Application of BHLHE22 gene in triple negative breast cancer

Abstract

The invention discloses application of a BHLHE22 gene in triple negative breast cancer, and belongs to the technical field of biological medicines. Data analysis shows that in triple negative breast cancer (TNBC), the expression of the BHLHE22 gene is reduced, and the low expression of the BHLHE22 gene is related to advanced pathological staging and poor prognosis. Meanwhile, experiments show that overexpression of the BHLHE22 can inhibit growth of tumor cells, and silence of the gene can promote malignant progression. Meanwhile, the deubiquitination enzyme OTUD3 stabilizes the BHLHE22 protein through the deubiquitination activity of the deubiquitination enzyme OTUD3, and the effect depends on the key C76 site of the deubiquitination enzyme OTUD3. In addition, the BHLHE22 inhibits a downstream target gene CDT1 through transcription to play an anti-proliferation function, and the effect can be reversed by CDT1 overexpression. The invention discloses the key cancer suppression effect of the OTUD3 / BHLHE22 / CDT1 signal axis in the TNBC, and provides a new potential target spot for the diagnosis and treatment of the TNBC.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of the BHLHE22 gene in triple-negative breast cancer. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by the loss of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). This subtype accounts for 15%-20% of all breast cancer cases and is significantly more aggressive, with higher recurrence and mortality rates compared to other subtypes. Because TNBC patients are not suitable for endocrine therapy or HER2-targeted therapy, chemotherapy and radiotherapy remain the cornerstone of systemic treatment. Elucidating the molecular mechanisms of TNBC progression is crucial for developing more effective treatment strategies.

[0003] e22 (BHLHE22), a fundamental member of the helical-loop-helical family of transcription factors, plays a role in cell differentiation during neuronal development. Recent evidence suggests that BHLHE22 may be involved in cancer progression, and its function varies depending on the cancer type. For example, in prostate cancer, BHLHE22 promotes bone metastasis by recruiting immunosuppressive neutrophils and monocytes, thereby fostering an immunosuppressive bone tumor microenvironment (Yin C, Wang M, Wang Y, Lin Q, Lin K, Du H, BHLHE22 drives immunosuppressive bone tumor microenvironment and associated bone metastasis in prostate cancer. 2023;11(3).). Conversely, in endometrial cancer, BHLHE22 expression is downregulated, while its high expression is associated with improved patient survival by inhibiting tumor cell proliferation and metastasis (Darmawi, Chen LY, Su PH, Liew PL, Wang HC, Weng YC, et al. BHLHE22 expression is associated with a pro-inflammatory immune microenvironment and predicts a good prognosis in endometrial cancer. International Journal of Molecular Sciences. 2022;23(13).). However, the function of BHLHE22 in TNBC remains unclear. Summary of the Invention

[0004] Based on the aforementioned problems in existing technologies, this invention, through analysis of the GSE45827 and GSE 113865 datasets, discovered downregulated BHLHE22 expression in TNBC tissues. Among various BC subtypes, BHLHE22 RNA expression was lowest in TNBC subtypes, and BHLHE22 expression levels in stage 3 and 4 patients were often lower than in stage 1 and 2 patients. Patients with high BHLHE22 expression had better survival prognosis than those with low expression. Functional studies showed that BHLHE22 overexpression impaired cell growth both in vitro and in vivo. However, BHLHE22 gene silencing enhanced the malignant behavior of cancer cells. The deubiquitinating enzyme OTUD3, known to inhibit TNBC progression, was found to enhance BHLHE22 protein stability through deubiquitination regulation. A mutation at the C76 site of OTUD3 eliminated OTUD3's catalytic activity but failed to regulate BHLHE22 protein stability. Furthermore, BHLHE22 enhanced the antitumor effect of OTUD3 in TNBC. mRNA sequencing analysis identified BHLHE22 as a potential anti-cancer gene involved in CDT1. Studies confirmed that BHLHE22 reduces CDT1 RNA expression levels by inhibiting CDT1 transcription. The anti-proliferative effect of BHLHE22 overexpression can be reversed by CDT1 overexpression. The OTUD3 / BHLHE22 / CDT1 axis may be a promising direction for TNBC treatment.

[0005] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: This invention provides the application of the BHLHE22 gene in the preparation of diagnostic and therapeutic products for triple-negative breast cancer.

[0006] Furthermore, the application is performed using a product for the detection and / or regulation of the BHLHE22 gene.

[0007] Furthermore, the diagnostic and therapeutic products include diagnostic products, testing products, screening products, prognostic assessment products, and / or prevention and treatment products.

[0008] Furthermore, the expression level of the BHLHE22 gene was downregulated in patients with triple-negative breast cancer.

[0009] Furthermore, the BHLHE22 gene detection product is used to prepare diagnostic products, detection products, screening products, and / or prognostic assessment products for triple-negative breast cancer.

[0010] Furthermore, the product forms of the diagnostic products, testing products, screening products, and / or prognostic assessment products include kits.

