Application of CDCA7 and RUNX1 in screening of drugs for promoting macrophage infiltration in esophageal squamous cell carcinoma immune microenvironment

Abstract

The invention belongs to the technical field of biological medicines, and provides application of CDCA7 and RUNX1 in screening of drugs for promoting macrophage infiltration in an esophageal squamous cell carcinoma immune microenvironment. The CDCA7 is low in expression, the RUNX1 is low in expression, and immune cell infiltration in an ESCC immune microenvironment is promoted. Experimental results show that the CDCA7 positively regulates the transcriptional level of the RUNX1, and the RUNX1 negatively regulates the transcriptional level of the CCL5, so that the CDCA7 indirectly negatively regulates the expression level of the CCL5. After the expression level of CDCA7 is increased, the expression level of CCL5 is reduced, and the chemotactic effect on macrophages, especially M0 and M1 type macrophages, is reduced, so that the infiltration of the M0 and M1 type macrophages is reduced. The CDCA7 can regulate and control the expression level of RUNX1 through transcription and indirectly influence the expression level of a chemotactic factor CCL5, so that the chemotactic effect of macrophages in a tumor microenvironment, especially M1 type macrophages, is influenced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of CDCA7 and RUNX1 in screening drugs that promote macrophage infiltration in the immune microenvironment of esophageal squamous cell carcinoma. Background Art

[0002] Esophageal cancer is mainly divided into two pathological types: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). The prognosis of esophageal cancer is poor. During the period from 2010 to 2014, the 5-year net survival rate in most countries was 10 - 30%. Although in the past decade, the 5-year survival rate of ESCC patients in China has increased from 20.9% to 30.3%, however, due to the lack of early specific symptoms, clinical specific tumor markers for early diagnosis, and effective treatment targets in ESCC, most ESCC cases are found at an advanced stage, and the prognosis is still very poor.

[0003] In China, at present, the treatment of esophageal cancer still mainly relies on surgical treatment. For patients with inoperable or locally recurrent esophageal cancer, the treatment mainly relies on radiotherapy and chemotherapy. Compared with some other solid tumors, the treatment targets of ESCC are very limited, and targeted therapy is still in the exploratory stage. Therefore, strengthening the research on the carcinogenesis mechanism of ESCC is of extraordinary significance for screening early diagnostic markers and prognostic prediction markers and finding new treatment targets.

[0004] CDCA7 is a cell division cycle-related protein, also known as JPO1, located at band 3 of the long arm of chromosome 2 (2q31), and is one of the members of the cell cycle division-related protein family. It shows periodic expression during the cell cycle process, and has the highest expression level during the G1-S phase.

[0005] Some previous studies have been conducted on the function and mechanism of action of CDCA7 in ESCC. The studies have shown that CDCA7 is highly expressed in ESCC tissues, and patients with high expression of CDCA7 have a poor prognosis. In vitro and in vivo experiments have shown that overexpression of CDCA7 can promote the proliferation, invasion, and migration of ESCC cells, while knockdown of CDCA7 can inhibit the proliferation, invasion, and migration of ESCC cells; Mechanism studies have shown that the effect of CDCA7 on the occurrence, development, invasion, and migration of ESCC is mainly achieved by regulating the transcriptional levels of genes such as CCNA2, SMAD4, and SMAD7 through a transcription factor-like effect.

[0006] Previous studies on CDCA7 mainly focused on the relationship between tumor occurrence and development. In recent years, studies have shown that the occurrence, growth, and metastasis of tumors are not only related to the structure, function, and metabolism of the tissues where tumor cells are located, as well as the tumor cells themselves, but also related to their external environment. The tumor microenvironment is a very complex and dynamic ecosystem and is the "soil" on which tumors depend for survival. The main participants include: tumor cells, immune cells, and supporting cells (such as fibroblasts). The tumor immune microenvironment refers to the presence of a large number of immune cells and immune regulatory factors on the basis of the tumor microenvironment. The tumor immune microenvironment plays an important role in the occurrence, development, metastasis, and prognosis of tumors. A full understanding and study of the impact of a certain gene on the tumor immune microenvironment, as well as the impact on various immune cells and immune factors in the microenvironment and their mechanisms of action, will provide sufficient theoretical basis and reference value for tumor treatment and its prognosis. Summary of the Invention

[0007] The present invention provides the use of CDCA7 and RUNX1 in screening drugs that promote macrophage infiltration in the immune microenvironment of esophageal squamous cell carcinoma.

[0008] The present invention is achieved by the following technical solutions: The use of CDCA7 and RUNX1 as target substances in screening drugs that promote macrophage infiltration in the immune microenvironment of esophageal squamous cell carcinoma.

[0009] Furthermore, low expression of CDCA7 and low expression of RUNX1 promote the infiltration of immune cells in the ESCC immune microenvironment.

[0010] Even further, low expression of CDCA7 promotes the expression and secretion of chemokines CCL5, CXCL10, and CXCL11, and promotes the chemotaxis of MO and M1 macrophages.

[0011] The chemokine CCL5 promotes the chemotaxis of M0 and M1 macrophages, and CXCL10 and CXCL11 promote the chemotaxis of M0 macrophages.

[0012] Low expression of CDCA7 and low expression of RUNX1 promote the high expression of chemokine CCL5 and promote the chemotaxis of M0 and M1 macrophages.

[0013] The drugs that promote macrophage infiltration in the immune microenvironment of esophageal squamous cell carcinoma are CDCA7 gene inhibitors, inhibitors that inhibit the cell proliferation of overexpressed CDCA7 genes, inhibitors that inhibit the overexpression of RUNX1 genes, or promoters that promote the high expression of chemokines CCL5, CXCL10, and CXCL11.

[0014] To explore the effect of the CDCA7 gene on the tumor immune microenvironment of ESCC and its mechanism of action, the present invention analyzed the transcriptomic sequencing (RNA-sequencing, RNA-seq) data of tumor tissues and adjacent normal tissues from 155 pairs of ESCC patients collected previously by bioinformatics methods, as well as the esophageal squamous cell carcinoma data in the TCGA database. Flow cytometry immunophenotyping experiments on animal tumor tissues were used to verify the content of various immune cells in tissues with different CDCA7 expression levels; molecular experimental techniques such as transcriptome sequencing, RT-qPCR, and ELISA of CDCA7 overexpression and knockdown stable strains were used to detect the expression and secretion of chemokines and cytokines at different CDCA7 expression levels; cell co-culture experiments were used to detect the polarization, growth, and chemotactic effects of the supernatants of stable strains with different CDCA7 expression levels and the downstream chemokines regulated by CDCA7 on M0, M1, and M2 macrophages; bioinformatics analysis was used to analyze the relationship between CDCA7 and its downstream target chemokines and the content of immune cells in the tumor microenvironment; ChIP-seq was used to screen the downstream target genes regulated by CDCA7 related to chemokine expression, RT-qPCR and Western Blot were used to detect the expression levels of downstream target genes, and dual-luciferase reporter experiments were used to verify the transcriptional regulatory effect of CDCA7 on them.

[0015] The results showed that CDCA7 positively regulated the transcriptional level of RUNX1, while RUNX1 negatively regulated the transcriptional level of CCL5. Therefore, CDCA7 indirectly negatively regulated the expression level of CCL5. After the CDCA7 expression level increased, the expression level of CCL5 decreased, and the chemotactic effect on macrophages, especially M0 and M1 macrophages, decreased. Therefore, the infiltration of M0 and M1 macrophages decreased. Description of the Drawings

