test; K represents the result of multicolor flow cytometry analysis. Results showed that C57BL / 6J mice had bone metastases infiltrated by CD4+. + T,CD8 + T,IFN-γ + CD8 + T and PD-1+ CD8 + The proportion of T cells; LM represents tumor-infiltrating CD4. + T,CD8 + T cells (L) and IFN-γ + CD8 + T, PD-1 + CD8 + Quantitative results of T cells (M). *P<0.05,**P<0.01,***P<0.001. t-test; NO is from BHLHE22 + MDSCs and CD8s sorted from BM mice + After 5 days of T cell co-culture, T cell proliferation inhibition was detected and quantitatively analyzed by multicolor flow cytometry (CFSE staining). ***P<0.001. One-way ANOVA.

[0026] Figure 5 AB represents the detection of tumor-infiltrating CD11b in an in vivo MDSCs depletion assay using multicolor flow cytometry. + Gr-1 + (MDSCs) cells (A) and CD4 + T,CD8 + T cell (B) proportion and quantification results. ***P<0.001. t-test; CF shows the proportion and quantification results of Treg cells (C), NK cells (D), macrophages (E), TAM-1 and TAM-2 cells (F) infiltrating bone metastases in C57BL / 6J mice, as detected by multicolor flow cytometry. ns, not significant. t-test.

[0027] Figure 6 A shows the RNA-seq results of RM-1 and PC-3 cell lines (BHLHE22 vs Vector), with volcano plots showing differentially expressed genes (FC > 1.5); B shows the multifactor protein chip analysis of RM-1-BHLHE22 and RM-1-Vector cell culture supernatants; CD shows the expression of CSF2 in BM tumor tissues of C57BL / 6J mice in the RM-1, PC-3, and BHLHE22 cell lines detected by Western blotting; E shows the BHLHE22, CSF2, DAPI staining and Gr-1 staining of bone marrow sections from C57BL / 6J mice with bone metastases injected into the left ventricle with RM-1 cells. + Representative multilabeled tissue immunofluorescence staining images (200×) of MDSC cell infiltration and quantitative CSF2 expression. Scale bar, 50 μm. ***P<0.001. t-test; F represents BHLHE22 and CD4+ expression in bone marrow sections of C57BL / 6J mice injected with RM-1 cells in the left ventricle.+ T(CD4), CD8 + Representative immunohistochemical staining images of T(CD8), MDSCs (Gr-1 and S100A9), CSF2, and Ki-67. Scale bar, 50 μm; G represents the number of positive cells per high-power field (400×). *P<0.05, **P<0.01, ***P<0.001. t-test; H represents the Ki-67 immunohistochemical staining score. **P<0.01. t-test; I represents CSF2. + With Gr-1 + Cellular correlation analysis. r = 0.645, P < 0.001; Spearman test; J represents the correlation analysis of CSF2 and BHLHE22 expression in the TCGA-PRAD database. r = 0.230, P < 0.001; Spearman test.

[0028] Figure 7 AB represents the BLI signal monitoring and H&E staining results of the C57BL / 6J mouse bone metastasis model after left ventricular injection of RM-1 cells in representative experimental groups; CD represents the BLI signal and immunostatistical results of bone metastasis and tumor lesions. ns, not significant, *P<0.05, ***P<0.001. One-way ANOVA; E represents the in vivo MDSCs infiltration analysis and statistical results detected by multicolor flow cytometry in each experimental group. ns, not significant. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA; F represents the co-culture experiment and statistical results of in vitro MDSCs amplification detected by multicolor flow cytometry in each experimental group (CFSE staining). ns, not significant. ***P<0.001. One-way ANOVA.

[0029] Figure 8A: Detection of CSF2 promoter activity using a dual-luciferase reporter gene assay. Normalized to firefly luciferase activity compared to Renilla luciferase activity (firefly luciferase activity / Renilla luciferase activity). ***P<0.001. One-way ANOVA; BC: ChIP-qPCR analysis of BHLHE22 and CSF2 promoter sequence co-precipitation results in RM-1 (B) and PC-3 (C) cells overexpressing BHLHE22. ***P<0.001. One-way ANOVA; D: DNA-binding motif illustrations of BHLHE22 in humans and mice; E: Image of silver staining of eluent from Co-IP assay, mass spectrometry (MS) analysis, and potential transcriptional cofactors interacting with BHLHE22. The red arrow indicates PRMT5; F is the predicted binding site of BHLHE22 in the CSF2 promoter region from the JASPAR database; G is a legend of the CSF2 promoter DNA probe and a silver-stained image of the elution buffer from the DNA pull-down assay. The red arrow indicates PRMT5; H is the expression of PRMT5 in the elution buffer from the DNA pull-down assay detected by Western blotting; I is the co-localization of BHLHE22 and PRMT5 in the RM-1-BHLHE22 cell line detected by immunofluorescence staining. Scale bar, 25µm; J is the dual-luciferase reporter gene assay for CSF2 promoter activity in RM-1-BHLHE22 cells transfected with pGL4-FL-BBS, pGL4-P1-BBS-WT, or pGL4-P1-BBS-Mut (endogenous assay) and HEK293T cells (exogenous assay, simultaneously transfected with BHLHE22 overexpression vector plasmid). Normalized to firefly luciferase activity compared to Renilla luciferase activity (firefly luciferase activity / Renilla luciferase activity). ***P<0.001. One-way ANOVA; KL represents the interaction between BHLHE22 and PRMT5 detected by endogenous (K) and exogenous (L) co-immunoprecipitation assays (Co-IP). In the exogenous assay, Flag labeled BHLHE22 and HA labeled PRMT5; M represents the expression level of PRMT5 in RM-1 and PC-3 cells after overexpression of BHLHE22 by qPCR. ns, not significant. t-test; N represents the results of ChIP-qPCR analysis of co-precipitation of BHLHE22 and PRMT5 with the CSF2 promoter and a schematic diagram of the BHLHE22 / PRMT5 complex binding to the CSF2 promoter. ***P<0.001. One-way ANOVA.

[0030] Figure 9A: qPCR detection of CSF2 expression level after PRMT5 knockdown. ***P<0.001. One-way ANOVA; B: Western blotting detection of CSF2 expression level after PRMT5 knockdown; C: Western blot detection of H4R3, H3R2, and H3R8 histone methylation levels; D: ChIP-qPCR analysis of co-precipitation results of H4R3, H3R2, and H3R8 methylated antibodies with the CSF2 promoter after PRMT5 knockdown. ***P<0.001. t-test; E: qPCR detection of CSF2 expression level after treatment with the PRMT5 inhibitor GSK591. ***P<0.001. One-way ANOVA; F represents the expression level of CSF2 detected by Western blotting after treatment with the PRMT5 inhibitor GSK591; G represents the co-precipitation results of H4R3, H3R2, and H3R8 methylated antibodies with the CSF2 promoter after treatment with the PRMT5 inhibitor GSK591 by ChIP-qPCR. ***P<0.001. t-test; HI represents the BLI signal monitoring and H&E staining images of the C57BL / 6J mouse bone metastasis model after left ventricular injection of RM-1 cells in each experimental group, and the statistical results of BLI signal intensity and tumor area of ​​bone metastasis lesions. ns, not significant, *P<0.05, ***P<0.001. One-way ANOVA; J represents the in vivo MDSCs infiltration analysis and statistical results detected by multicolor flow cytometry in each experimental group. ns, not significant. ***P<0.001. One-way ANOVA; K represents the co-culture assay and statistical results of in vitro MDSC amplification in each experimental group detected by multicolor flow cytometry (CFSE staining). ns, not significant. ***P<0.001. One-way ANOVA.

[0031] Figure 10A shows the experimental protocol and groupings for anti-CSF2 combined with ICT therapy: IgG group (n=10), anti-CSF2 group (n=10), anti-PD-1 group (n=10), and anti-CSF2 combined with PD-1 therapy group (n=10); B shows representative BLI signal monitoring images of bone metastases; C shows representative microCT images of bone metastases (arrows indicate osteolytic lesions). Scale bar, 1 mm. Representative H&E staining images (T, tumor; N, adjacent non-tumor tissue). Scale bar, 200 μm and 50 μm. Representative Ki-67 immunohistochemical staining images. Scale bar, 50 μm; D shows the incidence of bone metastases in each experimental group. ***P<0.001. Chi-square test; E shows the statistical results of BLI signal intensity of bone metastases in each experimental group. ns, not significant, ***P<0.001. One-way ANOVA; F shows the quantification results of osteolytic area by microCT. *P<0.05, ***P<0.001. One-way ANOVA; G represents the quantitative result of H&E staining of tumor lesion extent. *P<0.05, ***P<0.001. One-way ANOVA; HI represents the overall survival curve (H) and bone metastasis-free survival curve (I) of mice in each experimental group. *P<0.05, **P<0.01, ***P<0.001. Log-Rank test; J represents the statistical result of Ki-67 immunohistochemical staining score. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA.

[0032] Figure 11 A: Multicolor flow cytometry analysis of the proportion of MDSCs infiltrating bone metastases in each experimental group and statistical results. ns, not significant. ***P<0.001. One-way ANOVA; B: Multicolor flow cytometry analysis of CD4+ in bone metastases in each experimental group. + T and CD8 + T cell infiltration ratio and statistical results. ns, not significant, *P<0.05, ***P<0.001. One-way ANOVA; C represents the detection of IFN-γ in bone metastases in each experimental group by multicolor flow cytometry. + CD8 + T cell infiltration rate and statistical results. ns, not significant. *P<0.05,**P<0.01,***P<0.001. One-way ANOVA.