[0011] Furthermore, the BHLHE22 gene expression regulation product is used to prepare a product for the prevention and treatment of triple-negative breast cancer.

[0012] Furthermore, the BHLHE22 gene expression regulation product is a product that upregulates the expression level of the BHLHE22 gene.

[0013] Furthermore, the product that upregulates BHLHE22 gene expression levels works through the OTUD3 / BHLHE22 / CDT1 axis.

[0014] Furthermore, the product form of the prevention and treatment product includes drugs.

[0015] Compared with the prior art, the present invention has the following beneficial effects: Based on the GSE45827 and GSE 113865 datasets, this invention found that BHLHE22 expression is downregulated in TNBC tissues. Among various BC subtypes, BHLHE22 RNA expression levels were lowest in TNBC subtypes, and BHLHE22 expression levels in stage 3 and 4 patients were often lower than in stage 1 and 2 patients. Patients with high BHLHE22 expression had better survival prognosis than those with low expression. Functional studies showed that BHLHE22 overexpression impaired cell growth both in vitro and in vivo. However, BHLHE22 gene silencing enhanced the malignant behavior of cancer cells. OTUD3, a deubiquitinating enzyme known to inhibit TNBC progression, was found to enhance BHLHE22 protein stability through deubiquitination regulation. A mutation at the C76 site of OTUD3 eliminated OTUD3's catalytic activity but failed to regulate BHLHE22 protein stability. Furthermore, BHLHE22 enhanced the antitumor effect of OTUD3 in TNBC. mRNA sequencing analysis identified potential genes involved in the anticancer activity of BHLHE22, including CDT1. Studies have confirmed that BHLHE22 reduces CDT1 RNA expression levels by inhibiting CDT1 transcription. The antiproliferative effect of BHLHE22 overexpression can be reversed by CDT1 overexpression. The OTUD3 / BHLHE22 / CDT1 axis may be a promising direction for the treatment of TNBC. Attached Figure Description

[0016] Figure 1 This describes the screening process for the target gene in Example 1 of the present invention.

[0017] Figure 2 This study assesses the expression and prognostic value of the BHLHE22 gene in Example 1 of the present invention.

[0018] Figure 3 This describes the effect of BHLHE22 on the in vitro growth of TNBC cells in Example 1 of the present invention.

[0019] Figure 4This describes the effect of BHLHE22 on TNBC cell apoptosis and in vivo growth in Example 1 of the present invention.

[0020] Figure 5 The results show the effect of OTUD3 on the stability of BHLHE22 protein in Example 1 of this invention.

[0021] Figure 6 This is the mRNA-seq analysis result of the downstream target gene of BHLHE22 in Example 1 of the present invention.

[0022] Figure 7 This is a verification process of how BHLHE22 exerts its effect on TNBC cells through transcriptional regulation of CDT1 in Example 1 of the present invention.

[0023] Figure 8 This serves as a verification of the efficiency of BHLHE22 gene overexpression and knockdown in Example 1 of the present invention.

[0024] Figure 9 This is a verification process for the influence of BHLHE22-mediated OTUD3 on the TNBC phenotype in Embodiment 1 of the present invention.

[0025] Figure 10 This is the clinical relevance verification result of BHLHE22 and TNBC in Example 2 of the present invention. Detailed Implementation

[0026] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. The reagents, kits, and instruments used in the following examples are commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0027] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0028] Example 1 1. Experimental Materials and Methods 1.1 TNBC Database Mining Transcriptome and sample information were downloaded from The Gene Expression Omnibus, datasets GSE113865 and GSE45827. GSE113865 contained 3 TNBCs and 3 normal tissues, while GSE45827 contained 41 tumor tissues and 11 normal tissues. Genes with |log2 (fold change)| > 1 and p < 0.05 were defined as differentially expressed genes (DEGs). Bioinformatics analysis was performed on the DEGs in each dataset.

[0029] 1.2 Experimental cell lines MDA-MB-231 and MDA-MB-157 cells were purchased from Shanghai Saibaikang Biotechnology Co., Ltd.

[0030] 1.3 Reagents and Antibodies BHLHE22 antibody, cleaved caspase 3 antibody, OTUD3 antibody, CDT1 antibody, and internal control antibody β-actin were purchased from Proteintech (China); cleaved caspase 9 antibody was purchased from CST Biotech (China); flag antibody was purchased from Beyotime Biotechnology Co., Ltd. (China); and the EdU cell proliferation assay kit was purchased from Elabscience (China).

[0031] 1.4 Cell Culture and Transfection MDA-MB-231 and MDA-MB-157 cells were cultured in an L15 culture medium containing 10% fetal bovine serum at 37 ℃ and 5% CO2. After the cells were in good growth condition, they were used for experiments.

