Application of SOX12 inhibitor in reducing cisplatin tolerance of esophageal cancer cells

Abstract

The present invention relates to the field of biomedical technologies and provides an application of an SOX12 inhibitor in a drug for reducing the tolerance of esophageal cancer cells to cisplatin. The present invention applies the SOX12 inhibitor to the preparation of a drug for reducing the tolerance of esophageal cancer cells to cisplatin, and significantly reduces the tolerance of esophageal cancer cells to cisplatin by inhibiting the expression of SOX12 in esophageal cancer cells. In particular, the SOX12 inhibitor can be used in combination with cisplatin for the treatment of esophageal cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of SOX12 inhibitors in drugs for reducing the tolerance of esophageal cancer cells to cisplatin. Background Art

[0002] Esophageal cancer (ESCA) is divided into esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Currently, the main treatment methods for esophageal cancer include surgical intervention, chemotherapy, and radiotherapy. Chemotherapy is an effective treatment method for esophageal cancer, which can shrink tumor lesions and control local tumor progression. Cisplatin, as a first-line drug for the treatment of esophageal cancer, is widely used in the clinical treatment of esophageal cancer. However, the chemotherapy effect is significantly hindered by the emergence of drug resistance, and overcoming cisplatin resistance has become an urgent problem in the treatment of esophageal cancer. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the present invention provides the application of SOX12 inhibitors in drugs for reducing the tolerance of esophageal cancer cells to cisplatin, aiming to solve the problems mentioned in the background art.

[0004] Sex-determining region Y (SRY)-box transcription factor 12 (SOX12) belongs to the SOX transcription factor family. SOX12 mainly acts as a transcriptional activator and plays an important role particularly in embryonic development and cell fate determination.

[0005] In a first aspect, the present invention provides the application of SOX12 inhibitors in the preparation of drugs for reducing the tolerance of esophageal cancer cells to cisplatin.

[0006] Further, the SOX12 inhibitor includes at least one of RNA interference molecules or molecular compounds that inhibit the expression of SOX12.

[0007] Further, the RNA interference molecule includes at least one of shRNA, siRNA, dsRNA, or miRNA.

[0008] Further, the shRNA sequences are as shown in SEQ ID NO.1-2.

[0009] Further, the esophageal cancer cells are esophageal squamous cell carcinoma cells.

[0010] Further, the esophageal squamous cell carcinoma cells are Eca109 cells.

[0011] In a second aspect, the present invention provides a method for reducing the tolerance of esophageal cancer cells to cisplatin, which reduces the tolerance of esophageal cancer cells to cisplatin by inhibiting the expression of SOX12 in esophageal cancer cells.

[0012] Thirdly, the present invention provides a pharmaceutical composition for the combined treatment of esophageal cancer, which comprises a SOX12 inhibitor and cisplatin.

[0013] The present invention has the following technical effects:

[0014] (1) Applying a SOX12 inhibitor to the preparation of a drug for reducing the tolerance of esophageal cancer cells to cisplatin, the tolerance of esophageal cancer cells to cisplatin is reduced by inhibiting the expression of SOX12 in esophageal cancer cells. SOX12 can be used as a therapeutic target for overcoming chemotherapy resistance in esophageal cancer, especially the SOX12 inhibitor can be used in combination with cisplatin to treat esophageal cancer.

[0015] (2) Inhibiting the expression of SOX12 in esophageal cancer cells can promote the transcription of UBR5 and TRIP12, so as to inhibit the stability of RNF168 protein, thereby inhibiting the activation of the DNA damage repair pathway, and achieving the purpose of reducing the tolerance of esophageal cancer cells to cisplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] By referring to the following drawings, the exemplary embodiments of the present invention can be more fully understood:

[0017] Figure 1 It is a result diagram of detecting the effect of cisplatin on Eca109 cells and Eca109-CR (a cisplatin-resistant cell line derived from Eca109 cells) in Example 1 of the present invention; wherein:

[0018] Figure 1 In A, it is a result diagram of the doubling time of Eca109 cells and Eca109-CR cells measured by the CCK8 (Cell Counting Kit-8) detection method in the CCK8 proliferation experiment, and ns indicates p>0.05;

[0019] Figure 1 In B, it is a photograph taken by an inverted microscope of the cultured Eca109 cells and Eca109-CR cells at 40-fold magnification in the CCK8 proliferation experiment;

[0020] Figure 1 In C, it is a result diagram of detecting the drug resistance of Eca109 cells and Eca109-CR cells to cisplatin by the CCK8 cell viability assay;

[0021] Figure 1 In D, it is a result diagram of the comet assay of the Eca109-CR-cisplatin group, the Eca109-CR-control group, the Eca109-cisplatin group and the Eca109-control group. The comet assay photographs taken by a fluorescence microscope are analyzed by CASP (Comet Assay Image Analysis) software, *** indicates p<0.001, and **** indicates p<0.0001.