[0016] Figure 1 For the correlation between CDCA7 expression level and stroma, immune cell infiltration, and tumor purity in tumor tissues; * P<0.05; ** P<0.01; *** P<0.001; Figure 2 For the ssGSEA score clustering map of the transcriptome and clinical data of 155 cases of ESCC; in the figure: A is grouped by immune score; B is grouped by CDCA7 expression level; Figure 3 For the differences between 28 ssGSEA immune signaling pathways and high- and low-expression groups of CDCA7; * P<0.05; **P<0.01; *** P<0.001; Figure 4 For the linear correlation map between 28 ssGSEA immune signaling pathways and CDCA7 expression level; Figure 5Relationship between CDCA7 expression level and immune cell infiltration in TCGA database transcriptome data; In the figure: A: Correlation analysis of CDCA7 expression level and immune cell infiltration in various tumors in TCGA database, where esophageal cancer is marked in the black box. B: Relationship between CDCA7 expression level and immune subtypes. Note: C1 (wound healing); C2 (IFN-γ dominant); C3 (inflammatory); C4 (lymphocyte depleted); C6 (TGF-β dominant); Figure 6 Verification of the efficiency of mEC25-CDCA7-sh stable strain; In the figure: A is the verification of the knockdown efficiency of the mEC25-CDCA7-sh stable strain at the mRNA level; B is the verification of the knockdown efficiency of the mEC25-CDCA7-sh stable strain at the protein level; (* P<0.05; ** P<0.01; ***P<0.001); Figure 7 Effects of reduced CDCA7 expression level on immune cells and various T cells in the ESCC tumor microenvironment; In the figure: A: Effects of reduced CDCA7 expression level on total immune cells (CD45 + ) in the ESCC tumor microenvironment; B: Effects of reduced CDCA7 expression level on total T cells (CD45 + ) in the ESCC tumor microenvironment; C: Effects of reduced CDCA7 expression level on helper T cells (CD4 + ), cytotoxic T cells (CD8 + ), and γδ T cells (CD4 - CD8 - ) in the ESCC tumor microenvironment. (* P<0.05; ** P<0.01; *** P<0.001); Figure 8 Effects of reduced CDCA7 expression level on immune cells and various macrophages in the ESCC tumor microenvironment; In the figure: A: Effects of reduced CDCA7 expression level on total immune cells (CD45 + ) in the ESCC tumor microenvironment; B: Effects of reduced CDCA7 expression level on total monocytes (CD11b + ) in the ESCC tumor microenvironment; C: Effects of reduced CDCA7 expression level on total macrophages in the ESCC tumor microenvironment (F4 / 80 + ) D: Effects of reduced CDCA7 expression level on M1 macrophages and M2 macrophages in the ESCC tumor microenvironment. (* P<0.05; ** P<0.01; *** P<0.001); Figure 9 Identification of the expression of M0 macrophage markers; A: Expression levels of M0 macrophage markers; B: Expression levels of M1 macrophage markers; C: Expression levels of M2 macrophage markers (*P < 0.05; **P < 0.01; ***P < 0.001); Figure 10 Effect of CDCA7 expression levels on the chemotactic ability of M0, M1, and M2 macrophages; In the figure: A: Effect of the supernatant of KYSE150 - CDCA7 - sh stable strain on the chemotactic ability of M0 macrophages; B: Effect of the supernatant of KYSE150 - CDCA7 - sh stable strain on the chemotactic ability of M1 macrophages; C: Effect of the supernatant of KYSE150 - CDCA7 - sh stable strain on the chemotactic ability of M2 macrophages. (*P < 0.05; **P < 0.01; ***P < 0.001); Figure 11 Heatmap of differentially expressed genes of chemokines and cytokines in CDCA7 over - expressed and knocked - down stable strains; In the figure: A. Heatmap of KYSE150 - CDCA7 - sh knocked - down stable strain; B. Heatmap of KYSE180 - CDCA7 - EXP over - expressed stable strain; Figure 12 mRNA expression levels of chemokines in CDCA7 - knocked - down stable strains; In the figure: A. Expression level in KYSE150 - CDCA7 - sh stable strain; B. Expression level in KYSE410 - CDCA7 - sh stable strain; C. Expression level in KYSE450 - CDCA7 - sh stable strain (*P < 0.05; **P < 0.01; ***P < 0.001); Figure 13 mRNA expression levels of chemokines in CDCA7 - over - expressed stable strains; In the figure: A. Expression level in KYSE150 - CDCA7 - EXP stable strain; B. Expression level in KYSE410 - CDCA7 - EXP stable strain; C. Expression level in TE1 - CDCA7 - EXP stable strain (*P < 0.05; **P < 0.01; ***P < 0.001); Figure 14Protein expression levels of chemokines in CDCA7 knockdown and overexpression stable cell lines; In the figure: A. Expression levels in KYSE150 - CDCA7 - sh stable cell lines; B. Expression levels in KYSE410 - CDCA7 - sh stable cell lines; C. Expression levels in KYSE450 - CDCA7 - sh stable cell lines; D. Expression levels in KYSE150 - CDCA7 - EXP stable cell lines; E. Expression levels in KYSE180 - CDCA7 - EXP stable cell lines; F. Expression levels in TE1 - CDCA7 - EXP stable cell lines, (* P<0.05; ** P<0.01; *** P<0.001); Figure 15 Expression levels of RUNX1 and RUNX2 in CDCA7 overexpression stable cell lines; In the figure: A. Expression levels of RUNX1 and RUNX2 in KYSE180 - CDCA7 - EXP stable cell lines and their control cell lines; B. Expression levels of RUNX1 and RUNX2 in KYSE150 - CDCA7 - EXP stable cell lines and their control cell lines; C. Expression levels of RUNX1 and RUNX2 in TE1 - CDCA7 - EXP stable cell lines and their control cell lines. (* P<0.05; ** P<0.01; *** P<0.001); Figure 16 Expression levels of RUNX1 and RUNX2 in CDCA7 knockdown stable cell lines; In the figure: A. Expression levels of RUNX1 and RUNX2 in KYSE150 - CDCA7 - sh stable cell lines and their control cell lines; B. Expression levels of RUNX1 and RUNX2 in KYSE410 - CDCA7 - sh stable cell lines and their control cell lines, (* P<0.05; ** P<0.01; *** P<0.001); Figure 17 Dual - luciferase reporter assay of RUNX1 binding sequences in CDCA7 stable cell lines; In the figure: A. Dual - luciferase reporter assay of RUNX1 binding sequences in KYSE150 - CDCA7 - sh stable cell lines; B. Dual - luciferase reporter assay of RUNX1 binding sequences in KYSE410 - CDCA7 - sh stable cell lines; C. Dual - luciferase reporter assay of RUNX1 binding sequences in KYSE150 - CDCA7 - EXP stable cell lines. (* P<0.05; ** P<0.01; *** P<0.001); Figure 18Verification of the expression relationship between RUNX1 and CCL5 in ESCC cell lines; in the figure: A: Verification of the mRNA and protein expression levels of CCL5 after knocking down RUNX1 in KYSE150; B: Verification of the mRNA and protein expression levels of CCL5 after knocking down RUNX1 in the TE1-CDCA7-EXP stable cell line. (* P<0.05; ** P<0.01; *** P<0.001). Detailed implementation manners

[0017] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. The materials cited herein and their cited materials will be incorporated by reference.

[0019] Equivalent technologies of the specific embodiments described that can be understood by those of ordinary skill in the art through routine experiments will be included in this application.

[0020] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The instruments and equipment used in the following embodiments are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0021] I. Materials 1. Sample source: The 155 ESCC samples used in this invention were sourced from the Shanxi Cancer Hospital. All patients participating in the study were untreated. At the same time, in accordance with the requirements of the ethics committee, all patients signed the informed consent form. Transcriptome sequencing (RNA-sequencing, RNA-seq) studies were performed on the primary tumor tissues of 155 ESCC samples and their paired normal esophageal epithelial tissues.

[0022] 2. Cell lines: The human ESCC cell lines KYSE150, KYSE180, KYSE410, KYSE450, TE1, the murine ESCC cell line mEC25, and the human monocyte cell line THP-1 were preserved by the Key Laboratory of Esophageal Cancer Pathogenesis and Translational Research, Center for Translational Medicine.

[0023] Human CDCA7 knockdown stable cell lines KYSE150-CDCA7-sh1, KYSE150-CDCA7-sh2 and their control cell line KYSE150-sh-NC, KYSE410-CDCA7-sh1, KYSE410-CDCA7-sh2 and their control cell line KYSE410-sh-NC, KYSE450-CDCA7-sh1, KYSE450-CDCA7-sh2 and their control cell line KYSE450-sh-NC; human CDCA7 overexpression stable cell line KYSE180-CDCA7-EXP and its control cell KYSE180-EXP-NC, KYSE150-CDCA7-EXP and its control cell KYSE150-EXP-NC, TE1-CDCA7-EXP and its control cell TE1-EXP-NC; murine CDCA7 knockdown stable cell lines mEC25-CDCA7-sh1, mEC25-CDCA7-sh2 and their control cell line mEC25-sh-NC were constructed by the inventors.

[0024] 3. C57BL / 6N mice: All 4-6-week-old C57BL / 6N mice used were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.

[0025] 4. Primer sequences: All primers listed in Table 1 were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0026] Table 1: Primer sequences 5. Lentiviruses of CDCA7 gene: Lentiviruses for CDCA7 gene knockdown and overexpression were packaged and prepared by Hanheng Company. The transcript of human overexpressed CDCA7 gene is NM_145810.3, and the transcript of murine overexpressed CDCA7 gene is NM_025866. The CDCA7 knockdown sequences are shown in Table 2.