[0032] Cells were seeded in 6-well plates. After 24 h, the medium was replaced with serum-free basal medium. After 1 h of treatment, cell transfection was performed. A mixture of 2 μg plasmid, 6 μL liposome 2000 (Invitrogen, USA), and 200 μL transformation buffer (Invitrogen) was added dropwise to each well. The culture plate was shaken to mix gently. After culturing at 37 ℃ and 5% CO2 for 6 h, the supernatant of the transfection medium was discarded, and the medium was replaced with complete medium. The cells were then cultured at 37 ℃ and 5% CO2 for another 6 h.

[0033] 24 h after cell transfection, the cells are replaced with complete medium containing G418 (Invitrogen) and screened for another 1-2 weeks. Then, they are cultured in complete medium for another 2 weeks to form visible monoclonal cell clusters, and finally, a stable transfected cell line is obtained for subsequent experiments.

[0034] Prepare lentiviruses. Remove cells from the incubator and select cells in good condition for lentivirus infection. Use a pipette to add an accurate volume (virus to cell ratio of 50:1) of virus solution to the prepared culture medium. Gently shake the culture plate to mix well and incubate overnight at 37 ℃ and 5% CO2. After 24 h, replace the culture medium containing lentiviruses with fresh culture medium and continue culturing at 37 ℃ and 5% CO2 for another 48 h.

[0035] 1.5 MTT assay for cell viability Cells were seeded into 96-well culture plates according to the experimental groups, with 5 × 10⁶ cells per well. 3 Cells were seeded in 5 replicates per group and cultured at 37 ℃ in a 5% CO2 incubator. MTT assays were performed at 0, 24, 48, and 72 h after cell adhesion. The culture medium for each group that reached the specified time was discarded, and replaced with normal culture medium. 10 µL of MTT (Beyotime, China) mixture was added to each well, and the cells were incubated at 37 ℃ in a 5% CO2 incubator for 4 h. 100 μL of DMSO was added to dissolve the purple crystals formed by the cells, and the cells were allowed to stand in the dark for 10 min. The OD value at 570 nm was measured using a microplate reader for data analysis.

[0036] 1.6 Cloning Cells from each group were seeded in 60 mm cell culture dishes, with 300 cells seeded in each dish. The culture dishes were incubated at 37°C and 5% CO2 for 14 days, during which visible clones were formed. After fixation with 4% paraformaldehyde for 20 min, the cells were stained with Wright-Giemsa stain (Nanjing Jiancheng, China) for 5 min. The clones were photographed and counted. The clone formation rate was calculated as (number of clones / number of seeded cells) × 100%.

[0037] 1.7 EdU staining Cell slides were cultured at 37 ℃, 5% CO2, and saturated humidity. After transfection, EdU staining was performed using an EdU imaging detection kit (KGI Biotech, China). Cells in each group were added with 10 μM EdU staining solution and cultured at 37 ℃, 5% CO2 for 2 h. The culture medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. Then, 0.1 ml of Triton X-100 was added, and the cells were incubated at room temperature for 20 min. After washing the cells twice, Click-iT reaction solution was added, and the cells were incubated at room temperature in the dark for 30 min. After washing the cells twice, DAPI staining solution was added for 5 min, and the cells were washed twice. The cells were then photographed under a fluorescence microscope (OLYMPUS, Japan).

[0038] 1.8 Apoptosis Detection Cells from each group were collected, washed twice with PBS, and resuspended in each tube of cell sample with 500 μL Binding Buffer and 195 μL Annexin V-APC binding solution. 5 μL Annexin V-APC and 5 μL Propidium Iodide staining solution were added, mixed well, and incubated at room temperature in the dark for 15 min. Apoptosis was detected by flow cytometry (Agilent, USA).

[0039] 1.9 Reagent Kit Detection Cells from each group were collected, centrifuged, and the supernatant was collected. The protein concentration of the homogenate supernatant was determined using a BCA protein concentration assay kit (Beyotime, China). Caspase 3 and Caspase 9 activities were determined according to the instructions of the assay kit.

[0040] 1.10 Immunoprecipitation assay (Co-IP) Total protein was extracted from cell samples of each group, and protein concentration was quantified using a BCA protein assay kit (Beyotime, China). The total protein was diluted to approximately 1 μg / μL with PBS. 500 μL of the sample was then added to 60 μL of Protein A agarose beads to remove impurities. 30 μL of the supernatant from each group was taken as the input group, and 2 μL of the corresponding antibody was added to the corresponding group as the experimental group. 2 μL of IgG derived from the antibody species was added as the negative control group. The antigen-antibody mixture was incubated overnight at 4 °C. Then, 60 μL of Protein A agarose beads were added to capture the antigen-antibody complex, and the mixture was incubated at 4 °C for 2 h. Centrifuge to collect the agarose bead-antigen-antibody complex, discard the supernatant, add 5× loading buffer to resuspend the agarose bead-antigen-antibody complex, boil the loaded sample for 5 min to free the antigen, antibody and beads, centrifuge, take the supernatant for SDS-PAGE electrophoresis and transfer to a PVDF membrane (Millipore, USA), block with 5% (M / V) skim milk powder for 1 h, incubate overnight in primary antibody working solution at 4 ℃, wash the PVDF membrane with TBST buffer, incubate in secondary antibody working solution at 37 ℃ for 45 min, ECL substrate luminescence, analyze the optical density value of the target band using a gel image processing system (Gel-Pro-Analyzer software).