[0022] Figure 2 It is a graph showing the results of apoptosis analysis by flow cytometry after Eca109 cells and Eca109-CR cells were treated with or without cisplatin respectively.

[0023] Figure 3 It is a graph showing the results of detection by Western blotting in Examples 2 and 3 of the present invention, with GAPDH as the internal reference protein, where:

[0024] Figure 3 In it, A is a graph showing the results of detection by Western blotting in Example 2;

[0025] Figure 3 In it, B is a typical picture of detecting SOX12 protein in Eca109 cells by Western blotting after Eca109 cells in Example 3 were treated with different concentrations of cisplatin for 24 hours;

[0026] Figure 3 In it, C is a typical picture of detecting SOX12 protein in Eca109 cells by Western blotting after Eca109 cells in Example 3 were treated in 3 μM cisplatin for different times.

[0027] Figure 4 It is a graph showing the results of RT-qPCR (real-time quantitative polymerase chain reaction) detection in Example 4 of the present invention.

[0028] Figure 5 It is a graph showing the results of detecting the ubiquitination of SOX12 in Eca109 cells by immunoblotting in Example 5 of the present invention.

[0029] Figure 6 It is a graph showing the results of detecting cell viability by CCK8 assay in Example 6 of the present invention, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0030] Figure 7 It is a graph showing the results of detecting the viability of Eca109 cells by CCK8 assay in Example 7 of the present invention, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0031] Figure 8 It is a graph showing the results of the comet assay in Example 8 of the present invention. The comet assay photos taken by a fluorescence microscope were analyzed by CASP (comet assay image analysis) software, and **** indicates p < 0.0001.

[0032] Figure 9 It is a graph showing the results of the effect of increased SOX12 protein level on DNA repair efficiency in Example 9 of the present invention; where:

[0033] Figure 9 In A, it is the result graph of detecting the expression of GFP (green fluorescent protein) and m-Cherry (red fluorescent protein) in cells by flow cytometry;

[0034] Figure 9 In B, it is the result graph of statistical analysis of DNA DSBs repair efficiency. *** indicates p < 0.001, and **** indicates p < 0.0001.

[0035] Figure 10 It is the result graph of the influence of SOX12 protein in Example 10 of the present invention on NER repair efficiency;

[0036] Figure 10 In A, it is the result graph of detecting the expression of GFP (green fluorescent protein) and RFP (red fluorescent protein) in cells by flow cytometry;

[0037] Figure 10 In B, it is the result graph of statistical analysis of DNA NER repair efficiency. ns indicates p > 0.05.

[0038] Figure 11 It is the result graph of detecting the expression of proteins related to DNA damage response by Western blotting in Example 11 of the present invention, with GAPDH as the internal reference protein.

[0039] Figure 12 It is the result graph of detecting the expression by immunofluorescence in Example 12 of the present invention. Images were taken using a Leica laser confocal microscope, including DAPI (4',6-diamidino-2-phenylindole), γ-H2AX protein, and the merged result of the two.

[0040] Figure 13 It is the result graph of RT-qPCR detection in Example 13 of the present invention.

[0041] Figure 14 It is the result graph of detecting by Western blotting in Example 14 of the present invention, with GAPDH as the internal reference protein.

[0042] Figure 15 It is the result graph of detecting by Western blotting in Example 15 of the present invention, with GAPDH as the internal reference protein.

[0043] Figure 16 It is the result graph of detecting by Western blotting in Example 16 of the present invention, with GAPDH as the internal reference protein.

[0044] Figure 17 It is the result graph of detecting by Western blotting in Example 17 of the present invention, with GAPDH as the internal reference protein.

[0045] Figure 18 It is the RT-qPCR detection result graph of Example 18 of the present invention.

[0046] Figure 19 It is the Western blot detection result graph of Example 19 of the present invention, with β-actin as the internal reference protein.