[0027] Table 2: Knockdown sequences II. Experimental methods 1. Cell culture A. Cell resuscitation: Immediately after taking out the cryopreserved cells, place them in a 37°C water bath to melt them, centrifuge at 900 rpm for 2 min to obtain cell pellets. Discard the supernatant, add 1 ml of complete cell medium, pipette repeatedly with a pipette to obtain a cell suspension, add the cell suspension to a culture dish containing fresh complete medium, and pipette evenly. Place it in a 37°C, 5% CO 2 cell incubator for culture. The formula of the complete cell medium is: FBS fetal bovine serum: RPMI 1640 = 1:9.

[0028] B. Cell passage: When the cell confluence reaches 80%-90% or more, cell passage is required. Discard the cell culture medium in the culture dish, add 2-3 mL of 1×PBS buffer that has been equilibrated to room temperature to wash the cells 1-2 times, then add an appropriate amount of 0.25% trypsin, and place it in a 37°C, 5% CO 2 incubator for 2-3 minutes. Observe under a microscope. When most cells become round and floating, digestion is complete. Immediately add complete medium to terminate digestion, transfer the cell suspension to a centrifuge tube of an appropriate volume, centrifuge at 800-1000 rpm for 3 minutes, discard the supernatant, add complete medium to resuspend the cells, and then aliquot them into several new culture dishes. The composition of 2L of 1×PBS buffer (pH = 7.4) is: 16 g of NaCl, 0.4 g of KCl, Na 2 HPO 4 2.84 g, KH 2 PO 4 1.248 g.

[0029] C. Cell cryopreservation: After digesting and centrifuging the cells in the logarithmic growth phase by the conventional method, discard the supernatant, add an appropriate amount of cell cryopreservation solution to resuspend the cell pellet. The cell cryopreservation solution is FBS fetal bovine serum: DMSO = 9:1. Aliquot it into several sterile cryotubes and cryopreserve it according to the principle of gradient cooling. The gradient cooling conditions are: 4°C for 30 minutes, -20°C for 2 hours, -80°C overnight, and long-term storage in liquid nitrogen.

[0030] D. THP-1 cell culture: The methods of cell resuscitation and cryopreservation are the same as those for ESCC cells.

[0031] Cell medium change and passage: Since THP-1 cells grow in suspension, the semi-medium change method is used for medium change to maintain the cell concentration at 0.5-1×10 6 cells. When the cell concentration is greater than 0.5-1×10 6 cells, add an appropriate amount of fresh complete medium (containing 15%-20% serum, and the serum concentration can be adjusted according to the cell state) to the cell culture flask, and divide it into two or three parts for passage.

[0032] E. Establishment of macrophage model: When the THP-1 cells are in good condition and cultured to the logarithmic growth phase, add phorbol 12-myristate 13-acetate (PMA) with a final concentration of 100 ng / ml to the complete medium, and continue to culture at 37°C, 5% CO 2Cultivate in a cell incubator for 48 hours. After observing that the suspended cells become adherent, the induction of M0 macrophages is successful. Add 100 ng / mL of LPS and 20 ng / mL of IL-4 to the successfully induced M0 macrophages respectively, and continue the drug induction for 48 hours to construct M1 macrophages and M2 macrophages respectively.

[0033] 2. Construct stable cell lines with CDCA7 knockdown and overexpression using lentivirus: (1)Cell plating: One day in advance, inoculate 0.76×10 5 cells into a 24-well plate, with each well containing 500 μL of high-glucose medium containing 10% FBS, and incubate overnight at 37°C and 5% CO 2 conditions.

[0034] (2)Calculate the volume of human and mouse CDCA7 knockdown / overexpression lentivirus required: Set different gradients of lentivirus MOI values, and calculate the volume of the required lentivirus stock solution according to the MOI value and virus titer using the following formula: Lentivirus volume = (number of cells inoculated × MOI) / virus titer.

[0035] (3)Prepare a complete medium containing Polybrene at a final concentration of 5 μg / mL, discard the old medium, add the complete medium containing Polybrene at a final concentration of 5 μg / mL, and add different volumes of lentivirus stock solution. At the same time, set up a knockdown control group, a knockdown experimental group, an overexpression control group, and an overexpression experimental group, and culture in a cell incubator at 37°C and 5% CO 2 After 12 - 24 hours of virus transfection, discard the medium containing the virus stock solution, add 1 mL of fresh complete medium, and continue to culture in a cell incubator at 37°C and 5% CO 2 conditions.

[0036] (4)When the cell confluence reaches 70% - 80%, discard the original medium, add the medium containing 4 μg / mL of puromycin and continue to expand the culture. During this process, collect cell precipitates, and at the same time use RT-qPCR and Western Blot experiments to verify the CDCA7 knockdown and overexpression efficiency. After successfully constructing the CDCA7 knockdown and overexpression stable cell lines, continue the culture and perform cell cryopreservation.

[0037] 3. Real-time fluorescence quantitative PCR (Real-time quantitative PCR, RT-qPCR) A. Extraction of total cellular RNA: (1)Remove the cell culture dish, wash it 2 - 3 times with PBS to remove the medium attached to the cell surface, discard the PBS, add RNAiso Plus to lyse the cells according to the cell quantity. After standing for 5 - 10 min, pipette up and down repeatedly and collect the cell lysate into a 1.5 mL RNA-free EP tube; or digest the cells with trypsin, collect an appropriate amount of cell precipitate into a 1.5 mL RNA-free EP tube, wash it 1 - 2 times with PBS to remove the residual medium, add RNAiso Plus to lyse the cells, and pipette up and down repeatedly until there is no obvious precipitate in the lysate, then stand for 5 - 10 min to completely lyse it; (2)Add chloroform to the EP tube, with a volume of 1 / 5 of the amount of RNAiso Plus used. Vigorously shake it until there is no obvious phase separation, and stand for 5 - 10 min; centrifuge at 12000 rpm for 15 min using a centrifuge pre-cooled to 4°C in advance; (3)After centrifugation, it can be seen that the homogenate is divided into three layers. The RNA is dissolved in the upper colorless and transparent organic phase. Gently transfer the organic phase to a new 1.5 mL RNA-free EP tube with a pipette, taking care not to touch the middle white substance layer with the pipette tip. After the transfer, roughly estimate the volume of the supernatant, add isopropanol with the same volume as the supernatant, invert and mix well, stand at -20°C for 10 min, and centrifuge at 12000 rpm for 10 min at 4°C.

[0038] (4)After centrifugation, a small amount of white precipitate can be observed at the bottom of the EP tube. Discard the supernatant and retain the white precipitate at the bottom. Slowly add 1 mL of 75% ethanol (prepared with RNA-free water) along the tube wall, invert and wash the tube wall, centrifuge at 12000 rpm for 10 min at 4°C, and then discard the supernatant. Open the lid of the EP tube and air-dry it at room temperature until the ethanol completely evaporates. Add 30 μL of RNA-free water to dissolve the white precipitate at the bottom and place it on ice; detect the concentration of the extracted RNA with a micro ultraviolet spectrophotometer.

[0039] B. Reverse transcription: Prepare the reaction system according to Table 3 and perform the reverse transcription reaction according to the reaction conditions in Table 4.

[0040] Table 3: Reaction system (10 μL system) Table 4: Reaction program C. Real-time fluorescence quantitative PCR: Use the TB Green® Premix Ex TaqTM kit for RT-qPCR. Set three replicates for each experimental group. The reaction system and reaction conditions are shown in Table 5 and Table 6.

[0041] Table 5: Reaction system Table 6: Reaction procedure D, Result analysis: Using the housekeeping gene GAPDH of the control group as an internal reference, relative quantitative analysis of the results was performed using the 2 -ΔΔCt method.

[0042] 4. Western Blot (WB) A. Total cell protein extraction: Add RIPA protein lysate containing protease inhibitor according to the cell precipitation amount, gently pipette to mix evenly, and then ultrasonically lyse on ice; after ultrasonication, centrifuge at 4°C and 12,000 rpm for 30 min; after centrifugation, transfer the cell supernatant to a pre-cooled new EP tube.