[0033] Figure 12A shows the experimental protocol and grouping of GSK3326595 combined with ICT therapy: IgG group (n=10), GSK3326595 group (n=10), anti-PD-1 group (n=10), and GSK3326595 combined with PD-1 therapy group (n=10); B shows representative BLI signal monitoring images of bone metastases; C shows representative microCT images of bone metastases (arrows indicate osteolytic lesions). Scale bar, 1 mm. Representative H&E staining images (T, tumor; N, adjacent non-tumor tissue). Scale bars, 200 μm and 50 μm. Representative Ki-67 immunohistochemical staining images. Scale bar, 50 μm; D shows the incidence of bone metastases in each experimental group. ***P<0.001. Chi-square test; E shows the statistical results of BLI signal intensity of bone metastases in each experimental group. ns, not significant, ***P<0.001. One-way ANOVA; F represents the quantification result of osteolysis area by microCT. ns, not significant, *P<0.05, ***P<0.001. One-way ANOVA; G represents the quantification result of tumor lesion extent by H&E staining. ns, not significant, **P<0.01, ***P<0.001. One-way ANOVA; HI represents the overall survival curve (H) and the survival curve without bone metastasis (I) of mice in each experimental group. ns, not significant, *P<0.05, **P<0.01, ***P<0.001. Log-Rank test; J represents the statistical result of Ki-67 immunohistochemical staining score. ns, not significant, *P<0.05, ***P<0.001. One-way ANOVA.

[0034] Figure 13 A: Multicolor flow cytometry analysis of the proportion of MDSCs infiltrating bone metastases in each experimental group and statistical results. ns, not significant. ***P<0.001. One-way ANOVA; B: Multicolor flow cytometry analysis of CD4+ in bone metastases in each experimental group. + T and CD8 + T cell infiltration ratio and statistical results. ns, not significant, ***P<0.001. One-way ANOVA; C represents the detection of IFN-γ in bone metastases in each experimental group by multicolor flow cytometry. + CD8 + T cell infiltration rate and statistical results. ns, not significant. *P<0.05,**P<0.01,***P<0.001. One-way ANOVA.

[0035] Figure 14A shows the expression of BHLHE22 in human PCa bone metastasis tissue samples detected by IHC. 0 points (-), 1-4 points (+), 5-8 points (++), 9-12 points (+++); B shows the representative PRMT5 staining results and scoring statistics in human PCa bone metastasis tissue samples. Scale bar, 50µm. ns, not significant. t-test; C shows the expression of CD4+ in human PCa bone metastasis tissue samples detected by IHC. + T(CD4), CD8 + Infiltration of T(CD8) cells and MDSCs (CD33), and images stained with BHLHE22, CSF2, and Ki-67. Scale bar, 50 μm; D represents CD4. + T, CD8 + Statistical results of T cell and MDSC infiltration. *P<0.05,**P<0.01,***P<0.001. t-test; E represents the statistical results of CSF2 expression detected by IHC. ***P<0.001. t-test; F represents the statistical results of Ki-67 staining score. **P<0.01. t-test; GJ represents BHLHE22 and CD4. + T cells (r = -0.585, P < 0.001), CD8 + Correlation analysis of T cell (r = -0.593, P < 0.001), MDSCs (r = 0.688, P < 0.001) infiltration, and CSF2 expression (r = 0.678, P < 0.001). Spearman test; K is a schematic diagram of the molecular mechanism by which BHLHE22 drives the formation of an immunosuppressive bone microenvironment to promote PCa bone metastasis. Detailed Implementation

[0036] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0037] Example 1

[0038] 1. Materials and Methods

[0039] (1) Cell Culture

[0040] Prostate cancer cell lines PC-3 and RM-1 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. PC-3 cells were cultured in RPMI-1640 medium with 10% FBS and 1% penicillin and streptomycin (100 mg / ml). RM-1 cells were cultured in Dulbecco modified Eagle medium (DMEM) with 10% FBS and 1% penicillin and streptomycin (100 mg / ml). Incubation conditions were 5% CO2 at 37°C. Cell morphology was observed regularly under a microscope, and the medium was changed periodically as needed. Cells at 70-90% confluence were selected for in vitro and in vivo experiments.

[0041] (2) Plasmids, plasmid transformation and plasmid extraction

[0042] Plasmids: Human BHLHE22 (NM_152414.5) cDNA was amplified by PCR and cloned into the pCDH-puro plasmid vector (pCDH-BHLHE22-Puro-Flag). The pCDH-puro plasmid vector was used as a control. Mouse BHLHE22 (NM_021560.4) cDNA was amplified by PCR and cloned into the pLVX-hyg plasmid vector (pLVX-BHLHE22-Hyg-Flag). The pLVX-hyg plasmid vector was used as a control. Plasmid transformation: Competent E. coli were thawed on ice. 50 μL of the suspension was added to a 1.5 mL EP tube, and 5 μL of the target plasmid solution (not exceeding 50 ng in concentration and 10 μL in volume) was added. The tube was incubated at 42 °C for 90 seconds, then immediately placed on ice for 2 min. 600 μL of LB liquid medium (antibiotic-free) was added to the EP tube, mixed well, and cultured at 37 °C with shaking for 1 h (225 rpm). Centrifuge, resuspend, and plate (LB agar plates containing Amp), incubate upside down at 37°C for 12-16 hours. Select single colonies and add to 1.5 ml EP tubes (LB medium containing Amp). Incubate at 37°C with shaking for 6 hours (225 rpm). Store the strain in 15% glycerol at -80°C.

[0043] Plasmid extraction: Plasmid mini-prep kits (HiPure Plasmid MiniPrep Kit) and maxi-prep kits (HiPure Plasmid MaxiPrep Kit) were used, and the procedures were followed according to the kit instructions. The experimental method is as follows: The bacterial culture was taken out of the -80℃ freezer. A small amount of bacterial culture was picked up with a 10ul pipette tip and added to a 1.5ml EP tube (LB medium containing Amp). The bacterial culture was activated (37℃, 225rpm, shaken for 1 hour). 200-500ul of the activated bacterial culture was added to a 15ml centrifuge tube (LB medium containing Amp, mini-prep) or a 100ml Erlenmeyer flask (LB medium containing Amp, maxi-prep). The plasmid was gently shaken or vigorously shaken (37℃, 225rpm, shaken for 12-16 hours). After the bacterial culture is fully turbid, centrifuge at 12000×g for 2 min, remove the supernatant, add RB, LB and NB from the kit in sequence, centrifuge at 12000×g for 15 min, aspirate the supernatant and add it to the centrifuge column (pass through the column multiple times), centrifuge at 8000×g for 2 min, add TB and WB from the kit in sequence, centrifuge at 8000×g for 5 min, incubate at room temperature for 10 min, add 1-2 ml of deionized water, centrifuge at 8000×g for 2 min to elute the DNA, determine the plasmid concentration, and store at -20℃.

[0044] (3) Cell transfection

[0045] After digestion and resuspending of logarithmically growing cells for counting, 5.0 × 10⁻⁶ cells were counted. 5 Cells were seeded per well into 6-well plates. After 24 hours, when cell confluence reached 70%-80%, the culture medium was replaced with serum-free Opti-MEM, and transfection was initiated. Two sterile 1.5ml EP tubes were prepared. The plasmid concentration was calculated. Tube A contained 125μl Opti-MEM + 7.5μl Lipofectamine 3000; tube B contained 125μl Opti-MEM + 5μl P3000 + 2.5μg plasmid. After incubation at room temperature for 5 minutes, the reagents from tubes A and B were mixed, and incubated again at room temperature for 20 minutes. The mixture was then added to the 6-well plates, the plates were gently shaken, and the cells were returned to the incubator for further culture. Forty-eight hours after transfection, RNA was extracted from the cells to verify the transfection efficiency, and the next step of the experiment was performed.

[0046] (4) Constructing stable cell lines

[0047] One day prior to transfection, 293T cells were seeded in 10cm cell culture dishes, achieving 70%-80% confluence on the day of transfection. The cell culture medium was then replaced with serum-free Opti-MEM. Two sterile 1.5ml EP tubes were prepared, and plasmid concentrations were calculated. Tube A contained 125μl Opti-MEM + 36μl Lipofectamine 3000; tube B contained 125μl Opti-MEM + 24μl P3000 + 3μg recombinant plasmid and 9ug virus packaging plasmid mixture. After incubation at room temperature for 5 minutes, the reagents from tubes A and B were mixed, and incubated again at room temperature for 20 minutes. The mixture was then transferred to a 10cm cell culture dish, gently agitated, and the cells were returned to the incubator for further culture. Twenty-four hours after transfection, the medium was replaced with DMEM containing 10% FBS. The virus-containing supernatant was harvested 48-72 hours after transfection. After centrifugation and filtration, the supernatant was stored at -80°C.

[0048] After digesting and resuspending logarithmically growing cells for counting, the cells were analyzed at a concentration of 5.0 × 10⁻⁶. 5 To determine the cell / well ratio, seed PC-3 and RM-1 cells into 6-well plates and culture until cell confluence reaches 50-60%. Replace the culture medium and add polybrene (5 μg / ml). Calculate the required amount of virus solution to add per well using the formula: (cell count × MOI / virus titer) × 10⁻⁶. 3 The MOI value of the PC-3 cell line was 10, and the MOI value of the RM-1 cell line was 100. Fresh medium was added 24 hours after infection. After subculturing, positive cells were selected using 2 μg / ml puro-containing medium or 200 μg / ml hygromycin-containing medium. After continuous selection for 5-7 days, RNA was extracted from the cells to verify transfection efficiency. Cells were then passaged, cryopreserved, and used for further experiments.

[0049] (5) Western blot

[0050] Extracting cellular proteins: Aspirate the culture medium from the cell culture dish, wash twice with pre-chilled PBS, place the dish on ice, and add cell lysis buffer. Scrape cells with a pre-chilled cell brush and collect the lysis buffer into 1.5 ml EP (ultrasound to disrupt cell nuclei, optional). After determining the protein concentration, add protein denaturing agent (5% mercaptoethanol), mix thoroughly, and denature at 98°C for 10 min for later use or store directly at -80°C. Extracting tissue proteins: After cutting and weighing the tissue, add lysis buffer at a ratio of 100 mg / 1 mL (50 μL system: 40 μL RIPA + 5 μL protease inhibitor + 5 μL phosphatase inhibitor + 0.5 μL PMSF). After cleaning the homogenizer head, prepare a tissue homogenate using a tissue homogenizer and transfer it into a 1.5 ml EP tube. Lyse on ice for 30 min. Centrifuge at 12000 rpm / min for 10-15 min at 4°C. Collect the supernatant into 1.5 ml of EP (Eppendorf gel), determine the protein concentration, and denature at 98℃ for 10 min for later use or store directly at -80℃. Prepare separating and stacking gels. Add 20-30 μg of protein sample to each lane. Perform electrophoresis at 80V for approximately 1 hour, followed by transfer at 300mA for 2 hours to transfer the protein onto a PVDF membrane. Block with 5% skim milk for 45 minutes, and simultaneously prepare the primary antibody (prepare different concentrations according to the requirements of different antibodies). After blocking, add the prepared primary antibody and incubate overnight on a shaker at 4℃. After 12-18 hours, remove from cold storage and wash three times with TBST for 10 minutes each time. Prepare a secondary antibody of the same species as the primary antibody (at a ratio of 1:3000), incubate at room temperature for 45 minutes, and wash three times with TBST for 10 minutes each time. Then, press into a film in a darkroom and expose for development. Select an appropriate internal control protein to normalize protein expression according to different experimental requirements.