[0047] Figure 20 It is the Western blot detection result graph of Example 20 of the present invention, with β-actin as the internal reference protein.

[0048] Figure 21 It is the Western blot detection result graph of Example 21 of the present invention, with β-actin as the internal reference protein.

[0049] Figure 22 It is the Western blot detection result graph of Example 22 of the present invention, with β-actin as the internal reference protein. Detailed implementation manners

[0050] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and do not limit the present invention.

[0052] In some embodiments, the present invention provides the use of a SOX12 inhibitor in the preparation of a drug for reducing the tolerance of esophageal cancer cells to cisplatin.

[0053] Specifically, the SOX12 inhibitor includes at least one of RNA interference molecules or molecular compounds that inhibit the expression of SOX12.

[0054] Specifically, the RNA interference molecule includes at least one of shRNA, siRNA, dsRNA or miRNA.

[0055] Specifically, the shRNA sequence is as shown in SEQ ID NO.1-2.

[0056] Specifically, the esophageal cancer cells are esophageal squamous cell carcinoma cells.

[0057] Specifically, the esophageal squamous cell carcinoma cells are Eca109 cells.

[0058] In some embodiments, the present invention provides a method for reducing the tolerance of esophageal cancer cells to cisplatin by inhibiting the expression of SOX12 in esophageal cancer cells, thereby reducing the tolerance of esophageal cancer cells to cisplatin.

[0059] In some embodiments, the present invention provides a pharmaceutical composition for the combined treatment of esophageal cancer, which comprises a SOX12 inhibitor and cisplatin.

[0060] Experimental materials:

[0061] The human esophageal epithelial cell line HEEC cells were purchased from Geneo Biotechnology Co., Ltd.; HEK293T cells were purchased from the American Type Culture Collection (ATCC); esophageal squamous cell carcinoma cell lines (Eca109, KYSE150, KYSE410) were purchased from Fenghui Biology. Among them, the human esophageal epithelial cell line HEEC cells and HEK293T cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 1% penicillin / streptomycin and 10% fetal bovine serum; esophageal squamous cell carcinoma cell lines (Eca109, KYSE150, KYSE410) were cultured in RPMI-1640 medium containing 1% penicillin / streptomycin and 10% fetal bovine serum; all cells were cultured in a constant temperature incubator at 37 °C, 95% humidity and 5% CO2.

[0062] The shRNA targeting SOX12 was designed and purchased by Suzhou GenePharma Co., Ltd. The SOX12 shRNA sequences used are as follows:

[0063] SOX12 shRNA1: GGCUGCGGCUCAAGCACAUTT (SEQ ID NO.1);

[0064] SOX12 shRNA2: GCCCGUCUAGCAUCGCAGATT (SEQ ID NO.2).

[0065] Example 1:

[0066] (1) Establish Eca109-CR (a cisplatin-resistant cell line derived from Eca109 cells) from Eca109 cells, and perform CCK8 (Cell Counting Kit-8) proliferation experiments on Eca109 cells and Eca109-CR cells respectively.

[0067] The results of the CCK8 proliferation experiment are shown in Figure 1 A and B as follows. It can be seen that the doubling time of Eca109-CR cells is 29.75 hours, slightly longer than the doubling time of Eca109 cells: 27.65 hours (as shown in Figure 1as shown in A of [reference]; and there were no significant morphological differences between Eca109 cells and Eca109-CR cells except that Eca109-CR cells were slightly smaller (as shown in B of [reference]). Figure 1 as shown in [reference].

[0068] (2) The drug resistance of Eca109 cells and Eca109-CR cells to cisplatin was detected by the CCK8 cell viability assay.

[0069] The results of the CCK8 cell viability assay were as shown in C of [reference]. It could be seen that the drug resistance of Eca109-CR cells to cisplatin was significantly higher than that of Eca109 cells. The IC50 (half maximal inhibitory concentration) of Eca109 cells was 1.478 μM, and the IC50 of Eca109-CR cells was 16.74 μM. Figure 1 as shown in C of [reference]. It could be seen that the drug resistance of Eca109-CR cells to cisplatin was significantly higher than that of Eca109 cells. The IC50 (half maximal inhibitory concentration) of Eca109 cells was 1.478 μM, and the IC50 of Eca109-CR cells was 16.74 μM.