[0041] 1.11 Dual-luciferase assay Cells were seeded in 12-well plates. After 24 h, the culture medium in the plate was replaced with serum-free basal medium, 2 mL per well. After 1 h, 1 μg Promoter + 1 μg empty vector or target plasmid + 1 μg pRL-TK + 6 μL p3000 + 9 μL liposome 3000 and 300 μL optimization solution were added dropwise to each well. The plate was gently shaken to mix. After culturing at 37 °C and 5% CO2 for 4 h, the supernatant of the transfection medium was discarded and replaced with complete culture medium. The plate was then cultured at 37 °C and 5% CO2 for another 48 h.

[0042] Discard the cell culture medium, wash twice with PBS, add 250 μL of cell lysis buffer, prepare the detection plate, add 100 μL of firefly luciferase detection reagent and 20 μL of sample to each well, mix well, and detect the Firefly value. Then add 100 μL of Renilla luciferase detection reagent to each detection well, mix well, and detect the Renilla value. Set up 3 replicates for each sample, and calculate the luciferase ratio based on the results.

[0043] 1.12 Real-time PCR Total RNA was extracted from the samples and cDNA was obtained using BeyoRT II M-MLV reverse transcriptase (Beyotime, China). A Real-time PCR reaction system was constructed according to the SYBR Green (Solepro, China) kit instructions. The constructed PCR reaction system was then placed in an Exicycler™ 96 real-time PCR instrument (BIONEER, Korea) for quantitative PCR. β-actin was used as an internal control. -△△CT The method is used to quantify mRNA.

[0044] 1.13 Western blot Total protein was extracted from the samples, and protein quantification was performed using a BCA protein assay kit (Beyotime, China). 20-40 μg of protein was loaded onto the sample, subjected to SDS-PAGE electrophoresis, and transferred to a PVDF membrane (Millipore, USA). After blocking with 5% (M / V) skim milk powder for 1 h, the membrane was incubated overnight at 4 °C in primary antibody working solution. The PVDF membrane was washed with TBST buffer and incubated at 37 °C for 45 min in IgG-HRP secondary antibody working solution (Beyotime, China). ECL substrate luminescence was observed, and the optical density values ​​of the target bands were analyzed using a gel imaging system (Gel-Pro-Analyzer software). Specific antibody information is shown in Table 1.

[0045] Table 1 Antibody Information Table

[0046] 1.14 Tumor formation in nude mice Six-week-old healthy nude mice (Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.) were selected and acclimatized for one week, with 12 hours of daylight / 12 hours of nightlight, at a temperature of 22±1 ℃ and humidity of 45-55%, with free access to food and water. Cells in the logarithmic growth phase were collected at 5×10⁻⁶ cells per group. 6Dox was injected subcutaneously into the right axilla of each nude mouse. The mice were observed daily from the date of inoculation to monitor tumor formation. Once the tumors became visible to the naked eye, the mice were fed drinking water containing Dox (1 mg / mL). The long and short diameters of the tumor nodules were measured every 5 days during the experiment: days 5, 10, 15, 20, 25, 30, and 35. Thirty-five days after tumor cell injection, the mice were sacrificed, and the tumors were harvested and photographed.

[0047] 1.15 Immunohistochemical staining (IHC) Paraffin sections of tissue were dewaxed to water, and 0.1 M sodium citrate antigen retrieval solution was added. Antigen retrieval was performed by microwave heating, followed by incubation at room temperature for 15 min with 3% H2O2. Bovine serum albumin (Sangon Biotech, China) was then added and incubated at room temperature for another 15 min. Primary antibody was diluted 1:100 and added until completely covering the tissue. The tissue was incubated overnight at 4°C in a humidified chamber. The primary antibody was removed, and HRP-labeled goat anti-rabbit IgG (Thermo Fisher, USA) secondary antibody was diluted 500-fold with PBS and added until completely covering the tissue. The tissue was incubated at 37°C for 60 min in a humidified chamber. DAB (Maisin Reagent, China) staining was performed, followed by hematoxylin (Solepro, China) counterstaining. The sections were then dehydrated, cleared, and mounted. The sections were observed and photographed under a microscope (OLYMPUS, Japan).