[0043] B. Protein content detection by BCA method: (1) Standard curve drawing: Dilute the protein standard with 1×PBS to a final concentration of 0.5 mg / ml; add 20 μL, 19 μL, 18 μL, 16 μL, 12 μL, 8 μL, 4 μL, 0 μL of 1×PBS buffer to the 96-well plate in sequence, and make up to 20 μL with the diluted protein standard in each well. Set three replicates for each concentration; add 200 μL of freshly prepared working solution (BCA working solution A: BCA working solution B = 50:1) to each well, mix well by shaking at 37°C for 30 minutes; detect the absorbance value of each well at 562 nm and draw the standard curve. The BCA kit was purchased from Boster Biological Technology, USA, product number AR0197.

[0044] (2) Determination of sample protein concentration: Add 19 μL of 1×PBS to the wells of the 96-well plate, then add 1 μL of the protein sample to be tested, and then add 200 μL of freshly prepared working solution to each well. Mix well by shaking at 37°C for 30 min, detect the absorbance value of each well at 562 nm wavelength, and calculate the protein concentration (μg / μL) of the sample according to the drawn standard curve. Set 3 replicates for each sample and take the average value.

[0045] C. Protein sample preparation: Calculate the loading volume of each sample according to a protein loading amount of 50 μg, make up to 15 μL with 1×PBS, add 5 μL of 4× loading buffer, mix well, boil the sample in boiling water at 100°C for 5 min, and centrifuge briefly for later use. The 4× loading buffer preparation system is: 6 ml of glycerol, 4 ml of β-mercaptoethanol, 6.66 ml of 20% SDS, 3.12 ml of 1 M Tris-HCL (pH 6.8), 1 ml of 0.04% bromophenol blue.

[0046] D. Primary antibody incubation: The prepared samples are subjected to polyacrylamide gel electrophoresis. After the transfer membrane is completed, turn off the electrotransfer instrument, take out the PVDF membrane, mark the front side of the membrane, and place it in the freshly prepared blocking solution. The blocking solution is placed in 5% skim milk powder (or BSA), and incubated at room temperature with 60 - 80 rotations for 1 hour. Refer to the antibody instruction manual, dilute the primary antigen solution with the blocking solution in proportion. After blocking, incubate the primary antibody overnight at 4°C with 60 - 80 rotations.

[0047] E. Secondary antibody incubation: After incubating the primary antibody overnight, recover the primary antibody solution, wash the membrane 3 times with 1×TBST eluent on a horizontal shaker at room temperature with 110 - 120 rotations per minute for 8 - 10 minutes each time. The 1×TBST eluent is prepared as follows: 2.42 g of sodium chloride, 17.55 g of Tris, 2 ml of Tween - 20, and made up to 2 L with triple-distilled water. After the elution is completed, add the secondary antibody solution corresponding to the property of the primary antibody, and incubate on a horizontal shaker at room temperature with 60 - 80 rotations for 2 hours. After incubating the secondary antibody, recover the secondary antibody solution, wash the membrane 3 times with 1×TBST eluent on a horizontal shaker at room temperature with 110 - 120 rotations per minute for 8 - 10 minutes each time.

[0048] F. Development and result analysis: Develop using a dual-color infrared laser imaging system or an ECL imager. Use ImageJ software to perform grayscale analysis on the pictures saved after development. Take β-actin or GAPDH as the internal reference to calculate the content of the target protein.

[0049] 5. Animal subcutaneous tumor-bearing experiment: Female C57BL / 6N mice at 4 - 6 weeks old are raised in an SPF-class mouse cage with an independent air supply system. Inject 4.0×10 6 stable transfected strains of murine ESCC cells mEC25-sh-NC, mEC25-CDCA7-sh1, and mEC25-CDCA7-sh2 subcutaneously under the inguinal region of the mice, 5 mice in each group. Dilute 4.0×10 6 murine ESCC cells with 50 μL of basal medium and mix them with an equal volume of Matrigel and place on ice. Measure the tumor volume once a week. When the tumor diameter does not exceed 1 cm, take live photos of the small animals. After taking the photos, sacrifice the animals by cervical dislocation and dissect the tumor tissues. Divide the tumors into two parts. One part is dissociated into single-cell suspensions for subsequent flow cytometry immunophenotyping experiments, and the other part is snap-frozen in liquid nitrogen and stored at -80°C for RNA-seq to analyze the relationship between CDCA7 expression and the immune microenvironment of murine tumors.

[0050] 6. Flow cytometry immunophenotyping experiment: After the tumor mass is separated, weigh it, wash it with PBS, and place it in PBS containing 0.05% FBS or 1640. If the ex vivo time is long, it needs to be placed in tissue preservation solution; In a clean bench, take a clean EP tube and add 1 ml of 1640 containing 0.05% FBS. Cut the tissue into small pieces of 1 mm³, transfer them to a 15 ml centrifuge tube, and rinse the dish with RPMI-1640 cell culture medium, and transfer them into the tube together.

[0051] After centrifuging to discard 1640, add 3 ml of diluted enzyme working solution. Place the 15 ml centrifuge tube in a constant temperature shaker at 37 °C and 200 rpm for 1 - 2 h. Take it out every 30 min and shake the tissue to disperse it. If necessary, it can be blown and suspended with a pipette.

[0052] Filter the cell suspension after enzymatic digestion through a 300-mesh cell sieve, rinse the sieve mesh and the tube wall with PBS, centrifuge at 300 g for 5 min, discard the supernatant, and resuspend the cells until the cell concentration is 10 6 ~10 7 cells / ml, and record the number of viable cells by trypan blue staining. Take 1 million cells and add them to a 1.5 ml EP tube. After centrifuging to discard PBS, add 100 ul of cell staining buffer to resuspend the cells, add 1 ug of 2.4G2 purified antibody (CD16 / 32) that blocks Fc II / III receptors, and incubate at room temperature for 15 minutes.

[0053] Centrifuge to discard the blocking antibody, add 200 ul of cell staining buffer, and wash the cells once. Centrifuge to discard the buffer, resuspend the cells with 100 ul of buffer, add surface staining antibody (1 Test corresponding to 1 million cells) in the dark and mix well, and incubate at 4 °C in the dark for 30 min.

[0054] Centrifuge to discard the surface antibody, add 200 ul of cell staining buffer, and wash the cells once. Centrifuge to discard the buffer, add 500 ul of cell staining buffer to resuspend the cells, and prepare for loading onto the machine; Cells that require intracellular antibody staining continue with subsequent permeabilization and fixation staining.

[0055] Permeabilization and fixation staining: Add 100 ul of permeabilization solution A and fix at room temperature for 15 min; Centrifuge to discard permeabilization solution A, add 200 ul of cell staining buffer, and wash the cells once. Centrifuge to discard the buffer, add 100 ul of permeabilization solution B and intracellular antibody, mix well or vortex for 1 - 2 s, and place at room temperature or 4 °C for 30 min. Centrifuge to discard permeabilization solution B, add 200 ul of cell staining buffer, and wash the cells once. Centrifuge to discard the buffer, add 500 ul of cell staining buffer to resuspend the cells, and prepare for loading onto the machine.

[0056] 7. Enzyme-linked immunosorbent assay (ELISA) Cell plating: Digest the cells in the logarithmic growth phase of each group with 0.25% trypsin and count them. Take 2.5×106 Cells were seeded in 6-cm dishes and cultured overnight in an incubator at 37 °C with 5% CO2.

[0057] ELISA sample preparation: After the cells adhered to the wall, the complete medium was discarded. The same volume (2 ml) of basal medium was added to each dish of cells, and the cells were cultured overnight (48 h) in an incubator at 37 °C with 5% CO 2 After 48 h, the supernatant was collected. The supernatant was centrifuged at 1000 rpm for 3 min at room temperature to remove precipitates such as cell debris, and the supernatant was collected for subsequent ELISA reaction experiments.

[0058] ELISA reaction experiment: Reagents, samples, instruments, and consumables were prepared according to the product manual, and an ELISA experiment was performed. The absorbance was measured at 450 nm.

[0059] 8. Cell co-culture experiment CCK8 experiment: The successfully induced macrophages in the logarithmic growth phase were digested with 0.25% trypsin and counted. 3000 cells per well were seeded in 96-well plates, and 6 replicates were set for the cells under each group of conditioned media. Five plates were seeded for each group.

[0060] After culturing for 24 h, 48 h, 72 h, 96 h, and 120 h respectively, the original medium was discarded, and fresh medium containing 10% CCK8 was added. The plates were placed in an incubator at 37 °C with 5% CO 2 and incubated for another 0.5 - 1 h. The OD value was measured at 450 nm using a microplate reader.