[0051] (6) RNA extraction

[0052] RNA was extracted using the EZ-press RNA Purification Kit, following these steps: a. Sample lysis: Discard the culture medium, wash cells with PBS, add 500 μl of lysis buffer to each sample, mix thoroughly by pipetting, and transfer to a new EP tube after complete cell lysis. b. RNA binding: Add an equal volume of anhydrous ethanol to the lysate, mix thoroughly, transfer to a centrifuge column, and centrifuge at 4000×g for 1 min. c. DNase treatment: Add 2 μl of g DNARemover to 10 μl of double-distilled water and transfer to a centrifuge column. d. Washing: Add 500 μl of wash buffer to the centrifuge column, centrifuge at 12000×g for 1 min, and transfer the column to a new 1.5 ml EP tube (RNase-free). RNA elution: Add 20-30 μl of elution buffer to the centrifuge column, incubate at room temperature for 2 minutes, centrifuge at 12000×g for 1 min, discard the centrifuge column, determine the RNA concentration, and store at -80℃ for later use.

[0053] (7) Reverse transcription and real-time quantitative PCR (RT-qPCR)

[0054] Reverse transcription of RNA: RNA reverse transcription experiments were performed using the EZBioscience RNA Reverse Transcription Kit (Color Reverse Transcription Kit). RNA was treated with gDNA Remover: 2 μL of gDNA Remover was added to 100 ng - 2 u of RNA, mixed thoroughly, and reacted for 5 min. Preparation of the reverse transcription reaction system: Enzyme-free PCR tubes were prepared, and the reaction system was prepared according to the table below. The mixture was then thoroughly mixed.

[0055] Element Volume (20 μl system) Total RNA treated with gDNA Remover The above volume (X μl) 4X RT Master Mix 5μl <![CDATA[Nuclease free ddH2O]]> Make up to 20μl

[0056] Reverse transcription reaction conditions: 42℃ for 15 min, 95℃ for 30 s, collect and store cDNA at -80℃ for later use. Real-time quantitative PCR: Perform real-time quantitative PCR using a 2x Color SYBR Green qPCR Master Mix kit. Take out the prepared cDNA, place it on ice, prepare the reaction system according to the table below, mix well by pipetting, seal the plate, centrifuge, and then place it in a real-time quantitative PCR instrument.

[0057] Element 20μl system 2x Color SYBR Green qPCR Master Mix 10μl Forward primer (10 μM) 0.4μl Reverse primer (10 μM) 0.4μl cDNA 2μl <![CDATA[ddH2O]]> Make up to 20μl

[0058] Amplification conditions: pre-denaturation at 95℃ for 5 min. Subsequently, denaturation at 95℃ for 15 s, followed by annealing and extension at 60℃ for 45 s, for a total of 40 cycles. GAPDH was used as an internal reference gene for normalization. All primer sequences are shown in Table 1.

[0059] Table 1 RT-PCR primer sequences

[0060]

[0061]

[0062] (8) Immunohistochemistry (IHC)

[0063] All samples used in this experiment were paraffin-embedded sections. Immunohistochemistry was performed using the Zhongshan Jinqiao reagent kit. The specific steps were as follows: baking in a 60℃ oven (2-3 hours); dewaxing with xylene (5 min × 3 times); hydration (100% ethanol × 2 times, 95% ethanol × 1 time, 75% ethanol × 1 time; 3 min each time); removal of endogenous catalase (3% H₂O₂, 37℃, shaker for 15 min); microwave retrieval in citric acid retrieval solution (high heat for 5 min, medium heat for 20 min, then natural cooling); after drying, marking with an immunohistochemical pen; membrane perforation (containing 0.3% Triton). Incubate X-100 with PBS for 15-30 min (optional); rinse twice with PBS-T, then once with PBS; place the slides in a humidified chamber and block with blocking buffer (Solution A, goat anti-rabbit blocking buffer) for 30 min at room temperature; discard the blocking buffer and add primary antibody (diluted with antibody diluent), incubate overnight at 4°C (12-24 hours); remove the humidified chamber from the cold storage, warm to room temperature for 30 min, and rinse three times with PBS-T; incubate with Solution B for 30 min (biotin-labeled goat anti-mouse / rabbit IgG); rinse twice with PBS-T, then once with PBS; incubate with Solution C for 30 min. n (streptavidin-peroxidase); rinse twice with PBS-T, then once with PBS; observe under a microscope for DAB staining (experiment with optimal concentration and time); rinse with double-distilled water to stop DAB staining; hematoxylin staining for 1-2 min; differentiate with 95% hydrochloric acid alcohol for 10 s; return to blue (rinse with tap water for 30-45 min); observe the hematoxylin staining effect under a microscope; dehydrate (60% ethanol, 80% ethanol, and 100% ethanol for 3 min each); replace alcohol with xylene (5 min × 3 times); place in a fume hood for ventilation overnight; mount with neutral resin. Calculate the percentage of positive cells and staining intensity under a microscope to assign an IHC score. Percentage of positive cells: 0 (<5%), 1 (5%–25%), 2 (25%–50%), 3 (50%–75%), 4 (>75%). Staining intensity: 0 (no staining), 1 (pale yellow), 2 (brownish yellow), 3 (brownish brown). IHC scoring formula = percentage of positive cells × staining intensity score. 0 points is negative (-), 1-4 points is weakly positive (+), 5-8 points is moderately positive (++), and 9-12 points is strongly positive (+++).

[0064] (9) Migration and invasion experiments

[0065] Remove the Matrigel from the container and refrigerate overnight at 4°C. For invasion assays, use 50 mg / ml Matrigel to coat the Transwell chamber polycarbonate filter membrane (6.5 mm Transwell, 8 μm pore size). For migration assays, Matrigel coating is not required. Add 200 μl of serum-free culture medium to the Transwell chamber and 500 μl of complete culture medium containing 10% FBS to the lower chamber. Digest and collect cells in logarithmic growth phase, and then rehydrate at a rate of 1 × 10⁻⁶ cells / mL. 5 Seed cells at a density of 10 cells / ml into the Transwell chambers and incubate for 24-48 hours. Remove the colloid from the chambers (this step is not required for migration experiments), add 1 ml of 4% paraformaldehyde for fixation for 30 minutes, rinse once with PBS, stain with crystal violet for 10-20 minutes, rinse thoroughly with tap water, wipe off the upper layer of cells with a cotton swab, and air dry overnight. The next day, observe the inverted Transwell chambers under a microscope, take photos, and count the number of cells that have passed through.

[0066] (10) Animal experiments and drug treatment

[0067] The welfare and ethics of animal experiments were approved by the Laboratory Animal Management and Ethics Committee of Sun Yat-sen University (approval number SYSU-IACUC-2022-000178). The Laboratory Animal Barrier Center of Hemu Building, Sun Yat-sen University (SYXK Yue 2019-0209) completed animal feeding and related experiments. The 4-6-week-old C57BL / 6J and BALB / c-nu mice used in the experiment were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. Modeling and evaluation of bone metastasis status: In this experiment, under the induction of inhalation anesthesia with 2% isoflurane and maintenance of intraperitoneal anesthesia with 1% sodium pentobarbital, luciferase-labeled PCa cells were injected into the left ventricle to model PCa bone metastasis. After successful modeling, in vivo bioluminescence imaging (BLI) was performed twice a week to monitor the bone metastasis of mice. The specific steps are as follows: C57BL / 6J or BALB / c-nu mice were inhaled with 2% isoflurane to induce anesthesia, and intraperitoneal injection of 1% sodium pentobarbital was used to maintain the anesthesia state; RM-1 or PC-3 cells were resuspended in sterile PBS buffer to configure a cell concentration of 2×10 5 cells / ml, and 100 μl was aspirated with an insulin syringe for standby. Locate and disinfect the mouse heart injection site, and after seeing blood backflush when inserting the needle vertically, inject all the cells into the left ventricle. After 2 minutes, 100 μl of fluorescein sodium salt solution (15 mg / ml, Yeasen) was injected intraperitoneally; after 5 minutes, the anesthetized mice were placed in a live imaging instrument (Caliper Life Sciences, USA) to detect the fluorescein signal to confirm whether the modeling was successful. After the mice showed cachectic states such as hunchback in the later stage of the experiment, the mice were sacrificed by cervical dislocation under anesthesia, and the two legs of the mice were dissected and collected, fixed with formalin and stored. Small animal microCT scanning (Bruker, Skyscan1276) was used and the osteolytic area was measured on the CT image. The mouse leg bones were paraffin-embedded and sectioned for use in experiments such as IHC and H&E. ImageJ V1.52 was used to analyze and calculate the osteolytic and tumor lesion areas.

[0068] Drug treatment: For in vivo MDSCs depletion experiments, Anti-Gr-1 was intraperitoneally injected (200 μg / animal, twice a week, BioXcell). For in vivo MDSCs expansion experiments, recombinant mouse CSF2 was subcutaneously injected (0.6 μg / animal, twice a week, PeproTech); Anti-CSF2 was intraperitoneally injected (20 μg / animal, twice a week, BioXcell). For in vitro MDSCs expansion experiments, recombinant mouse CSF2 was added to the medium containing 10% FBS (concentration 20 ng / ml, PeproTech); Anti-CSF2 antibody was added to the medium containing 10% FBS (concentration 20 ng / ml, BioXcell). The same dose and the same concentration of isotype IgG antibody were used as controls.