[0048] 2. Experimental Results 2.1 Screening of target genes Figure 1 The study revealed a co-expression module in triple-negative breast cancer (TNBC), with branches of a clustering tree corresponding to different gene modules. Each leaf in the tree corresponds to a gene. Figure 1 b shows the numerical values ​​of module-trait relationships between normal and tumor tissues. Figure 1 c is a scatter plot showing the characteristic genes of the blue and turquoise color modules. Figure 1 The Venn plot in d shows that there are 824 overlapping genes in the turquoise and blue modules with the GSE113865 and GSE45827 datasets. Figure 1 The volcano plot in e shows the distribution of differentially expressed genes in the GSE45827 and GSE3865 datasets. Figure 1 The Venn plot in f shows that 51 transcription factors were identified as differentially expressed in triple-negative breast cancer from the transcription factors obtained from the AnimalTFDB website. Figure 1 Data sets GSE45827 and GSE113865 in g showed that, compared with the Normal group, the expression levels of BHLHE22 and BHLHE41 mRNA were significantly reduced in the TNBC group samples. p <0.001 or p <0.01 orp <0.05). Figure 1 h showed that, compared with patients with low BHLHE22 expression, patients with high BHLHE22 expression had significantly increased overall survival (OS) and recurrence-free survival (RFS). p <0.001 or p <0.05); compared with patients with low BHLHE41 expression, there was no significant difference in overall survival (OS) and recurrence-free survival (RFS) among patients with high BHLHE41 expression. Therefore, BHLHE22 was selected for this study.

[0049] 2.2 Expression and prognostic value of BHLHE22 Figure 2 The UALCAN website in a shows that, compared with the Normal group, the BHLHE22 RNA level in the Tumor group samples was significantly reduced ( p <0.001). Figure 2 b shows the BHLHE22 mutation status in pan-cancer data obtained from the Sangerbox website analysis. Figure 2 The box plot in c shows the relationship between BHLHE22 expression and breast cancer (BC) stage and lymph node metastasis, as analyzed by the UALCAN website. Figure 2 Image d shows the expression of BHLHE22 in tumor tissues of different BC patients, retrieved from the Human Protein Atlas website. Figure 2 Analysis of the GSCA website in e showed that BHLHE22 expression was associated with cell cycle and apoptosis activity in BC.

[0050] 2.3 Effects of BHLHE22 on the in vitro growth of TNBC cells Figure 3 a shows that, compared to EV (Dox+) Group comparison, BHLHE22 OE(Dox+) The viability of MDA-MB-231 and MDA-MB-157 cells was reduced; compared with BHLHE22 OE(Dox-) Group comparison, BHLHE22 OE(Dox+) The viability of MDA-MB-231 and MDA-MB-157 cells was reduced; compared with NC sh(Dox+) Group comparison, BHLHE22 sh-1(Dox+) BHLHE22sh-2 (Dox+) Increased viability of MDA-MB-157 cells in group BHLHE22; sh-1(Dox-) Group comparison, BHLHE22 sh-1(Dox+) Increased viability of MDA-MB-157 cells in group BHLHE22; sh-2(Dox-) Group comparison, BHLHE22 sh-2(Dox+)The viability of MDA-MB-157 cells in the group increased. Figure 3 b shows, with NC sh(Dox+) Group comparison, BHLHE22 sh-1(Dox+) BHLHE22sh-2 (Dox+) The number of EdU-positive MDA-MB-157 cells was significantly increased in the group ( p <0.001); and BHLHE22 sh-1(Dox-) Group comparison, BHLHE22 sh-1(Dox+) The number of EdU-positive MDA-MB-157 cells was significantly increased in the group ( p <0.001); and BHLHE22 sh-2(Dox-) Group comparison, BHLHE22 sh-2(Dox+) The number of EdU-positive MDA-MB-157 cells was significantly increased in the group ( p <0.001); and EV (Dox+) Group comparison, BHLHE22 OE(Dox+) The number of EdU-positive MDA-MB-231 and MDA-MB-157 cells was significantly reduced in the group. p <0.001 or p <0.01); with BHLHE22 OE(Dox-) Group comparison, BHLHE22 OE(Dox+) The number of EdU-positive MDA-MB-231 and MDA-MB-157 cells was significantly reduced in the group. p <0.001 or p <0.01). Figure 3 c displays, with NC sh(Dox+) Group comparison, BHLHE22 sh-1(Dox+) BHLHE22sh-2 (Dox+) Increased colony formation rate in MDA-MB-157 cells compared to BHLHE22 sh-1(Dox-) Group comparison, BHLHE22 sh-1(Dox+) Increased colony formation rate in MDA-MB-157 cells compared to BHLHE22 sh-2(Dox-) Group comparison, BHLHE22 sh-2(Dox+) Increased colony formation rate of MDA-MB-157 cells in group; compared with EV (Dox+) Group comparison, BHLHE22 OE(Dox+) The colony formation rate of MDA-MB-231 and MDA-MB-157 cells was significantly reduced. p <0.05); with BHLHE22 OE(Dox-) Group comparison, BHLHE22 OE(Dox+) The colony formation rate of MDA-MB-231 and MDA-MB-157 cells was significantly reduced. p <0.01 orp <0.05).