[0061] Transwell migration experiment: The successfully induced macrophages in the logarithmic growth phase were digested with 0.25% trypsin and counted. The macrophages were resuspended in RPMI-1640 medium to a concentration of 3×10 5 cells / 200 μL. 200 μL of the diluted cell suspension was added to the upper chamber of the Transwell, and 600 μL of different conditioned media was added to the lower chamber. The cells were cultured in a cell incubator at 37 °C with 5% CO 2 for 48 - 72 h. The 24-well plate was taken out of the cell incubator, and the cells in the upper chamber were wiped clean. The chamber was fixed with 4% paraformaldehyde for 30 min, washed with triple-distilled water, observed under a microscope, and randomly selected 5 fields of view for photography and counting.

[0062] 9. Construction of dual-luciferase reporter vector Obtaining the sequence: According to the ChiP-seq results in the UCSC database, the DNA sequence at the binding position of CDCA7 protein and RUNX1 gene was obtained.

[0063] Design primers: Select the modified pGL4.10 vector as the detection vector, combine the vector and the target sequence, select Kpn I and Nhe I as the double digestion sites, design primers according to the sequence of the target fragment and the selected digestion sites, and send the designed primers to Sangon Biotech for synthesis.

[0064] Obtain the target fragment by PCR: Dissolve and dilute the synthesized primer dry powder to the required concentration, use gDNA as the template to amplify the required target fragment according to the PCR system shown in Table 7 and the program shown in Table 8, then perform agarose gel electrophoresis, quickly cut the agarose gel at the position of the target DNA fragment size under ultraviolet irradiation, put it into a 1.5 mL EP tube, and use a gel recovery kit to purify and recover the DNA fragment. Double digest the purified and recovered target fragment with the vector, and the double digestion system is shown in Table 9; after incubating at 37°C in a water bath for 1 - 2 h, perform agarose gel electrophoresis again. After the electrophoresis is completed, perform gel cutting and recovery again, and the steps are the same as the purification of the target fragment.

[0065] Table 7: PCR system Table 8: PCR reaction program Table 9: Double digestion reaction system Ligation, transformation and identification: Measure the DNA concentrations of the target fragment and the vector recovered by enzyme digestion with a micro ultraviolet spectrophotometer, repeat the measurement of each DNA fragment 2 - 3 times, and after determining the DNA concentrations of the target fragment and the vector, prepare the reaction system according to the ligase instruction manual, ligate at 16°C for 30 min or at 4°C overnight, and the ligation reaction system is shown in Table 10.

[0066] Table 10: Ligation system Transformation reaction: Take 9 μL of the ligation product into a clean EP tube, add 1 μL of Solution Ⅲ (this step is optional), mix well by pipetting, then add it to 50 μL of DH-5α competent cells, gently flick or pipette to mix well, place on ice for 30 min, heat shock at 42°C for 90 s (accurately time), and place on ice for 2 - 3 min. Add 900 μL of antibiotic-free LB medium to the competent cells transfected with the plasmid, and shake the bacteria at 37°C and 200 rpm in a constant temperature shaking incubator for 1 h.

[0067] After the incubation at room temperature was completed, centrifuge at 2000 rpm for 1 min, discard 800 μL of the supernatant, and evenly pipette the remaining 200 μL of the bacterial solution onto an LB solid plate containing the corresponding antibiotic. Spread it evenly with a pre-prepared sterile glass spreader and incubate it upside down at 37 °C for 12 - 16 h.

[0068] Identification: Use a sterile inoculation loop to pick several plump single colony clones on the plate and place them in 2 - 3 mL of LB liquid medium containing the corresponding antibiotic. Shake the bacteria overnight for 12 - 16 h. Use colony PCR or double digestion to verify whether the target sequence is inserted into the vector. Positive samples are further sent to the company for sequencing to verify whether there are mutations in the sequence.

[0069] 10. Dual-luciferase reporter assay Cell plating: Count and plate the cells one day before transfection to ensure that the cell density reaches about 60% - 70% when transfected the next day. Each group of cells has 3 replicate wells and incubate them overnight in a cell culture incubator at 37 °C with 5% CO 2 in the incubator.

[0070] Plasmid transfection: Calculate the mass of the plasmids required for each group of cells according to 800 ng of pGL4.10 recombinant plasmid and 2.4 ng of pRL-TK internal reference plasmid per well. The amounts of Opti-MEM and transfection reagent EL required per well are 50 μL and 1.6 μL respectively. After the system is prepared, gently mix it and let it stand for 15 - 20 min, then dropwise add it into the cells containing 500 μL of antibiotic-free complete medium and culture it in a cell culture incubator at 37 °C with 5% CO2.

[0071] Cell lysis: 48 h after plasmid transfection, discard the medium, wash it twice with 1×PBS buffer, add 100 μL of 1×Cell Lysis Buffer cell lysis solution to each well, lyse the cells on ice for 10 min, scrape the cells with a cell scraper, transfer them to a 1.5 mL sterilized centrifuge tube, centrifuge at 4 °C and 12000 rpm for 10 min, and transfer the supernatant to another new EP tube.

[0072] Dual-luciferase reporter assay: Operate according to the instructions of the TransDetect® Dual-Luciferase Reporter Assay Kit. Aliquot 50 μL of Luciferase Reaction Reagent Ⅰ into each EP tube, add 10 μL of cell lysate supernatant to Luciferase Reaction Reagent Ⅰ, and detect the activity value RLU1 of firefly luciferase in a chemiluminescence analyzer. Take out the EP tube and then add 50 μL of Luciferase Reaction Reagent Ⅱ to detect the activity value RLU2 of Renilla luciferase. The value of RLU1 / RLU2 is the result of the dual-luciferase reporter assay.

[0073] 11. RNA-seq and Its Data Analysis RNA extraction: Collect cell pellets, perform RNA extraction and detection, and perform quality control on the extracted RNA samples using an Agilent 2100 bioanalyzer.

[0074] Library construction: Using random oligonucleotide chains as primers and fragmented mRNA as a template, synthesize the first strand of cDNA under the action of M-MuLV reverse transcriptase. Subsequently, remove RNA from the product mixture (RNaseH degradation method). After RNA removal, synthesize the second strand of cDNA using DNA polymerase I with dNTPs as raw materials. Purify the synthesized product. After end repair, A-tailing, and ligation of sequencing adapters to the purified double-stranded cDNA, screen cDNA around 250 - 300 bp for PCR amplification and purify the PCR product again to finally obtain the library. The kit used for library construction is NEBNext® Ultra™ RNA Library Prep Kit for Illumina®. After library construction, first perform preliminary quantification using a Qubit 2.0 Fluorometer, dilute the library to 1.5 ng / ul, and then use an Agilent 2100 bioanalyzer to detect the insert size fragment size distribution of the library to ensure library quality.

[0075] Sequencing on the machine: Use the Illumina sequencing platform for sequencing to obtain the original sequencing data, which is called raw data or raw reads, and perform quality control on the original raw data using fastqc. Subsequently, filter the original test data to remove reads with adapters, low quality, and ambiguous base information (reads with the number of bases with Qphred <= 20 accounting for more than 50% of the entire read length) to obtain Clean Reads.

[0076] Reference genome alignment: The HISAT2 software was used to align the Clean Reads with the reference genome to obtain the positioning information of the Reads on the reference genome. The overall alignment rate of this sample reached over 94%.

[0077] Quantitative analysis: The featureCounts in the subread software was used to perform quantitative analysis on the reads aligned to the genome to obtain gene expression quantification and the original read count value of each gene in the sample. Subsequently, after correcting for sequencing depth and gene length using FPKM, the expression values (FPKM) of all genes were obtained.

[0078] Differential analysis: The R package DESeq2 was used to screen for differential genes in different groups. The screening criteria were |log2(FoldChange)| > 1 & padj <= 0.05. The screened differential genes were subjected to GO functional enrichment analysis, KEGG pathway enrichment analysis, clustering analysis, etc.

[0079] Data statistical method: All experiments were independently repeated three times, and the data were expressed as x ± s. The t-test was used for comparison of experimental data between two groups, and one-way ANOVA was used for comparison among multiple groups. P < 0.05 was considered to have significant statistical differences. The GraphPad Prism 8.0 software was used for data collation and graphing, the Image J software was used for quantifying the pictures, and the AI software was used for combining the pictures.