[0069] (11) Multilabeled tissue immunofluorescence (IF)

[0070] Immunofluorescence staining of tissues was performed using the PANOVUE four-color multi-label kit (human, TSA-RM) and the four-color multi-label kit (mouse, TSA-Rab), following the instructions for use. The specific steps are as follows: First round of single staining: baking slides at 60℃ for 2-3 hours; dewaxing with xylene for 5 minutes, repeated 3 times; gradient ethanol hydration: 100% ethanol × 2 times, 95% ethanol × 1 time, 75% ethanol × 1 time, 3 minutes each time; removal of endogenous catalase at 37℃ with 3% H₂O₂, shaken for 15 minutes; microwave retrieval with citrate retrieval solution, high for 5 minutes, medium for 20 minutes; natural cooling, spin-drying, and histochemical staining with a pen; staining with 0.3% Triton... Incubate X-100 with PBS for 15-30 min (to break the membrane, optional); rinse twice with TBST; place the slide in a humidified chamber and block with blocking solution (goat anti-rabbit blocking solution) for 20 min at room temperature; incubate with primary antibody for 1 h at room temperature under humid conditions, then rinse twice with TBST; incubate with secondary antibody for 10 min at room temperature under humid conditions, then rinse twice with TBST; for fluorescence staining to amplify the signal, add 100 μL of 1× dye working solution (diluted 1:100 with signal amplification solution) to the slide, incubate for 10 min at room temperature under humid conditions, then rinse twice with TBST. Follow-up staining: Repeat all steps from microwave repair to fluorescence staining to amplify the signal three times (using a different fluorescent dye each time, with the last stain being DAPI). After rinsing twice with TBST, rinse once with sterile water. Add a strong quenching-resistant mounting medium to the slide, cover with a coverslip, mount with nail polish, and store in a dark room or observe and interpret the results under a fluorescence microscope. Analyze and calculate the fluorescence intensity grayscale value using ImageJ V1.52.

[0071] (12) Multicolor flow cytometry

[0072] Mice were euthanized by cervical dislocation, disinfected with 75% alcohol, and the leg bones were dissected, removing the leg bone muscles and soft tissues. The mice were then placed in P60 culture dishes. A digestion solution (composition: 1.25 mg / ml collagenase D, 0.85 mg / ml collagenase V, 50 μg / ml DNase I, 1 mg / ml dispersin II, 1% penicillin antibody, 10% FBS, and 10 mM HEPES) was added to the culture dish. The leg bone was cut into several small segments and ground to release bone marrow cells. The cells were incubated at 37°C and 80 rpm for 40 min on a shaker. The cells were filtered through a cell strainer, and the cell suspension was collected and centrifuged (1500 rpm, 5 min). 1 ml of erythrocyte lysis buffer was added, and the cells were lysed at room temperature for 2 min. The cells were resuspended and centrifuged (1500 rpm, 5 min). PBS containing 2% FBS was added, and the cells were resuspended and aliquoted into 1.5 ml EP tubes (100 μl / tube). Add 1 μL of anti-mouse CD16 / 32 antibody and 1 μL of anti-mouse Ig2G antibody to each tube, and block on ice for 30 min. Add 0.5 μL of staining antibody to each tube and stain in the dark for 30 min. Rinse once, resuspend, and centrifuge (1500 rpm, 5 min), discarding the supernatant. Add 300 μL of PBS containing 2% FBS to each tube, resuspend, and then analyze using a flow cytometry (CytoFLEX, BECKMAN). Analyze the data using FlowJo V10.6.2. See Table 2 for relevant colorimetric schemes and antibodies.

[0073] Table 2 Color schemes for multicolor flow cytometry

[0074]

[0075] (13) Cell sorting

[0076] Using CD8 + T-cell sorting kit (STEMCELL, Catalog #19853) was used to sort CD8 cells from the spleen of healthy mice. + T cells. MDSCs were obtained from the spleen of tumor-bearing mice using an MDSCs sorting kit (STEMCELL, Catalog #19867). The specific steps were as follows: a single-cell suspension was prepared, and rabbit blocking serum (CD8+) was added. + T-sorting (50 μL / ml) or FcR blocker (MDSCs sorting: 40 μL / ml), add Cocktail (50 μL / ml), mix well by pipetting, and incubate for 10 min; shake for 30 s, then add streptavidin RapidSpheres (CD8). +T-sorting: 125ul / ml; MDSCs sorting: 75ul / ml), mix by pipetting and incubate for 5 minutes; bring each tube to 2.5ml, place on a magnetic rack, and let stand for 3 minutes; invert the magnetic rack and pour the liquid from the tube into a new flow cytometer tube for analysis (CytoFLEX, BECKMAN) or prepare for the next experiment.

[0077] (14) T cell proliferation inhibition experiment

[0078] Pre-experiment preparation (Coating): Prepare PBS with a concentration of 1 μg / ml anti-CD3 antibody. Add 300 μl to each well of a 12-well plate and incubate overnight at 4°C. Reserve uncoated wells. Cell labeling (CFSE): Prepare and count sorted MDSCs and CD8+ T cells. Resuspend cells in PBS containing 10% FBS and prepare 5 × 10⁻⁶ cells / well plate. 6 Cells / ml concentration; add 2 uM CFSE, vortex to mix, let stand for 10 min; centrifuge (1500 rpm, 5 min), discard the supernatant, rinse twice, and resuspend the cells in fresh medium containing 10% FBS.

[0079] Co-culture: Remove the coated plate and resuspend CD8. + T cells were added to anti-CD28 antibody to a final concentration of 1 μg / ml. The antibody was administered at ratios of 1:0, 1:1, and 1:5 (CD8). + T:MDSCs) will CD8 + T cells and MDSCs were added to each well for culture; CD8 cells were not stimulated with anti-CD3 and anti-CD28 antibodies. + T cells were used as a control. Cells were incubated in an incubator (5% CO2, 37°C), with the culture medium replaced periodically as needed. Data were analyzed on days 4-5 of co-culture. FlowJo V10.6.2 was used for data analysis.

[0080] (15) Multifactor protein chip detection

[0081] The Mouse Cytokine Array C2 (Raybiotech, AAM-CYT-2) multifactor protein chip was used for detection, and the procedure was followed according to the instructions. The specific steps are as follows: Sample preparation: Collect the supernatant (2% FBS medium) of RM-1-BHLHE22 and RM-1-Vector cells cultured for 48 hours under low-concentration serum medium. Add 2 ml of blocking buffer to the incubation chamber, completely covering the membrane surface, and incubate at room temperature with shaking for 1 hour. Discard the blocking buffer, add 700 μL of sample, and incubate overnight at 4°C with shaking. Discard the sample and rinse. Prepare biotin-labeled antibody, rapidly centrifuge the tubes containing the biotin-labeled antibody, add 2 ml of 1× blocking buffer to each tube, mix well, and then add 1 ml to each of the two membranes for incubation at room temperature for 2 hours. After rinsing, add 2 ml of HRP-streptavidin and incubate at room temperature with shaking for 2 hours. After rinsing, prepare a mixture of detection solutions C and D. Add 0.5 mL of the mixture to each membrane, incubate for 2 min, and then blot dry with filter paper. Perform analysis using an ImageQuant LAS4000 chemiluminescence imaging system or store at -20°C. Multifactor protein detection indicators are shown in Table 3.

[0082] Table 3. Detection Indicators of Multifactor Protein Chip

[0083]

[0084] (16) Cellular transcriptome sequencing (RNA-seq)

[0085] RM-1 cells (3×3) and PC-3 cells (3×3) from the logarithmic growth phase BHLHE22 overexpression group and the Vector group were collected. RNA extraction quality control: RNA was extracted using the Tianmo#TR205-200 kit. Total RNA integrity was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), and total RNA concentration and purity were determined using a Qubit 3.0 Fluorometer (Life Technologies, CA, USA) and a Nanodrop One Spectrophotometer (Thermo Fisher Scientific Inc, USA). RNA library construction: mRNA isolation → mRNA fragmentation → first-strand cDNA synthesis → second-strand cDNA synthesis → strand end repair → 3' end A addition → adapter addition → strand enrichment → sequencing sample library construction completed. The concentration of the constructed library was assessed using a Qubit 3.0 Fluorometer, and the library size was assessed using an Agilent 2100. Only libraries meeting the construction standards were allowed for subsequent sequencing.

[0086] Sequencing: According to the standard procedure of the cBot User Guide, cluster generation and hybridization of the first-strand sequencing primers were performed on the cBot attached to the Illumina NovaSeq 6000 sequencer. Prepare the sequencing reagents in advance as shown in the Illumina NovaSeq6000 UserGuide, and load the flow cell with clusters onto the machine. Select the paired-end program for paired-end (PE) sequencing. The sequencing process was controlled by the data collection software provided by Illumina, and the sequencing result data was analyzed in real time.

[0087] (17) Tissue sample collection

[0088] In this study, biopsy samples, surgical samples, normal prostate tissue samples, and clinical pathological data of prostate cancer patients were collected from the Department of Pathology of Guangzhou First People's Hospital and the First Affiliated Hospital of Sun Yat-sen University from May 2008 to March 2020. The tissue samples were embedded in paraffin, sectioned, and stored, or stored in a liquid nitrogen tank. A total of 132 prostate cancer in situ tissue samples without bone metastasis and 60 prostate cancer in situ tissue samples with bone metastasis, as well as 30 prostate cancer bone metastasis tissue samples were used in this study. The clinical samples and clinical pathological data of prostate cancer patients in this study were approved by the patients' consent and the ethics committee. The total number of followed-up patients and related clinical pathological data are shown in Table 4.

[0089] Table 4 Clinical pathological data of prostate cancer patients

[0090]

[0091] Note: PSA, Prostate-specific Antigen; BM, Bone Metastasis; n-BM: Non-bone Metastasis; x 2 test, chi-square test.