[0051] 2.4 Effects of BHLHE22 on TNBC cell apoptosis and in vivo growth Figure 4 a shows that, compared to EV (Dox+) Group comparison, BHLHE22 OE(Dox+) The apoptosis rate of MDA-MB-231 and MDA-MB-157 cells was significantly increased. p <0.001); and BHLHE22 OE(Dox-) Group comparison, BHLHE22 OE(Dox+) The apoptosis rate of MDA-MB-231 and MDA-MB-157 cells was significantly increased. p <0.001). Figure 4 b shows that, compared to EV (Dox+) Group comparison, BHLHE22 OE(Dox+) The activities of Caspase 3 and Caspase 9 were significantly increased in MDA-MB-231 and MDA-MB-157 cells. p <0.001); and BHLHE22 OE(Dox-) Group comparison, BHLHE22 OE(Dox+) The activities of Caspase 3 and Caspase 9 were significantly increased in MDA-MB-231 and MDA-MB-157 cells. p <0.001). Figure 4 c shows, compared to EV (Dox+) Group comparison, BHLHE22 OE(Dox+) The expression levels of cleaved caspase 3 and cleaved caspase 9 proteins were increased in MDA-MB-231 and MDA-MB-157 cells; compared with BHLHE22 OE(Dox-) Group comparison, BHLHE22 OE(Dox+) The expression levels of cleaved caspase 3 and cleaved caspase 9 proteins were increased in MDA-MB-231 and MDA-MB-157 cells. Figure 4 (de) shows, with BHLHE22 OE(Dox-) Group comparison, BHLHE22 OE(Dox+) The tumor weight in the group was significantly reduced ( p <0.001), tumor volume decreased; compared with BHLHE22 sh(Dox-) Group comparison, BHLHE22 sh(Dox+) The tumor weight in the group increased significantly ( p <0.001), tumor volume increased. Figure 4 f shows, compared to EV (Dox+)Group comparison, BHLHE22 OE(Dox+) The number of BHLHE22-positive cells and the number of Ki67-positive cells increased in the tumor tissue of the group; compared with BHLHE22 OE(Dox-) Group comparison, BHLHE22 OE(Dox+) The number of BHLHE22-positive cells and the number of Ki67-positive cells decreased in the tumor tissue group.

[0052] 2.5 Effect of OTUD3 on the stability of BHLHE22 protein Figure 5 a is a protein interaction network with BHLHE22 as the core protein, obtained from the HitPredict website. Figure 5 Co-IP assay results in b showed that OTUD3 and BHLHE22 proteins were bound in 293T cells. Figure 5 c displays, with NC sh Group comparison, OTUD3 sh The expression level of BHLHE22 protein was reduced in MDA-MB-157 cells. Figure 5 In d, cycloheximide (CHX) was used to treat each group of cells individually. The results showed that, compared with NC sh Group comparison, OTUD3 sh The level of BHLHE22 protein was decreased in MDA-MB-157 cells; compared with OTUD3 cells treated with CHX alone. sh Group cell comparison, OTUD3 cells treated with CHX and MG132 together sh The level of BHLHE22 protein increased in the group cells. Figure 5 Co-IP assay results in e showed that, compared with the His-BHLHE22+HA-Ubi group, the BHLHE22 ubiquitination level in 293T cells of the flag-OTUD3+His-BHLHE22+HA-Ubi group was reduced. Figure 5 f showed that, compared with the EV group, the expression level of BHLHE22 protein in MDA-MB-157 cells was increased in the flag-OTUD3-WT group; compared with the flag-OTUD3-WT group, the expression level of BHLHE22 protein in MDA-MB-157 cells was decreased in the flag-OTUD3-C76A group. Figure 5 Co-IP assay results in g showed that the OTUD3 C76A mutation did not significantly interfere with its binding to the BHLHE22 protein. Figure 5 Co-IP analysis in h showed that BHLHE22 ubiquitination level increased after OTUD3 C76A mutation. Figure 5 i represents the molecular docking results of OTUD3 and BHLHE22 obtained from the GRAMM website analysis.

[0053] 2.6 mRNA-seq analysis to identify downstream target genes of BHLHE22 Figure 6 PCA analysis in (ab) showed BHLHE22 OE(Dox-) Group, BHLHE22 OE(Dox+) The low similarity between sample groups indicates that the sequencing data is reasonable and the sequencing results are reliable. Figure 6 The volcano diagram in c shows BHLHE22. OE(Dox-) Group, BHLHE22 OE(Dox+) Distribution of differentially expressed genes in the group sample. Figure 6 GO enrichment analysis was performed on differentially expressed genes in d, and the results showed that these genes are related to GO functions such as DNA replication. Figure 6 KEGG enrichment analysis of differentially expressed genes in e showed that these genes are related to pathways such as the cell cycle. Figure 6 The Cluego network diagram in f illustrates the relationships and overlaps among molecules associated with proliferation-related GO functions. Figure 6 The Venn diagram in g shows that there are 6 overlapping genes in the four GO functions related to proliferation. Figure 6 The heatmap in h shows the expression of six overlapping genes.