[0080] III. Experimental results 1. The expression level of CDCA7 is negatively correlated with the infiltration of immune cells in the ESCC tumor microenvironment: 155 ESCC samples were divided into two groups according to the high and low CDCA7 expression levels at the median, named CDCA7-High and CDCA7-Low. Subsequently, the scores of stroma, immune cell infiltration in tumor tissues, and tumor purity in the tumor tissues of the two groups were analyzed respectively. The results are as Figure 1 shown. It was found that the expression level of CDCA7 showed a negative correlation trend with these three scores.

[0081] Furthermore, 155 cases were stratified and clustered into a high-immunity group and a low-immunity group according to the ssGSEA scores of 28 immune signals. The changes in immune signals and the CDCA7 expression level in the two groups were observed. The results are as Figure 2 shown in Figure A. The results showed that in the high-immunity score group, patients with low CDCA7 expression were mainly aggregated, while in the low-immunity score group, patients with high CDCA7 expression were mainly aggregated. 155 ESCC cases were divided into two groups of high and low CDCA7 according to the CDCA7 expression level, and the differences in immune signals between the two groups were studied. The results are asFigure 2 As shown in Figure B, analysis revealed that the immune score was low in the high CDCA7 expression group and high in the low CDCA7 expression group, indicating that the expression level of CDCA7 may be negatively correlated with tumor immunity.

[0082] The transcriptome data of 155 patients were divided into two groups according to the high and low CDCA7 expression levels, and the differences in 28 immune signaling pathways and the correlations between these 28 immune signaling pathways and the CDCA7 expression level were studied in these two groups of data. The results are as Figure 3 、 4 shown. It is suggested that in patients in the low CDCA7 expression group, the expression of markers of various immune cells such as macrophages, various T cells, dendritic cells, natural killer cells (NK cells), and MDSC cells was significantly increased, indicating that in the tumor immune microenvironment with low CDCA7 expression, the content of various immune cells such as macrophages, various T cells, dendritic cells, natural killer cells (NK cells), and MDSC cells was rich and the infiltration degree was high; while in the tumor immune microenvironment with high CDCA7 expression, the content of various immune cells such as macrophages, various T cells, dendritic cells, natural killer cells (NK cells), and MDSC cells was low and the infiltration degree was low. The expression level of CDCA7 was significantly correlated with 21 of the 28 ssGSEA immune signaling pathways, and was also significantly correlated with the central memory CD4 T cell, central memory CD8 T cell, helper T cell, regulatory T cell, γδ T cell, and macrophage pathways among them. Therefore, multiple immunofluorescence and mouse tumor flow cytometry experiments will continue to be used to focus on verifying the infiltration of various T cells and macrophages in ESCC tumor tissues.

[0083] 2. TCGA ESCC transcriptome data indicate that CDCA7 is negatively correlated with immune cell infiltration in the ESCC microenvironment To verify the relationship between the CDCA7 gene expression level and tumor immune infiltration, the transcriptome data of esophageal cancer tissues in the TCGA database (http: / / cis.hku.hk / TISIDB / index.php) were used for correlation analysis of the CDCA7 expression level and tumor immune cell infiltration. The analysis found that CDCA7 was negatively correlated with the infiltration of most immune cells, such as Macrophages, NK cells, Immature B cells, Mast cells, pDC, iDC, NK CD56bright cells, Tregs, Tem_CD4, Tem_CD8, TFH, Th1, Tgd, Eosinophils (p<0.05), and positively correlated with the infiltration of Act_CD4 and Th2 (p<0.05) ( Figure 5 A).

[0084] Further immune subtype analysis revealed that the expression level of CDCA7 in esophageal cancer was highest in tumors of the IFN-gamma dominant subtype (C2 type) and the lymphocyte depleted subtype (C4 type), and lowest in tumors of the TGF-β dominant subtype (C6 type). Figure 5 B).

[0085] 3. Low expression of CDCA7 promotes infiltration of T cells and macrophages in the tumor microenvironment of C57 mice A. Successfully constructed a stable knockdown strain of murine esophageal squamous cell line mEC25: To verify the effect of CDCA7 expression level on the infiltration of T cells and macrophages in the ESCC tumor immune microenvironment, the murine ESCC cell line mEC25 was infected with lentivirus to construct cell lines mEC25-CDCA7-sh1, mEC25-CDCA7-sh2 with stable knockdown of murine CDCA7 and their control cell line mEC25-sh-NC. RT-qPCR and Western Blot assays were used to detect the knockdown of CDCA7 at the mRNA and protein levels. The results are as Figure 6 shown, indicating that the expression of CDCA7 was decreased at both the mRNA and protein levels in the stable knockdown strains, suggesting that the stable strains were successfully constructed and subsequent subcutaneous tumorigenesis experiments in mice could be carried out.

[0086] B. Flow cytometry immunophenotyping experiments showed that low expression of CDCA7 promoted the infiltration of T cells and macrophages in the microenvironment: 4×10 6 cells of mEC25-sh-NC and mEC25-CDCA7-sh were respectively injected subcutaneously into the groin of 4-6-week-old C57 mice, 5 mice in each group. 2-3 weeks after subcutaneous injection of tumor cells into C57 mice, the mice were sacrificed and the tumor masses were dissected. Subsequently, the tumor masses were dissociated into single-cell suspensions. The dissociated single-cell suspensions were divided into two groups and stained with flow cytometry antibodies to label T cells and macrophages respectively. The markers for the T cell group were CD45, CD3, CD4, and CD8, and the markers for the macrophage group were CD45, CD11b, F4 / 80, MHC II, and CD206.

[0087] The flow cytometry results of the antibody-labeled T cell group are as Figure 7 shown, showing that: compared with the mice injected with the mEC25 control cell line, the immune cells, namely CD45 + cell content in the tumor tissues of C57 mice injected with the mEC25-CDCA7-sh stable strain was significantly increased ( Figure 7 A), and at the same time, the content of T cells, namely CD3 + cells was significantly increased.Figure 7 B); Continuing the analysis of cells labeled with CD4 and CD8 antibodies, it was found that the content of helper T cells (Th cells) of CD4 + increased in the tumors of mice injected with the mEC25-CDCA7-sh stable strain, but the P value was not significant. For cytotoxic T cells (Tc or CTL) of CD8 + in the tumors of mice injected with the mEC25-CDCA7-sh stable strain, only the sequence of CDCA7-sh1 showed a significant increase, while the sequence of CDCA7-sh2 had no significant change. However, compared with the mEC25 control cell line, for both the CDCA7-sh1 sequence and the CDCA7-sh2 sequence, the content of γδ T cells (CD4 - CD8 - ) was significantly increased in the tumor tissues of C57 mice injected with the mEC25-CDCA7-sh stable strain ( Figure 7 C).

[0088] Flow cytometry results of antibodies labeling macrophages showed that: compared with mice injected with the mEC25 control cell line, the content of monocytes, that is, cells of CD11b + , was significantly increased in the tumor tissues of C57 mice injected with the mEC25-CDCA7-sh stable strain ( Figure 8 B); Continuing to analyze the content of cells of F4 / 80 + in the cells of CD11b + , it was found that after the CDCA7 expression level decreased, the content of total macrophages (F4 / 80 + ) in the tumor tissues of C57 mice was significantly increased ( Figure 8 C); Continuing to analyze the cells labeled with MHC II and CD206 antibodies in the cells positive for CD11b + and F4 / 80 + , it was found that compared with mice injected with the mEC25 control cell line, in the tumor tissues of the sequence of CDCA7-sh1, the content of both M1 macrophages (MHC II + ) and M2 macrophages (CD206 + ) was significantly increased. However, in the tumor tissues of the sequence of CDCA7-sh2, the content of M2 macrophages (CD206 + ) had no change compared with the control group ( Figure 8 D).

[0089] The above results indicate that after the CDCA7 expression level decreases, it can promote the infiltration of T cells, especially γδ T cells (CD4 - CD8 - ) and macrophages, especially M1 macrophages.

[0090] C. Low expression of CDCA7 promotes the chemotaxis of ESCC cells towards M0 and M1 macrophages: Both bioinformatics and flow cytometry experiments on in vivo tumor tissues formed by subcutaneous injection of ESCC cells in animals showed that the expression level of CDCA7 can affect T cells and macrophages, especially the content of M0 and M1 macrophages in the tumor immune microenvironment of ESCC. As is well known, macrophages play an important role in the regulation of the immune microenvironment. Therefore, we focused on verifying the regulation of macrophages by the expression level of CDCA7, including aspects such as the growth and chemotaxis of M0, M1, and M2 macrophages.