[0092] (18) Statistical analysis

[0093] Statistical analysis and graphs were performed using SPSS 17.0 and GraphPad Prism 8. The t-test was used for two groups of continuous normally distributed data, and the Mann-Whitney U test was used for continuous non-normally distributed data. One-way ANOVA was used for three or more groups of normally distributed data. Spearman correlation analysis was used to analyze the correlation between non-normally distributed and ordinal data; Kaplan-Meier analysis and log-rank test were used for survival data. The significance level was set at 0.05, two-tailed. Experimental data are expressed as mean ± standard deviation, and each experiment was independently repeated three times. *P<0.05, **P<0.01, ***P<0.001.

[0094] 2. Experimental Results

[0095] (1) As Figure 1 As shown in Figure A, the expression level of BHLHE22 in PCa primary lesions with bone metastasis (PCa / BM) was significantly increased compared to that in PCa primary lesions without bone metastasis (PCa / nBM), and the expression level was further increased in PCa bone metastasis (BM). Next, we analyzed the expression profile sequencing results from the GEO database GSE77930, as shown... Figure 1 As shown in Figure B, BHLHE22 expression was significantly upregulated in PCa bone metastases compared to the primary PCa lesion and other organ metastases. Further analysis based on data from TCGA-PRAD revealed that BHLHE22 levels were significantly higher in PCa / BM compared to PCa / nBM. Figure 1 C). Furthermore, Kaplan-Meier analysis based on TCGA-PRAD showed that high BHLHE22 expression predicted shorter disease-free survival. Figure 1 D). Consistent with our PCa patient follow-up results, high BHLHE22 expression predicted poor clinicopathological features and shorter overall survival and bone metastasis-free survival (Table 4). Figure 1 E and Figure 1 F). The above results suggest that BHLHE22 is upregulated in PCa tissues with bone metastases, and its expression level is further increased in PCa bone metastasis tissues, and it is associated with the bone metastasis status and poor prognosis of PCa patients.

[0096] (2) Bone metastasis mouse models (immunogenic C57BL / 6J mice and immunodeficient BALB / c nude mice) were established by injecting luciferase-labeled PCa cells (RM-1 and PC-3 cell lines) into the left ventricle. Bone metastasis was monitored using in vivo bioluminescence (BLI). We observed that overexpression of BHLHE22 in murine prostate cancer RM-1 cells did not significantly differ in tumor bone metastasis ability between immunodeficient BALB / c nude mice. Figure 2 A- Figure 2 C). Meanwhile, overexpression of BHLHE22 in human prostate cancer PC-3 cells did not significantly alter the tumor bone metastasis rate in immunodeficient BALB / c nude mice. Figure 2 D- Figure 2 F). Interestingly, in syngeneic, immunocompetent C57BL / 6J mice, BHLHE22 overexpression significantly promoted RM-1 cell tumor bone metastasis (F). Figure 2 G). Subsequently, we further analyzed the osteolytic area and tumor lesion extent of bone metastases in C57BL / 6J mice using MicroCT scanning and H&E staining. MicroCT scanning and H&E staining analysis showed that in immune-active C57BL / 6J mice, the group with RM-1 cells overexpressing BHLHE22 had a larger area of ​​osteolytic lesions and tumor lesions (G). Figure 2 H- Figure 2 K). Survival analysis of immunocompetent C57BL / 6J mice showed that overexpression of BHLHE22 predicted shorter overall survival and bone metastasis-free survival. Figure 2 L and Figure 2 M). Homology analysis of the UCSC genome database (PhyloP algorithm) showed that BHLHE22 has good conservation in mice and humans during evolution. Figure 3 A). The high conservation of BHLHE22 (89% sequence identity at the cDNA level and 94% sequence identity at the protein level) suggests that BHLHE22 plays a similar regulatory role in humans and mice. Furthermore, we evaluated the biological function of BHLHE22 in vitro. Transwell migration / invasion assays showed no significant difference in migration and invasion abilities between the BHLHE22 overexpression group and the vector group in RM-1 and PC-3 cells. Figure 3 B and Figure 3 C). In summary, we found that BHLHE22 exhibited different tumor-promoting bone metastasis capabilities between immunocompetent and immunodeficient mice. Furthermore, different performances were observed between in vivo and in vitro experiments assessing tumor cell metastasis capabilities.

[0097] Based on BHLHE22 expression, we performed gene set enrichment analysis (GSEA) on the TCGA-PRAD dataset. Interestingly, GSEA analysis showed that the negative regulatory pathways of immune response and IFN-γ production were activated in the high BHLHE22 expression group. Figure 3 D). Furthermore, analysis of our BHLHE22-overexpressing and control PCa cell RNA sequencing (RNA-seq) results showed that BHLHE22-overexpressing PCa cells activated negative regulatory pathways of the immune response and T cell-mediated negative immune regulation. Figure 3 E). Meanwhile, Gene Ontology results (GO analysis) indicate that the set of genes related to immune response is enriched in the Top 10 (E). Figure 3 F). The above results suggest that BHLHE22 may promote PCa bone metastasis in an immune-related manner.

[0098] (3) The multi-label tissue immunofluorescence assay (IF) was used, and human MDSCs cell markers (CD33) and CD8 were selected. + T cell markers (CD8), BHLHE22, and DAPI were used to detect PCa / BM and BM tissues, respectively. Figure 4 A and Figure 4 B). Our results suggest that, in both PCa / BM and BM tissues, the number of MDSCs infiltrating in the high BHLHE22 expression group was significantly higher than that in the low BHLHE22 expression group. + The number of infiltrating T cells was significantly higher in the high BHLHE22 expression group ( Figure 4 D and Figure 4 E). Subsequently, we further examined the infiltrating MDSCs (Gr-1) in the bone marrow of C57BL / 6J mice after left ventricular injection of RM-1-BHLHE22 and RM-1-Vector cells using IF. + CD4 + T and CD8 + T cell count. Our results showed that, compared with the RM-1-Vector group, the RM-1-BHLHE22 group significantly increased the number of MDSCs infiltrating the bone marrow and decreased the number of CD4+ cells in the bone marrow. + T and CD8 + The number of infiltrating T cells ( Figure 4 C and Figure 4 F).

[0099] Bone marrow cell specimens from C57BL / 6J mice after left ventricular injection of RM-1-Vector and RM-1-BHLHE22 cells were collected, and immune cell population analysis was performed using multicolor flow cytometry. Firstly, CD45 in the bone marrow of RM-1-BHLHE22 and RM-1-Vector cells was analyzed. + 7AAD - Cells and CD3 + The proportion of CD11b- cells was relatively high. Figure 4 G and Figure 4 H). Subsequently, we measured CD45. + 7AAD - Mononuclear MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) in cells, CD4 + T cells, CD8 + The infiltration rates of T cells, Tregs, macrophages, tumor-associated macrophage-1 (TAM-1), tumor-associated macrophage-2 (TAM-2), and NK cells were observed. We found that compared to the RM-1-Vector group, the RM-1-BHLHE22 group significantly increased the infiltration rate of MDSCs (especially M-MDSCs), but significantly reduced CD4 counts. + T and CD8 + The proportion of T cell infiltration ( Figure 4 I- Figure 4 L). In addition, we investigated the factor Arg-1, which is associated with MDSC function, and its relationship with CD8. + T cell function-related factors IFN-γ and PD-1. Results showed that Arg-1 was present in RM-1-BHLHE22 bone metastasis samples. + The proportion of MDSCs increased ( Figure 4 I and Figure 4 J), IFN-γ + CD8 + The proportion of T cells decreased, PD-1 + CD8 + The proportion of T cells increased ( Figure 4 K and Figure 4 M). Furthermore, to confirm these CD11b... + Gr-1 + The cells were indeed functional MDSCs, and we conducted an in vitro co-culture proliferation inhibition experiment with MDSCs and T cells. The sorted CD11b cells were then... + Gr-1 + Cells and CD8 + T cells were co-cultured in different proportions. The results showed that CD11b... + Gr-1 +The cells strongly inhibited the proliferation and activation of T cells induced by CD3 and CD28 antibodies. Figure 4 N and Figure 4 O). Subsequently, C57BL / 6J mice with bone metastasis modeling were injected with Anti-Gr-1 antibody to deplete MDSCs in the mice. The results showed that after depletion of MDSCs with Anti-Gr-1 antibody, the number of MDSCs in the RM-1-BHLHE22 group was significantly reduced, and the number of T cells was significantly increased. Figure 5 A and Figure 5 B). We also analyzed other immune cell populations in bone marrow cell specimens from bone metastases in the RM-1-Vector and RM-1-BHLHE22 groups, and the results showed no significant difference in the infiltration ratios of Treg, macrophages, TAM-1, TAM-2, and NK cells. Figure 5 C- Figure 5 F). In summary, we believe that PCa cells with high BHLHE22 expression induce MDSC infiltration and drive the formation of an immunosuppressive TME in PCa bone metastases through MDSC-exhausted T cells.

[0100] (5) Stable prostate cancer cell lines of mouse RM-1 and human PC-3 overexpressing BHLHE22, along with their corresponding Vector group cells, were analyzed by RNA-seq. The results showed that, compared to the Vector group, RM-1-BHLHE22 and PC-3-BHLHE22 co-expressed 103 genes were upregulated, and 37 genes were downregulated (FC > 1.5). Among these differentially expressed genes, we focused on identifying genes mediating direct cell-cell interactions and genes secreting cytokines. Surprisingly, colony-stimulating factor-2 (CSF2) was the only secreted cytokine co-upregulated in RM-1-BHLHE22 and PC-3-BHLHE22. Figure 6 A). Subsequently, we used a multifactor protein chip to analyze the culture supernatant of RM-1-BHLHE22 and RM-1-Vector cells. The results showed that CSF2 was significantly increased in the culture supernatant of RM-1-BHLHE22 cells. This suggests that RM-1 cells secrete more CSF2 after overexpression of BHLHE22. Figure 6 B). Meanwhile, Western blot results showed that CSF2 expression was increased in PC-3 and RM-1 cell lines overexpressing BHLHE22 and in BM tumor tissues of C57BL / 6J mice in the BHLHE22 group. Figure 6 C and Figure 6 D). The results above show that overexpression of BHLHE22 increases the expression and secretion of CSF2 in PCa cells.