[0054] 2.7 BHLHE22 exerts its effects on TNBC cells through transcriptional regulation of CDT1. Figure 7 a shows that, compared to EV (Dox+) Group comparison, BHLHE22 OE(Dox+) CDT1 RNA levels were significantly reduced in MDA-MB-157 cells. p <0.001); and BHLHE22 OE(Dox-) Group comparison, BHLHE22 OE(Dox+) CDT1 RNA levels were significantly reduced in MDA-MB-157 cells. p <0.001); and NC sh(Dox+) Group comparison, BHLHE22 sh (Dox+) CDT1 RNA levels were significantly increased in MDA-MB-157 cells. p <0.001); and BHLHE22 sh(Dox-) Group comparison, BHLHE22 sh (Dox+) CDT1 RNA levels were significantly increased in MDA-MB-157 cells. p <0.001). Figure 7 b represents two BHLHE22 recognition motifs predicted by the Jaspar website. Figure 7 c shows that, with pGL3-enhancer(2000 / +50) +vector group comparison, pGL3-enhancer (2000 / +50) +BHLHE22 OE Group luciferase activity was significantly reduced ( p <0.001). Figure 7 d shows, compared to EV (Dox+) +EV group comparison, BHLHE22 OE(Dox+) +EV group MDA-MB-157 cell viability was significantly reduced ( p <0.01); with BHLHE22 OE(Dox+) +EV group comparison, BHLHE22 OE(Dox+) +CDT1 OE The viability of MDA-MB-157 cells in the group was significantly increased ( p <0.05). Figure 7 e-display, compared to EV (Dox+) +EV group comparison, BHLHE22 OE(Dox+) The number of EdU-positive MDA-MB-157 cells was significantly reduced in the +EV group. p <0.01); with BHLHE22 OE(Dox+) +EV group comparison, BHLHE22 OE(Dox+) +CDT1 OE The number of EdU-positive MDA-MB-157 cells was significantly increased in the group ( p <0.05). Figure 7 f shows, compared to EV (Dox+) +EV group comparison, BHLHE22 OE(Dox+) The apoptosis rate of MDA-MB-157 cells in the +EV group was significantly increased ( p <0.001); and BHLHE22 OE(Dox+) +EV group comparison, BHLHE22 OE(Dox+) +CDT1 OE The apoptosis rate of MDA-MB-157 cells in the group was significantly reduced ( p <0.001).

[0055] 2.8 Validation of gene overexpression and knockdown efficiency Figure 8 a shows, with NC sh(Dox+) Group comparison, BHLHE22 sh-1(Dox+) BHLHE22sh-2 (Dox+) The level of BHLHE22 RNA was significantly reduced in MDA-MB-231 and MDA-MB-157 cells. p <0.001); and BHLHE22 sh-1(Dox-) Group comparison, BHLHE22 sh-1(Dox+)The level of BHLHE22 RNA was significantly reduced in MDA-MB-231 and MDA-MB-157 cells. p <0.001); and BHLHE22 sh-2(Dox-) Group comparison, BHLHE22 sh-2(Dox+) The level of BHLHE22 RNA in MDA-MB-157 cells was significantly reduced. p <0.001); and EV (Dox+) Group comparison, BHLHE22 OE(Dox+) The level of BHLHE22 RNA was significantly increased in MDA-MB-157 cells. p <0.001); and BHLHE22 OE(Dox-) Group comparison, BHLHE22 OE(Dox+) The level of BHLHE22 RNA was significantly increased in MDA-MB-157 cells. p <0.001). Figure 8 b shows that, compared to the EV group, CDT1 OE CDT1 RNA levels were significantly increased in MDA-MB-157 cells. p <0.001).