[0091] E. Successfully constructed in vitro models of M0, M1, and M2 macrophages: Monocytic cell line THP-1 is a suspension cell. When observing THP-1 cells with good growth status under the microscope, their morphology is uniform and round, without particulate matter such as cell debris, and the cell proliferation rate is good, showing logarithmic growth. After inducing THP-1 cells with 100 ng / ml phorbol 12-myristate 13-acetate (PMA) for 24 h, it was observed under the microscope that the cells gradually changed from suspension growth to adherent growth, and their morphology also changed from single, round cells to irregular shapes with obvious pseudopodia extended. The cell growth status was good. After treating THP-1 cells with 100 ng / ml phorbol 12-myristate 13-acetate (PMA) for 48 h, the successfully induced M0 macrophages were respectively added with 100 ng / mL lipopolysaccharide (LPS) and 20 ng / mL interleukin-4 (IL-4), and drug induction was continued for 48 h to construct M1 and M2 macrophages respectively. We used RT-qPCR technology to identify the surface markers of M0, M1, and M2 macrophages. The results are as Figure 9 shown. The marker CD68 of M0 macrophages showed increased expression compared with THP-1, the markers TNF and IL-12a of M1 macrophages showed increased expression compared with THP-1, and the markers CD163 and MCR-1 of M2 macrophages showed increased expression compared with THP-1, indicating that M0, M1, and M2 macrophages were successfully induced.

[0092] 4. Low expression of CDCA7 promotes the chemotaxis of M0 and M1 macrophages, and has no effect on M2 macrophages: Chemotaxis experiments of M0, M1, and M2 macrophages were carried out using the collected different conditioned media. The conditioned media were prepared with the supernatants of the collected KYSE150-CDCA7-sh cell line and its control cell line KYSE150-sh-NC, and used as the lower chamber media of the Transwell chamber. The upper chamber was the basal medium. After 72 h, crystal violet staining was performed and photographed for statistics. The results are as Figure 10 shown. After analysis, it was found that knockdown of CDCA7 can promote the chemotaxis of M0 and M1 macrophages, but has no effect on the chemotaxis of M2 macrophages.

[0093] 5. Low expression of CDCA7 promotes the expression and secretion of CCL5, and further promotes the chemotaxis of M0 and M1 macrophages: RNA-seq data analysis showed that the expression of CDCA7 affects chemokines such as CCL5, CXCL10, and CXCL11: The cell precipitates of the constructed KYSE150-CDCA7-sh knockdown stable strain and its control cell line KYSE150-sh-NC, KYSE180-CDCA7-EXP overexpression stable strain and its control cell line KYSE180-EXP-NC were subjected to RNA-seq, and the differentially expressed cytokines and chemokine genes screened by RNA-seq were analyzed by heat map. The results are as Figure 11 shown. The analysis showed that a decrease in CDCA7 can promote the expression of chemokines CXCL8, CCL5 and cytokines IL32, IL12A, IL6, IL18, IL1A, IL1B, and an increase in CDCA7 can promote chemokine CXCL14 and cytokine IL18, and inhibit the expression of chemokines CCL2, CXCL1, CCL20, CXCL2, CXCL3, CXCL11, CXCL10.

[0094] RT-qPCR and ELISA experiments showed that low expression of CDCA7 promoted the expression of CCL5, CXCL10, and CXCL11, and high expression inhibited the expression of CCL5, CXCL10, and CXCL11; To further verify the above results, the results of RNA-seq and Luminex multi-factor chips were comprehensively referred to, and the primers for fluorescence quantitative PCR of differentially expressed chemokines screened from them were selected, including CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCL8, CXCL10, CXCL11, and CXCL14.

[0095] RT-qPCR experiments were respectively performed in the KYSE150-CDCA7-sh, KYSE410-CDCA7-sh, KYSE450-CDCA7-sh knockdown stable strains and their corresponding control cell lines, as well as the KYSE180-CDCA7-EXP, KYSE150-CDCA7-EXP, TE1-CDCA7-EXP overexpression stable strains and their corresponding control cell lines to detect the mRNA expression levels of these chemokines. The experimental results are as Figure 12 、 13As shown, the results showed that in the KYSE150-CDCA7-sh, KYSE410-CDCA7-sh, and KYSE450-CDCA7-sh knockdown stable cell lines, the expression levels of chemokines CCL2 and CXCL8 showed a downward trend, while the expression levels of chemokines CCL5, CXCL1, CXCL2, CXCL10, CXCL11, and CXCL14 showed an upward trend; in the KYSE180-CDCA7-EXP, KYSE150-CDCA7-EXP, and TE1-CDCA7-EXP overexpression stable cell lines, the expression levels of chemokines CCL2, CCL5, CXCL3, CXCL8, CXCL10, and CXCL11 showed a downward trend, while the expression levels of chemokines CXCL1 and CXCL2 showed an upward trend.

[0096] Based on the RT-qPCR experimental results of the above four CDCA7 stable cell lines, only chemokines CCL5, CXCL10, and CXCL11 had a consistent correlation with the expression level of CDCA7, and the protein expression levels of these three chemokines were verified subsequently.

[0097] ELISA experiments showed a negative correlation between CDCA7 and CCL5, CXCL10, and CXCL11 at the protein level: After RT-qPCR detection of the chemokine expression levels in two CDCA7 knockdown stable cell lines and overexpression stable cell lines respectively, it was found that only chemokines CCL5, CXCL10, and CXCL11 had a consistent correlation with the expression level of CDCA7. Therefore, an ELISA kit was used to measure the protein expression levels of these three chemokines. The results were as Figure 15As shown in the figure, the results showed that the expression of chemokine CCL5 tended to increase in the three CDCA7 knockdown ESCC stable cell lines 150-CDCA7-sh, 410-CDCA7-sh, and 450-CDCA7-sh, while it tended to decrease in the three CDCA7 overexpressing ESCC stable cell lines 180-CDCA7-EXP, 150-CDCA7-EXP, and TE1-CDCA7-EXP, indicating that the expression level of chemokine CCL5 was negatively correlated with the expression level of CDCA7. The expression of chemokine CXCL10 tended to increase in the three CDCA7 knockdown ESCC stable cell lines, tended to decrease in the 150-CDCA7-EXP and TE1-CDCA7-EXP stable cell lines, and there was no significant change in the expression level of the 180-CDCA7-EXP stable cell line compared with the 180-EXP-NC stable cell line. The protein expression of chemokine CXCL11 could not be detected in the cell lines KYSE410 and KYSE180, and there was no significant change in the expression level of the KYSE450-CDCA7-sh stable cell line and the TE1-CDCA7-EXP stable cell line compared with their respective control stable cell lines. The expression showed an upward trend in the 150-CDCA7-sh stable cell line and a downward trend in the 150-CDCA7-EXP stable cell line, indicating that the expression level of CXCL11 was negatively correlated with the expression level of CDCA7.

[0098] The verification of the protein level in the ELISA experiment obtained results consistent with those of the mRNA level verification. The ELISA results are as Figure 14 shown, indicating that CDCA7 may affect the infiltration of immune cells by regulating the expression and secretion levels of chemokines CCL5, CXCL10, and CXCL11. Next, the relationships between these three chemokines and immune cell infiltration were analyzed separately in the esophageal cancer transcriptome data of the TCGA database to further verify the relationships between these three chemokines and immune cell infiltration.

[0099] The ESCC transcriptome data in the TCGA database showed that CCL5, CXCL10, and CXCL11 promoted the infiltration of macrophages in the tumor microenvironment: After bioinformatics screening and verification by RT-qPCR and ELISA experiments, it was found that the chemokines CCL5, CXCL10, and CXCL11, which may be negatively regulated by CDCA7. In the TCGA database, the transcriptome data was used to analyze the correlations between the expression levels of CCL5, CXCL10, and CXCL11 and the expression levels of various immune cells, especially macrophage markers. The analysis found that the expressions of these three chemokines were positively correlated with the expressions of various immune cells and macrophage markers.

[0100] 6. Co-culture with macrophages using antagonist dosing experiments of CCR3, CCR5, and CXCR3 respectively to observe the chemotactic effects of the three chemokines CCL5, CXCL10, and CXCL11 on macrophages.