[0101] Bone marrow sections were collected from C57BL / 6J mice after left ventricular injection. BHLHE22, CSF2, tumor cell proliferation marker (Ki-67), and MDSCs (Gr-1, S100A9) and CD4+ were detected by multi-label tissue immunofluorescence (IF) and immunohistochemistry (IHC) staining. + T, CD8 + The relationship between T cell infiltration and tumor invasiveness. Our results suggest that, compared with the Vector group, the BHLHE22 group had a greater number of tumor-infiltrating MDSCs and CD4+. + T and CD8 + Fewer T cell infiltrations and higher CSF2 expression ( Figure 6 E- Figure 6 G). Positive staining with Ki-67 indicated that bone metastases in the BHLHE22 group mice proliferated at a faster rate. Figure 6 F and Figure 6 H). Based on IHC staining results, CSF2 + PCa cells and MDSCs (Gr-1) + Correlation analysis of cells showed that CSF2 was positively correlated with the number of MDSCs infiltrating (r = 0.645, P < 0.001). Figure 6 I). Interestingly, in the TCGA-PRAD database, BHLHE22 and CSF2 were also positively correlated (r = 0.230, P < 0.001); Figure 6 J). The above results suggest that CSF2 is BHLHE22. + PCa induces the expansion of MDSCs and is a potential functional factor driving the formation of immunosuppressive bone TME.

[0102] In the in vivo MDSC infiltration analysis experiment, C57BL / 6J mice were injected into the left ventricle with RM-1-Vector and RM-1-BHLHE22 cells, respectively, with non-tumor-bearing C57BL / 6J mice serving as the Vehicle group. Then, non-tumor-bearing mice were treated with recombinant mouse CSF2 (with isotype IgG as a control), and mice injected into the left ventricle were treated with Anti-CSF2 antibody (with isotype IgG as a control). Thirty days after injection, BLI results showed that in mice with bone metastases treated with Anti-CSF2 antibody, there was no significant difference in BLI signal between the Vector group and the control group (IgG treatment group), while the BHLHE22 group showed a smaller BLI signal and smaller bone metastatic tumor lesion size compared to the control group (IgG treatment group). Figure 7 A- Figure 7D). Bone marrow was collected from each group of mice for flow cytometry analysis. Similar to non-tumor-bearing mice treated with recombinant mouse CSF2, BHLHE22 overexpression strongly promoted MDSC infiltration. Treatment with Anti-CSF2 antibody significantly inhibited MDSC infiltration. Figure 7 E). In the in vitro MDSCs amplification analysis experiment, we will sort the mouse MDSCs (CD11b) + Gr-1 + Cells were stained with CSFE and then co-cultured with RM-1-Vector or RM-1-BHLHE22 cells. Non-co-cultured CD11b cells... + Gr-1 + Cells were used as the Vehicle group. Then, we treated non-co-cultured cells with recombinant mouse CSF2 (with isotype IgG as a control) and co-cultured cells with Anti-CSF2 antibody (with isotype IgG as a control). Four days later, flow cytometry analysis of CFSE staining showed that, similar to the recombinant mouse CSF2 treatment group, co-culture of RM-1-BHLHE22 cells with MDSCs strongly promoted the in vitro proliferation of MDSCs, while Anti-CSF2 significantly inhibited the in vitro proliferation of MDSCs. Figure 7 F). The above results confirm that CSF2 is a key regulator of BHLHE22-driven immunosuppressive TME in bone metastases.

[0103] Example 2

[0104] 1. Materials and Methods

[0105] (1) Plasmid

[0106] The cDNA of mouse PRMT5 (NM_013768.3) was amplified by PCR and cloned into the pLVX-hyg plasmid vector (pLVX-PRMT5-hyg), which served as a control. Two short hairpin RNAs (shRNAs) targeting the PRMT5 (NM_013768.3) sequence were cloned into the pLKO.1 plasmid vector. The targeting sequences were: shRNA PRMT5#1,5′-GGTGAACACAGTGCTTCATGG-3′; shRNA PRMT5#2,5′-CCATGAAGCACTGTGTTCACC-3′.

[0107] (2) DNA pull-down

[0108] Based on the predicted BHLHE22 binding site sequence to the CSF2 promoter region from the JASPAR database, four 5′ labeled biotinylated DNA probes (P1, P2, P3, and P4, Focobio) with sizes approximately 50-150 bp were designed. Streptavidin magnetic beads without probes served as a negative control. All DNA probe sequences are shown in Table 5. DNA pull-down experiments were performed using a magnetic bead assay kit (Focobio), following the instructions. The main steps are as follows: Add 100 μL of streptavidin magnetic beads to a 1.5 mL enzyme-free EP tube. Wash the magnetic beads twice with washing buffer (0.1 M NaOH, 50 mM NaCl). Adsorb the magnetic beads onto a magnetic rack and discard the washing buffer. Lyse cells and obtain protein lysis buffer. Couple the biotin-labeled DNA probe to the magnetic beads, binding 100 pmol of DNA probe to 50 μL of magnetic beads, and incubate at room temperature for 15-30 min. Thoroughly mix the 100 pmol-coupled DNA probe with 200 μL of protein-DNA binding premix (20 μL protein-DNA binding buffer, 60 μL 50% glycerol, 60 μL protein lysis buffer, 60 μL enzyme-free water) and incubate at room temperature for 1 h. Place the EP tube on a magnetic rack and wash the DNA-binding protein complex, discarding the supernatant. Repeat twice. Add 100 μL of elution buffer and incubate at room temperature for 15-30 min. Place the EP tube on a magnetic rack and retain the supernatant. Heat the eluted sample at 98 °C for 10 min. Gel electrophoresis or store at -80°C.

[0109] Table 5. CSF2 promoter region DNA probe sequences

[0110]

[0111] (3) Luciferase reporter gene assay

[0112] After PCR amplification of the CSF2 promoter and the CSF2 promoter P1 fragment (including wild-type and mutant), the fragments were inserted into the PGL4.10-basic (Addgene, Plasmid #46387) luciferase reporter plasmid, with PGL4.10-basic serving as a negative control. Positive clones were screened, amplified, and purified for later use. One day before the experiment, cells in logarithmic growth phase were digested and seeded, and cultured in a 37°C incubator with 5% CO2. When cell confluence reached 70-80%, plasmid transfection was prepared (transfection steps were the same as in Part I: Materials and Methods). 36 hours after transfection, cells were lysed, and proteins were extracted. Using the Dual Luciferase Reporter Assay Kit (Promega), following the instructions, luciferase signals were detected and calculated. The results were normalized to firefly luciferase activity / Renilla luciferase activity and compared with the empty vector control group. The CSF2 promoter P1 fragment DNA sequence is shown in Table 6.

[0113] Table 6 Wild-type and mutant sequences of segment P1

[0114]

[0115]

[0116] (4) Chromatin Immunoprecipitation (ChIP)

[0117] ChIP experiments are used to detect the binding of proteins and DNA, and the bound DNA fragments are verified by subsequent PCR experiments. The EZ ChIP Chromatin Immunoprecipitation Kit (Millipore, Bedford, MA) was used for ChIP experiments, following the instructions. The main steps are as follows: 1% formaldehyde solution was added to the cell culture dish to maintain the interaction of the DNA-protein complex; 1 ml of cell lysis buffer containing 5 μL of protease inhibitor was added, and the cell suspension was harvested after thorough lysis; the cell suspension was sonicated to break down the DNA into small chromatin fragments; the DNA-protein complex was bound with the target protein antibody (isotype IgG was used as a control), and incubated overnight at 4°C; 60 μL of Protein A was added to precipitate the complex, and the mixture was incubated at 4°C for 1 h; the complex was eluted, the cross-links were decross-linked, and the DNA fragments were recovered; specific PCR primers were designed to amplify the CSF2 promoter. The ChIP-qPCR primer sequences are shown in Table 7.

[0118] Table 7 ChIP-qPCR Primer Sequences

[0119]

[0120]

[0121]

[0122] (5) Co-immunoprecipitation (Co-IP)

[0123] After washing cells with pre-chilled PBS, add 1 ml of lysis buffer (50 mM Tris-HCl pH 7.4 + 150 mM NaCl + 1 mM EDTA + 1% Triton X-100) containing 1% Cocktail (broad-spectrum protease inhibitor) and 0.5% PMSF to the cell culture dish to lyse the cells. Incubate at 4°C with shaking for 30 min. Place the dish on ice and scrape the collected protein into a 1.5 ml EP tube. Centrifuge at 12000 × g for 10 min at 4°C. Collect the supernatant. Remove Anti-Flag and Anti-HA beads (SIGMA) from the -20°C freezer; or prepare IgG beads (by adding Protein... (G beads and IgG antibodies were conjugated, with blank beads used as a control); the cleaned beads or prepared IgG beads were added to the protein sample and incubated overnight at 4°C; the next day, the sample was centrifuged at 12000×g for 30 seconds at 4°C, and the supernatant was discarded; 500 μL of RIPA was added to each tube for 5 min, centrifuged at 12000×g for 30 seconds at 4°C, and the supernatant was discarded, repeating 3 times; protein lysis buffer was added to the precipitated beads or IgG beads; the sample was centrifuged at 12000×g for 30 seconds at 4°C, the supernatant was collected, and the protein concentration was determined; the sample was stored at -80°C or used for the next experiment.

[0124] (6) Silver Dye

[0125] Silver staining experiments were performed using the Pierce Silver Stain Kit (Thermo Scientific, 24612) according to the instruction manual. The main steps were as follows: fixation overnight on a shaker at room temperature (60-70 rpm) using 20 ml anhydrous ethanol + 4 ml acetic acid + 16 ml sterile water); washing with 100 ml 30% ethanol for 10 min; washing with 200 ml double-distilled water for 10 min; sensitization with 30 ml 1× sensitizing solution at room temperature for 10 min; washing with 200 ml double-distilled water for 10 min, repeated once; staining with 30 ml 1× silver staining solution for 10 min; washing with 100 ml double-distilled water for 1 min; adding developing solution for 3-7 min; stopping the development process after observing a suitable color intensity. Differential bands were excised with a scalpel and sent for protein chromatography analysis.