[0070] (3) Eca109-CR cells and Eca109 cells were set as follows: Eca109-CR-cisplatin group: Eca109-CR cells were treated with 6 μM cisplatin for 12 hours; Eca109-CR-control group: Eca109 cells were not treated with cisplatin; Eca109-cisplatin group: Eca109 cells were treated with 6 μM cisplatin for 12 hours; Eca109-control group: Eca109 cells were not treated with cisplatin. Then, the comet assay was performed on each group.

[0071] The results of the comet assay were as shown in D of [reference]. It could be seen that after treatment with cisplatin for 12 hours, the degree of DNA damage in Eca109-CR cells was lower than that in Eca109 cells. Figure 1 as shown in D of [reference]. It could be seen that after treatment with cisplatin for 12 hours, the degree of DNA damage in Eca109-CR cells was lower than that in Eca109 cells.

[0072] (4) After Eca109 cells and Eca109-CR cells were treated with or without 3 μM cisplatin for 24 hours respectively, cell apoptosis analysis was performed by flow cytometry.

[0073] The results of cell apoptosis analysis were as shown in Figure 2 [reference]. It could be seen that after treatment with cisplatin for 24 hours, 21% of Eca109 cells had early apoptosis, 62.9% had late apoptosis and cell death; only 8.74% of Eca109-CR cells had early apoptosis, and 4.77% had late apoptosis and cell death.

[0074] In summary, Eca109-CR cells have developed drug resistance to cisplatin.

[0075] Example 2: The expression of SOX12 protein in Eca109 cells and Eca109-CR cells was detected by Western blotting.

[0076] The results of Western blotting were as shown inFigure 3 As shown by A in [reference], it can be seen that the expression level of SOX12 protein in Eca109-CR cells is significantly higher than that in Eca109 cells.

[0077] Example 3:

[0078] After treating Eca109 cells with different concentrations (0 μM, 1.5 μM, 3 μM, 6 μM, 12 μM, and 24 μM) of cisplatin for 24 hours, the effect of cisplatin concentration on the expression of SOX12 protein in Eca109 cells was analyzed by Western blotting.

[0079] The results of Western blotting detection are as Figure 3 shown by B in [reference]. It can be seen that within a certain concentration range, cisplatin treatment significantly increases the expression of SOX12 protein in Eca109 cells.

[0080] (2) Treat Eca109 cells with 3 μM cisplatin for different times (0 hours, 8 hours, 12 hours, 24 hours, 36 hours, and 48 hours), and analyze the effect of cisplatin treatment time on the expression of SOX12 protein in Eca109 cells by Western blotting.

[0081] The results of Western blotting detection are as Figure 3 shown by C in [reference]. It can be seen that after cisplatin treatment, the expression level of SOX12 protein in Eca109 cells is time-dependent, and after 24 hours of cisplatin treatment, the expression level of SOX12 protein in Eca109 cells reaches the peak.

[0082] Example 4: After treating Eca109 cells with different concentrations (0 μM, 3 μM, and 10 μM) of cisplatin for 24 hours, the mRNA expression of SOX12 in Eca109 cells was detected by RT-qPCR (real-time quantitative polymerase chain reaction).

[0083] The results of RT-qPCR detection are as Figure 4 shown. It can be seen that cisplatin treatment did not significantly upregulate the mRNA expression of SOX12 in Eca109 cells.

[0084] Example 5: After co-transfecting Eca109 cells with Flag-SOX12 and HA-ub plasmids, after 24 hours, the cells were treated with or without 3 μM cisplatin for 24 hours, cell lysates were prepared, and immunoprecipitation was performed using anti-IgG and anti-Flag antibodies respectively. Finally, the ubiquitination of SOX12 was detected by immunoblotting.

[0085] The results of immunoblotting detection are as Figure 5As shown, it can be seen that cisplatin treatment significantly reduces the ubiquitination of SOX12 protein in Eca109 cells.

[0086] According to the detection results of Examples 2 - 5, the increased protein expression of SOX12 in cisplatin-induced Eca109 cells may be achieved through the ubiquitin-proteasome system.

[0087] Example 6: Eca109 cells were set as follows: control group; SOX12-shRNA-1 group: SOX12 was knocked down using SOX12 shRNA1; SOX12-shRNA-2 group: SOX12 was knocked down using SOX12 shRNA2; after treatment with different concentrations (0 μM, 3 μM, 6 μM, and 12 μM) of cisplatin for 24 hours, the viability of Eca109 cells was detected by the CCK8 assay.