[0101] Receptor antagonist dosing experiments showed that the chemokine CCL5 promoted the chemotactic ability of M0 and M1 macrophages, CXCL10 and CXCL11 promoted the chemotactic ability of M0 macrophages, and had no effect on the chemotaxis of M1 macrophages. To further explore the effects of CCL5, CXCL10, and CXCL11 on the chemotactic ability of macrophages, antagonist dosing experiments of CCR3, CCR5, and CXCR3 were used to co-culture with macrophages respectively to observe the chemotactic effects of the three chemokines CCL5, CXCL10, and CXCL11 on macrophages. Since the supernatant of the 150 knockdown stable strain only affected the chemotaxis of M0 and M1 macrophages, the effects of the antagonists of CCR3, CCR5, and CXCR3 on M0 and M1 macrophages were only verified. The results were as shown in 15 and Figure 16 As shown, after adding the antagonists of CCR3, CCR5, and CXCR3 to M0 macrophages, the number of cells passing through the chamber decreased, indicating that CCL5, CXCL10, and CXCL11 promoted the chemotaxis of M0 macrophages. After adding the antagonists of CCR3 and CCR5 to M1 macrophages, the number of cells passing through the chamber decreased, while after adding the CXCR3 antagonist, the number of cells passing through the chamber did not change significantly, indicating that CCL5 promoted the chemotaxis of M1 macrophages, while CXCL10 and CXCL11 had no effect on the chemotaxis of M1 macrophages.

[0102] 7. Low expression of CDCA7 inhibits the expression of RUNX1, a transcriptional inhibitor of CCL5, and thus promotes the expression of CCL5. CDCA7 can bind to the promoter regions of RUNX1 and RUNX2. To explore the relationship between the expression of CDCA7 and the RUNX family in ESCC, proteins of the RUNX family that bind to CDCA7 were screened in ChIP-seq data, and it was found that RUNX1 and RUNX2 could bind to CDCA7, and CDCA7 had several binding sites with them in the promoter regions of the RUNX1 and RUNX2 genomes respectively.

[0103] CDCA7 positively regulates the expression of RUNX1: To further explore the correlation between the expression of CDCA7 and RUNX1 and RUNX2, RT-qPCR and Western Blot assays were used to detect the expression levels of RUNX1 and RUNX2 in the KYSE150-CDCA7-sh, KYSE410-CDCA7-sh knockdown stable cell lines and their corresponding control cell lines, as well as the KYSE180-CDCA7-EXP, KYSE150-CDCA7-EXP, TE1-CDCA7-EXP overexpression stable cell lines and their corresponding control cell lines. The results are as Figure 15 and Figure 16 shown. In the CDCA7 overexpression stable cell lines, the expression levels of RUNX1 and CDCA7 were positively correlated at both the protein and mRNA levels. In the TE1-CDCA7-EXP overexpression stable cell line, there was no change in the expression level of RUNX2 compared to its control cell line at the protein level. In the CDCA7 knockdown stable cell lines, the expression level of RUNX1 decreased at both the protein and mRNA levels compared to the control cell lines, while the expression level of RUNX2 showed inconsistent changes or no significant significance, indicating that CDCA7 can positively regulate the expression level of RUNX1.

[0104] CDCA7 positively regulates the expression of RUNX1 at the transcriptional level: The previous experimental results showed that CDCA7 can bind to the promoter region of RUNX1 and positively regulate its expression level. Therefore, three DNA sequences that bind CDCA7 to the promoter region of RUNX1 analyzed from the ChIP-seq results were inserted into the modified pGL4.10 vector and named RUNX1-1, RUNX1-2, and RUNX1-3, respectively. The successfully constructed recombinant vectors and their internal reference vector pBEC22 were co-transfected into the KYSE150-CDCA7-sh stable cell line, KYSE410-CDCA7-sh stable cell line and their control cells, as well as the KYSE150-CDCA7-EXP stable cell line and its control cell line to verify whether CDCA7 regulates the transcription of RUNX1 through the above binding sequences. The results are as Figure 17 shown. In the KYSE150-CDCA7-sh stable cell line and KYSE410-CDCA7-sh stable cell line, the expression level of the luciferase reporter gene decreased compared to their control cells, indicating that after the CDCA7 expression level decreased, it could downregulate the expression of RUNX1. Conversely, in the KYSE150-CDCA7-EXP stable cell line, the expression level of the luciferase reporter gene increased compared to its control cells, indicating that after the CDCA7 expression level increased, it could promote the expression of RUNX1. In summary, CDCA7 can positively regulate the expression of RUNX1 at the transcriptional level.

[0105] RUNX1 inhibits the expression level of CCL5 in ESCC: In the KYSE150 blank cell line and the TE1-CDCA7-EXP stable cell line, small interfering RNAs targeting RUNX1 were used to perform knockdown experiments. RT-qPCR and ELISA assays were respectively used to detect the mRNA and protein expression levels of CCL5. The results are as Figure 18 shown. Whether in the ESCC blank cell line KYSE150 or the CDCA7-overexpressing stable cell line TE1-CDCA7-EXP, knockdown of RUNX1 led to an increase in the expression level of CCL5, indicating that there is also a negative regulatory relationship between RUNX1 and CCL5 in ESCC.

[0106] In summary, CDCA7 can positively regulate the transcriptional level of RUNX1, a transcriptional repressor of CCL5, and indirectly negatively regulate the expression level of CCL5. In the ESCC microenvironment with high CDCA7 expression, the expression level of CCL5 is reduced, so the infiltration of M0 and M1 macrophages is decreased.

[0107] In this invention, the transcriptome data of 155 ESCC samples and the transcriptome data in the TCGA database on the TISIDB website were used to analyze the correlation between the CDCA7 expression level and 28 immune signaling pathways, and it was found that CDCA7 was negatively correlated with most immune cells. Animal experiments and flow cytometry immunophenotyping experiments were used to analyze the relationship between the CDCA7 expression level and T cells and macrophages, with a focus on the effect of the CDCA7 expression level on macrophages. The immunophenotyping results of animal tumors showed that the expression level of CDCA7 was negatively correlated with M0 macrophages and M1 macrophages. The co-culture experiment of the supernatant of the CDCA7 stable cell line and macrophages showed that low CDCA7 expression promoted the chemotaxis of M0 and M1 macrophages, obtaining results consistent with those of bioinformatics analysis and animal tumor immunophenotyping experiments. Further, three chemokines, CCL5, CXCL10, and CXCL11, were identified.

[0108] After identifying these three chemokines, experiments with three chemokine receptor antagonists were conducted. The final experimental results showed that the chemokine CCL5 could promote the chemotaxis of both M0 macrophages and M1 macrophages. To further clarify the mechanism by which CDCA7 regulates CCL5 expression, subsequent explorations were carried out. It was found through research that CDCA7 can positively regulate the transcriptional level of the upstream transcription factor RUNX1 of CCL5.

[0109] In summary, CDCA7 positively regulates the transcriptional level of RUNX1, while RUNX1 negatively regulates the transcriptional level of CCL5. Therefore, CDCA7 indirectly negatively regulates the expression level of CCL5. After the increase in the expression level of CDCA7, the expression level of CCL5 decreases, and the chemotactic effect on macrophages, especially M0 and M1 macrophages, decreases. Therefore, the infiltration of M0 and M1 macrophages decreases. CDCA7 indirectly affects the expression level of chemokine CCL5 by transcriptionally regulating the expression level of RUNX1, thereby affecting the chemotaxis of macrophages, especially M1 macrophages, in the tumor microenvironment.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of CDCA7 and RUNX1 as targets in screening drugs that promote macrophage infiltration in the immune microenvironment of esophageal squamous cell carcinoma.

2. The use according to claim 1, characterized in that: The low expression of CDCA7 and RUNX1 promotes the infiltration of immune cells in the immune microenvironment of ESCC.

3. The use according to claim 2, characterized in that: The low expression of CDCA7 promotes the expression and secretion of chemokines CCL5, CXCL10 and CXCL11, and promotes the chemotaxis of MO and M1 macrophages.

4. The use according to claim 3, characterized in that: The chemokine CCL5 promotes the chemotaxis of M0 and M1 macrophages, and CXCL10 and CXCL11 promote the chemotaxis of M0 macrophages.

5. The use according to claim 2, characterized in that: The low expression of CDCA7 and RUNX1 promotes the high expression of the chemokine CCL5 and promotes the chemotaxis of M0 and M1 macrophages.

6. The use according to any one of claims 1 to 5, characterized in that: The drug that promotes macrophage infiltration in the immune microenvironment of esophageal squamous cell carcinoma is a CDCA7 gene inhibitor, an inhibitor that inhibits proliferation of cells overexpressing the CDCA7 gene, an inhibitor that inhibits overexpression of the RUNX1 gene, or a promoter that promotes high expression of chemokines CCL5, CXCL10 and CXCL11.

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