[0056] 2.9 Effects of BHLHE22-mediated OTUD3 on TNBC phenotype Figure 9 (ab) shows that, compared with EV+NC sh(Dox+) Group comparison, flag-OTUD3 WT +NC sh(Dox+) The number of EdU-positive MDA-MB-157 cells was significantly reduced in the group ( p <0.001), flag-OTUD3 C76A +NC sh(Dox+) There was no significant difference in the number of EdU-positive MDA-MB-157 cells compared to the flag-OTUD3 group. WT +NC sh(Dox+) Group comparison, flag-OTUD3 WT +BHLHE22 sh(Dox+) The number of EdU-positive MDA-MB-157 cells was significantly increased in the group ( p <0.05). Figure 9 c shows that, with EV+NC sh(Dox+) Group comparison, flag-OTUD3 WT +NC sh(Dox+) The viability of MDA-MB-157 cells in the group was significantly reduced ( p <0.01), flag-OTUD3 C76A +NC sh(Dox+)There was no significant difference in cell viability between the MDA-MB-157 group and the flag-OTUD3 group; WT +NC sh(Dox+) Group comparison, flag-OTUD3 WT +BHLHE22 sh(Dox+) The viability of MDA-MB-157 cells in the group was significantly increased ( p <0.05). Figure 9 (de) shows, with EV+NC sh(Dox+) Group comparison, flag-OTUD3 WT +NC sh(Dox+) The apoptosis rate of MDA-MB-157 cells in the group was significantly increased ( p <0.001), flag-OTUD3 C76A +NC sh(Dox+) There was no significant difference in apoptosis rate between the MDA-MB-157 group and the flag-OTUD3 group; WT +NC sh(Dox+) Group comparison, flag-OTUD3 WT +BHLHE22 sh(Dox+) The apoptosis rate of MDA-MB-157 cells in the group was significantly reduced ( p <0.001). Figure 9 f shows that, with EV+NC sh(Dox+) Group comparison, flag-OTUD3 WT +NC sh(Dox+) The activities of Caspase 3 and Caspase 9 were significantly increased in MDA-MB-157 cells. p <0.001), flag-OTUD3 C76A +NC sh(Dox+) There was no significant difference in the activities of Caspase 3 and Caspase 9 in MDA-MB-157 cells compared to flag-OTUD3. WT +NC sh(Dox+) Group comparison, flag-OTUD3 WT +BHLHE22 sh(Dox+) The activities of Caspase 3 and Caspase 9 in MDA-MB-157 cells were significantly reduced (p<0.01 or p<0.05).

[0057] In summary, based on the GSE45827 and GSE 113865 datasets, this invention found that BHLHE22 expression is downregulated in TNBC tissues. Among various BC subtypes, BHLHE22 RNA expression levels were lowest in TNBC subtypes, and BHLHE22 expression levels in stage 3 and 4 patients were often lower than in stage 1 and 2 patients. Patients with high BHLHE22 expression had better survival prognosis than those with low expression. Functional studies showed that BHLHE22 overexpression impaired cell growth both in vitro and in vivo. However, BHLHE22 gene silencing enhanced the malignant behavior of cancer cells. OTUD3, a deubiquitinating enzyme known to inhibit TNBC progression, was found to enhance BHLHE22 protein stability through deubiquitination regulation. A mutation at the C76 site of OTUD3 eliminated OTUD3's catalytic activity but failed to regulate BHLHE22 protein stability. Furthermore, BHLHE22 enhanced the antitumor effect of OTUD3 in TNBC. mRNA sequencing analysis identified potential genes involved in the anticancer activity of BHLHE22, including CDT1. Studies have confirmed that BHLHE22 reduces CDT1 RNA expression levels by inhibiting CDT1 transcription. The antiproliferative effect of BHLHE22 overexpression can be reversed by CDT1 overexpression. The OTUD3 / BHLHE22 / CDT1 axis may be a promising direction for the treatment of TNBC.

[0058] Example 2 Paraffin-embedded tissue sections of triple-negative breast cancer were collected from 51 patients at Liaoning Cancer Hospital. Informed consent was obtained from all participants. All experiments were approved by the Ethics Committee of Liaoning Cancer Hospital. For correlation analysis of clinical factors, a BHLHE22-positive tumor cell count / total tumor cell count >10% was considered positive (+), and <10% was considered negative (-). BHLHE22 expression was associated with TNM stage and lymph node metastasis. Compared with patients with positive BHLHE22 expression, patients with negative BHLHE22 expression were more likely to be in T2-4 stage and had a higher incidence of lymph node metastasis. Figure 10 This further demonstrates that BHLHE22 may suppress the malignant phenotype of triple-negative breast cancer cells.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of BHLHE22 gene in the preparation of diagnostic and therapeutic products for triple-negative breast cancer.

2. The application according to claim 1, characterized in that, The application is performed using products for the detection and / or regulation of the BHLHE22 gene.

3. The application according to claim 2, characterized in that, The diagnostic and therapeutic products include diagnostic products, testing products, screening products, prognostic assessment products, and / or prevention and treatment products.

4. The application according to claim 3, characterized in that, The expression level of the BHLHE22 gene is downregulated in patients with triple-negative breast cancer.

5. The application according to claim 4, characterized in that, The BHLHE22 gene detection product is used to prepare diagnostic products, detection products, screening products and / or prognostic assessment products for triple-negative breast cancer.

6. The application according to claim 5, characterized in that, The diagnostic products, testing products, screening products, and / or prognostic assessment products include reagent kits.

7. The application according to claim 4, characterized in that, The BHLHE22 gene expression regulation product is used to prepare a product for the prevention and treatment of triple-negative breast cancer.

8. The application according to claim 7, characterized in that, The BHLHE22 gene expression regulation product is a product that upregulates the expression level of the BHLHE22 gene.

9. The application according to claim 8, characterized in that, The product that upregulates BHLHE22 gene expression levels works through the OTUD3 / BHLHE22 / CDT1 axis.

10. The application according to claim 9, characterized in that, The product form of the prevention and treatment products includes drugs.

Images