[0126] (7) Protein spectroscopy (MS)

[0127] After enzymatic digestion of the bands, the supernatant was collected. 100 μL of extraction buffer (67% acetonitrile, containing 2% formic acid) was added to the remaining gel block. The mixture was incubated at room temperature for 30 min, sonicated for 15 min, and then centrifuged. The supernatants were combined, centrifuged again, concentrated, and dried for mass spectrometry analysis. The centrifuged and dried sample was redissolved in Nano-LC mobile phase A (0.1% formic acid / water), bottled, and loaded for online LCMS analysis. 6 μL of the dissolved sample was added to a nanoViper C18 pre-column (3 μm, 100), followed by a 20 μL wash for desalting. The liquid chromatography system was an Easy nLC1200 nano-liquid chromatography system (ThermoFisher). After desalting and retention (pre-column), separation (analytical column) was performed. The gradient used was mobile phase B (80% acetonitrile, 0.1% formic acid), increasing from 5% to 38% over 30 min. Mass spectrometry was performed using a ThermoFisher QExactive system combined with a nano-spray Nano Flex ion source (ThermoFisher), with a spray voltage of 1.9 kV and an ion transmission tube heating temperature of 275 °C. The mass spectrometry scan was performed in information-dependent acquisition (DDA) mode, with a primary mass spectrometry scan resolution of 70,000 m / s, a scan range of 350–2000 m / s, and a maximum injection time of 100 ms. A maximum of 20 secondary mass spectra with charges ranging from 2+ to 5+ were acquired per DDA cycle, with a maximum ion injection time of 50 ms for secondary mass spectrometry.

[0128] (8) Cellular immunofluorescence (IF)

[0129] After adding cell crawling slides to 24-well plates, incubate cells in a 5% CO2 incubator at 37°C. When cell confluence reaches 50%, remove the 24-well plate; rinse three times with pre-warmed PBS; fix with 4% paraformaldehyde for 30 min; rinse once with PBS after fixation; perforate the membrane with 0.3% Triton X-100 PBS for 15 min; rinse once with PBS; block with blocking buffer for 30 min; dilute the primary antibody with blocking buffer (when there are two genes to be detected, antibodies from different species should be selected), add 200 μL of diluted primary antibody to each well, and incubate overnight at 4°C on a shaker; the next day, remove the plate and rinse three times with PBS; dilute the fluorescent secondary antibody with blocking buffer (use antibodies from the same species as the primary antibody), add 200 μL of diluted fluorescent secondary antibody to each well, and incubate at room temperature in the dark for 1 h; rinse three times with PBS; add 200 μL of DAPI to each well for nuclei staining (in the dark, for 20 min); pick out the cell crawling slides, add 5 μL of anti-quenching mounting medium, mount, and photograph.

[0130] (9) MDSCs co-culture experiment

[0131] RM-1-BHLHE22 and RM-1-Vector cells were pre-seeded in 6-well plates; sorted MDSCs were stained with CFSE (MDSCs sorting and CFSE staining procedures are the same as in Part I, Materials and Methods), and 1×10⁶ cells were added to each well. 6 Labeled MDSCs were co-cultured with RM-1 cells. Six-well plates were placed in a 37°C incubator with 5% CO2. After 4 days, MDSCs were collected, stained with CD45, CD11b, and Gr-1, and analyzed by flow cytometry.

[0132] (10) Animal experiments and drug treatment

[0133] The methods for establishing mouse models and evaluating bone metastasis status were the same as those described in Part I, Materials and Methods. Drug treatment: In the in vitro co-culture experiment, the PRMT5 inhibitor GSK591 was added to the cell culture medium (5 μM, TargetMol). In the in vivo animal experiment, mice were administered the PRMT5 inhibitor GSK3326595 (50 mg / kg / day, TargetMol) by gavage, or Anti-CSF2 (20 μg / mouse, twice / week, BioXcell) by intraperitoneal injection, or Anti-PD-1 (200 μg / mouse, twice / week, BioXcell) by intraperitoneal injection. Isotype IgG antibodies of the same dose and concentration were used as controls.

[0134] 2. Experimental Results

[0135] (1) The results of the luciferase reporter gene assay showed that, compared with the Vector group, the luciferase activity of the CSF2 promoter was activated in stable cell lines overexpressing BHLHE22 in RM-1 and PC-3. Figure 8 A). Simultaneously, to investigate whether BHLHE22 binds to the CSF2 promoter, we designed a chromatin immunoprecipitation (ChIP) assay, finding that CSF2 promoter DNA and anti-BHLHE22 antibody could be co-precipitated, which was verified by qPCR. Figure 8 B and Figure 8 C). Furthermore, based on the JASPAR and CIS-BP databases, we compared the DNA-binding motif of BHLHE22 in humans and mice, finding that humans and mice share the same DNA-binding motif (C). Figure 8 D).

[0136] After co-incubating the protein lysis buffer with Anti-flag beads, protein eluates were obtained for each experimental group. Silver staining revealed several distinct differentially expressed bands between 60-75 kDa and 35-45 kDa. The eluates and differentially expressed bands were sent for MS proteomic analysis, which identified 10 potential transcriptional cofactors. Figure 8 E). Meanwhile, to further determine the specific BHLHE22 binding sites (BBSs) and key transcriptional cofactors on the CSF2 promoter, we analyzed the JASPAR database and found six high-confidence BBSs for BHLHE22 in the CSF2 promoter region. Figure 8 F). Subsequently, we designed four 5′-labeled DNA biotinylated probes covering 80–180 bp of these BBS sequences (Table 5). These four DNA probes were coupled to magnetic beads as experimental groups (P1, P2, P3, and P4), with uncoupled magnetic beads serving as the negative control group (control), and a group with equal amounts of the four coupled probes serving as the positive control group (P1-4). DNA pull-down assays yielded eluents, which were then subjected to silver staining. The results showed that lanes P1 and P1-4 exhibited the same differential bands as in the Co-IP assay between 60 and 75 kDa, while lanes P2, P3, and P4 did not show the same differential bands. Figure 8 G). Therefore, we believe that BHLHE22 binds to the P1 segment of the CSF2 promoter, and the binding motif sequence is -47 to -38 bp upstream of the predicted CSF2 transcription start site (TSS) (CAAATATGCC). Further Western blotting analysis of the DNA probe elution buffer showed that the differential band between 60 and 75 kDa was protein arginine methyltransferase-5 (PRMT5). Figure 8 H). Furthermore, we performed immunofluorescence staining (IF) on RM-1-BHLHE22 cells, and the results showed that BHLHE22 and PRMT5 were mainly distributed in the nucleus and exhibited co-localization. Figure 8I). To further verify whether BHLHE22 binds to the P1 segment of the CSF2 promoter region and transcribes and activates CSF2, we constructed three CSF2 promoter luciferase vector plasmids: pGL4-FL-BBS (full-length), pGL4-P1-BBS-WT (P1 segment), or pGL4-P1-BBS-Mut (P1 segment mutation). These three vector plasmids were then transfected into RM-1-BHLHE22 cells and HEK293T cells (when transfecting HEK293T cells, the BHLHE22 overexpression vector plasmid was also transfected). The luciferase reporter gene assay results showed that mutation of the BHLHE22 binding site sequence (CAAATATGCC) in the P1 segment significantly reduced CSF2 promoter luciferase activity (Table 6 and...). Figure 8 J). To further verify the interaction between BHLHE22 and PRMT5 proteins, we conducted endogenous and exogenous co-immunoprecipitation assays (Co-IP). We found that BHLHE22 protein could precipitate PRMT5 protein, and conversely, PRMT5 protein could also precipitate BHLHE22 protein. Figure 8 K and Figure 8 The above results indicate that BHLHE22 forms a transcriptional complex with PRMT5 and binds to the CAAATATGCC sequence in the P1 segment of the CSF2 promoter region, thereby transcribedly activating CSF2 expression.

[0137] The expression level of PRMT5 in RM-1 and PC-3 cell lines was detected after overexpression of BHLHE22. The results showed that the expression level of PRMT5 did not change significantly after overexpression of BHLHE22. Figure 8 Therefore, we believe that BHLHE22 and PRMT5 do not have a regulatory relationship. Next, we tested them in three cell lines: RM-1-Vector (BHLHE22). - / PRMT5 + ), RM-1-BHLHE22 (BHLHE22) + / PRMT5 + ) and RM-1-BHLHE22-PRMT5-sh(BHLHE22 + / PRMT5 -We designed a ChIP experiment targeting the CSF2 promoter sequence with BHLHE22 and PRMT5 and validated it using qPCR. We found that neither BHLHE22 nor PRMT5 co-precipitated with the CSF2 promoter in RM-1-Vector cells. However, BHLHE22 co-precipitated with the CSF2 promoter in both RM-1-BHLHE22 and RM-1-BHLHE22-PRMT5-sh cells. PRMT5 co-precipitation with the CSF2 promoter was only detected in RM-1-BHLHE22 cells, while no co-precipitation was detected in RM-1-BHLHE22-PRMT5-sh cells. Figure 8 N). As shown in the schematic diagram of the action of the BHLHE22 / PRMT5 complex ( Figure 8 When BHLHE22 is absent, PRMT5 cannot bind to the CSF2 promoter. The ability of PRMT5 to bind to the CSF2 promoter is dependent on BHLHE22. These results reveal the specific mode of binding of the BHLHE22 / PRMT5 complex to the CSF2 promoter: BHLHE22 binds to the CSF2 promoter and recruits PRMT5 to form a transcription complex; PRMT5 cannot directly bind to the CSF2 promoter.

[0138] (2) After knocking down PRMT5, the expression level of CSF2 in the RM-1-BHLHE22 cell line was detected by qPCR and Western blot. The results showed that knocking down PRMT5 significantly reduced the expression level of CSF2. Figure 9 A and Figure 9 B). As previously mentioned, PRMT5 has the function of epigenetic regulation of gene expression through histone methylation. We then examined the methylation levels at different histone sites (H4R3, H3R2, and H3R8). Notably, in the RM-1-BHLHE22 cell line with PRMT5 knockdown, Western blot detected reduced levels of H4R3me2a and H3R2me2s. Figure 9 C). Previous studies have shown that dimethylation of H4R3 (H4R3me2a) and H3R2 (H3R2me2s) can activate gene transcription in tumor cells. Therefore, to investigate whether PRMT5 catalyzes methylation of H4R3 and H3R2 residues in the CSF2 promoter region, we performed ChIP-qPCR experiments in RM-1-BHLHE22 cells. The results showed that H4R3me2a and H3R2me2s were enriched at the CSF2 promoter, and knocking down PRMT5 reduced the enrichment of H4R3me2a and H3R2me2s. Figure 9D). Furthermore, by qPCR and Western blot analysis, we found that the PRMT5 inhibitor GSK591 significantly reduced the expression level of CSF2 in RM-1-BHLHE22 cells after inhibiting PRMT5 activity. Figure 9 E and Figure 9 F). Consistently, in ChIP-qPCR experiments, we observed that GSK591 significantly reduced the enrichment of H4R3me2a and H3R2me2s on the CSF2 promoter of RM-1-BHLHE22 cells. Figure 9 G).

[0139] In in vivo MDSC infiltration analysis, C57BL / 6J mice were induced to develop MDSC infiltration by injecting RM-1-vector, RM-1-BHLHE22, or RM-1-BHLHE22-PRMT5-sh cell lines into the left ventricle. Two RM-1-BHLHE22 groups were included, with one group receiving GSK3326595 treatment (isotype IgG treatment served as a control). After 30 days, BLI results showed that the PRMT5 knockdown and GSK3326595-treated mice had smaller BLI signal and smaller bone metastatic tumor lesions compared to the RM-1-BHLHE22 control group. Figure 9 H and Figure 9 I). Bone marrow from the above groups of mice was collected for flow cytometry analysis. The results showed that PRMT5 knockdown and treatment with the PRMT5 inhibitor GSK3326595 significantly reduced the infiltration of MDSCs in BHLHE22-overexpressing bone metastatic tumor tissues. Figure 9 J). In the in vitro MDSCs amplification analysis, the sorted mouse MDSCs (CD11b) were first... + Gr-1 + Cells were labeled with CSFE, and then the labeled mouse MDSCs were co-cultured with RM-1-Vector, RM-1-BHLHE22, or RM-1-BHLHE22-PRMT5-sh cells. In addition, one of the two groups co-cultured with RM-1-BHLHE22 cells received GSK591 treatment (isotype IgG as a control). Four days later, flow cytometry and CFSE staining showed that BHLHE22 overexpression strongly promoted MDSC expansion, while PRMT5 knockdown and the PRMT5 inhibitor GSK591 significantly inhibited MDSC expansion. Figure 9 In summary, PRMT5 catalyzes methylation of the CSF2 promoter sites H4R3me2a and H3R2me2s, epigenetically activating CSF2 expression and ultimately promoting tumor MDSCs infiltration.

[0140] (3) Three days after successful left ventricular injection of RM-1-BHLHE22 cells to establish the model, we began treating mice with anti-CSF2 and / or anti-PD-1 antibodies (isotype IgG as a control). Figure 10 A). Bone metastasis was monitored using BLI, and BM lesions were measured using MicroCT scans and H&E staining. Figure 10 B and Figure 10 C). The experimental endpoint was set at day 70. There was no significant difference in the incidence of bone metastases between the control and anti-CSF2 or anti-PD-1 treatment alone. However, the anti-CSF2 combined with ICT treatment significantly reduced the incidence of bone metastases and was the most effective in inhibiting bone metastasis among all treatment groups. Figure 10 D). Further analysis revealed that although there was no significant difference in the incidence of bone metastases, unlike the rapid progression of bone metastases in the IgG group, anti-CSF2 and anti-PD-1 therapy alone could slow tumor progression and reduce the area of ​​bone metastases. Figure 10 E- Figure 10 G). Notably, among all treatment groups, the combination therapy group was the most effective in slowing tumor progression and reducing the area of ​​BM lesions. Figure 10 E- Figure 10 G). Survival analysis showed that the combined treatment group significantly prolonged the overall survival and bone metastasis-free survival of mice. Figure 10 H and Figure 10 I). Positive staining results for Ki-67 proliferation markers showed that the combined treatment significantly inhibited the growth of RM-1-BHLHE22 prostate tumors metastasizing in mouse bone. Figure 10 C and Figure 10 J).

[0141] Multicolor flow cytometry was used to detect BHLHE22 after treatment. + MDSCs and T cell infiltration in bone metastases. Compared with the control group, anti-CSF2 therapy alone reduced MDSC infiltration and increased CD4+. + T and CD8 + T cell infiltration, but it does not promote IFN-γ + CD8 + T cell expansion ( Figure 11 A- Figure 11 C). Anti-PD-1 therapy alone promotes IFN-γ. + CD8 + T cell expansion, but with respect to MDSCs and CD4 in bone metastases. + T and CD8 + T cell infiltration was not significantly affected. Figure 11 A- Figure 11C). Notably, anti-CSF2 combined with ICT therapy significantly reduced MDSC infiltration and increased CD4 levels. + T and CD8 + T cell infiltration, while simultaneously promoting IFN-γ + CD8 + T cell expansion ( Figure 11 A- Figure 11 C). In summary, our findings indicate that anti-CSF2 combined with ICT therapy effectively enhances the response of ICT to BHLHE22 by relieving the immunosuppressive TME driven by MDSCs. + Treatment efficacy of PCa bone metastases.

[0142] (4) Three days after successful left ventricular injection of RM-1-BHLHE22 cells to establish the model, we began treating mice with GSK3326595 and / or anti-PD-1 antibody (isotype IgG as a control). Figure 12 A). Bone metastasis was monitored using BLI, and BM lesions were measured using MicroCT scans and H&E staining. Figure 12 B and Figure 12 C). The experimental endpoint was set at day 70. There was no significant difference in the incidence of bone metastases between GSK3326595 treatment alone and the control group. However, the combination therapy of GSK3326595 and ICT effectively inhibited the occurrence of bone metastases. Figure 12 D). Compared with the control group, GSK3326595 treatment alone slowed tumor progression and reduced the area of ​​BM lesions. Figure 12 E- Figure 12 G). Importantly, among all experimental groups, the combination therapy group most effectively slowed tumor progression and reduced the area of ​​BM lesions (G). Figure 12 E- Figure 12 G). Survival analysis showed that the combination therapy group significantly prolonged the overall survival and bone metastasis-free survival of mice. Figure 12 H and Figure 12 I). Positive staining results for Ki-67 proliferation markers showed that the combination therapy significantly inhibited the growth of RM-1-BHLHE22 prostate tumors in mouse bone metastases. Figure 12 C and Figure 12 J). Multicolor flow cytometry results showed that GSK3326595 treatment reduced MDSC infiltration in bone metastases and increased CD4 counts. + T and CD8 + T cell infiltration, but unable to promote IFN-γ + CD8 + T cell expansion ( Figure 13 A- Figure 13C). However, when combined with ICT, GSK3326595 reduced MDSCs and increased CD4. + T, CD8 + T cell infiltration, while simultaneously promoting IFN-γ + CD8 + T cell expansion ( Figure 13 A- Figure 13 C). The above results indicate that the combination therapy of GSK3326595 and ICT relieved the immunosuppressive TME driven by MDSCs and effectively improved the response of ICT to BHLHE22. + Treatment efficacy of PCa bone metastases.

[0143] (5) The expression levels of BHLHE22, PRMT5, and CSF2, the tumor proliferation marker Ki-67, and the number of immune infiltrating cells were detected in human PCa bone metastasis tissue samples (BM) using IHC. BHLHE22 staining results showed that 26.7% of BM samples were negative, 20% were weakly positive, 23.3% were moderately positive, and 30% were strongly positive. Figure 14 A). Previous studies have shown that PRMT5, as an oncogene, is widely expressed in PCa cells and promotes PCa cell growth. Consistently, our results showed that PRMT5 was positively stained in BM samples, and there was no significant difference between the BHLHE22-low and BHLHE22-high groups. Figure 14 B). Further analysis revealed that, compared to the BHLHE22-low group, the BHLHE22-high group had a greater number of tumor-infiltrating MDSCs and CD4+. + T and CD8 + Fewer T cells and higher CSF2 expression ( Figure 14 C- Figure 14 E). Ki-67 staining, a tumor proliferation marker, suggests that BHLHE22-induced bone infiltration MDSCs favor the growth of PCa bone metastases. Figure 14 C and Figure 14 F). Furthermore, correlation analysis showed that BHLHE22 and CD4... + T cell infiltration (r = -0.585, P < 0.001); Figure 14 G), CD8 + T cell infiltration (r = -0.593, P < 0.001); Figure 14 H) showed a negative correlation. Conversely, BHLHE22 was negatively correlated with MDSCs infiltration (r = 0.688, P < 0.001); Figure 14 I) CSF2 expression (r = 0.678, P < 0.001; Figure 14J) shows a positive correlation.

[0144] In summary, our findings reveal the molecular mechanisms by which the BHLHE22 / PRMT5 / CSF2 pathway drives the formation of immunosuppressive bone metastases in PCa bone metastases. Figure 14 K). BHLHE22, as a potential biomarker, could help screen PCa patients suitable for ICT treatment. (The last part, "BHLHE22," appears to be unrelated and likely refers to a separate topic.) + In patients with PCa, the PRMT5 inhibitor GSK3326595 combined with ICT and anti-CSF2 combined with ICT are effective treatment options for inhibiting PCa bone metastasis.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of a reagent for detecting the expression level of BHLHE22 in the preparation of a product for predicting or detecting bone metastasis of prostate cancer, wherein the reagent comprises a reagent for detecting the expression level of BHLHE22 by real-time quantitative PCR, immunodetection or chip detection.

2. Use according to claim 1, characterized in that, The reagent for detecting the expression level of BHLHE22 is a reagent for detecting the expression level of BHLHE22 in a sample.

3. Use according to claim 2, characterized in that, The sample is a human tissue sample.

4. Use according to claim 3, characterized in that, The human tissue sample is a prostate cancer primary lesion or bone metastasis tissue.

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