[0088] The detection results by the CCK8 assay are as Figure 6 shown. It can be seen that compared with the control group, the knockdown of SOX12 in Eca109 cells significantly reduces the tolerance of Eca109 cells to cisplatin.

[0089] Example 7: Eca109 cells were set as follows: control group; overexpression group of SOX12; after treatment with different concentrations (0 μM, 3 μM, 6 μM, and 12 μM) of cisplatin for 24 hours, the viability of Eca109 cells was detected by the CCK8 assay.

[0090] The detection results by the CCK8 assay are as Figure 7 shown. It can be seen that compared with the control group, the overexpression of SOX12 in Eca109 cells enhances the tolerance of Eca109 cells to cisplatin.

[0091] Example 8: Eca109 cells were set as follows: Eca109-SOX12-cisplatin group: after transfection with the SOX12 plasmid, treated with 12 μM cisplatin for 4 hours; Eca109-SOX12-control group: after transfection with the SOX12 plasmid, not treated with cisplatin; Eca109-empty vector-cisplatin group: after transfection with the empty vector, treated with 12 μM cisplatin for 4 hours; Eca109-empty vector-control group: after transfection with the SOX12 plasmid, not treated with cisplatin; the comet assay was performed separately.

[0092] The detection results of the comet assay are as Figure 8 shown. It can be seen that compared with the control group, Eca109 cells overexpressing SOX12 showed significantly less cisplatin-induced DNA damage in the comet assay.

[0093] According to the detection results of Examples 6-8, inhibiting SOX12 expression in Eca109 cells can reduce the tolerance of esophageal cancer cells to cisplatin, and overexpression of SOX12 in Eca109 cells can reduce cisplatin-induced DNA damage.

[0094] Example 9: One of the main mechanisms by which cancer cells develop resistance to cisplatin is the enhanced activation of DNA repair pathways. Therefore, the effect of elevated SOX12 protein levels on DNA repair efficiency was detected using an HR / NHEJ (homologous recombination / non-homologous end joining) repair efficiency detection system: HEK293T cells were co-transfected with an HR / NHEJ repair detection plasmid and an HA-SOX12 plasmid or an HA-SOX12(1-280) plasmid, and a control was set. After 72 hours, the expression of GFP (green fluorescent protein) and m-Cherry (red fluorescent protein) in the cells was detected by flow cytometry.

[0095] The results of the effect of elevated SOX12 protein levels on DNA repair efficiency are shown in Figure 9 A and B as follows. It can be seen that elevated SOX12 protein levels significantly improve the efficiency of both HR and NHEJ DNA repair mechanisms; exogenous expression of the SOX12(1-280) protein fragment lacking the transcriptional activation domain (TAD) does not affect HR / NHEJ repair efficiency. This result indicates that the SOX12 protein may affect HR / NHEJ repair efficiency through its transcriptional activity.

[0096] Example 10: Cisplatin-induced DNA damage is mainly repaired through the NER (nucleotide excision repair) pathway, and the NER pathway is closely related to tumor resistance to cisplatin. Therefore, the effect of SOX12 protein on NER repair efficiency was detected: HEK293T cells were co-transfected with an NER repair detection plasmid and an HA-SOX12 or an HA-SOX12(1-280) plasmid, and a control was set. After 72 hours, the expression of GFP (green fluorescent protein) and RFP (red fluorescent protein) in the cells was detected by flow cytometry.

[0097] The results of the effect of SOX12 protein on NER repair efficiency are shown in Figure 10 A and B as follows. It can be seen that overexpression of SOX12 protein or SOX12 truncated protein has no significant effect on NER repair efficiency.

[0098] Example 11: SOX12 was knocked down using SOX12 shRNA1 and SOX12 shRNA2 in Eca109 cells. After setting up the control, the cells were treated with or without 3 μM cisplatin for 24 hours, and then the expression levels of DNA damage response-related proteins (CHK1 / p-CHK1, CHK2 / p-CHK2, and H2AX / γH2AX) were detected by Western blotting.

[0099] The results of Western blotting detection are as Figure 11 shown. It can be seen that whether treated with cisplatin or not, the knockdown of SOX12 led to a significant decrease in the expression levels of H2AX and γ-H2AX proteins, as well as CHK1 / p-CHK1 and CHK2 / p-CHK2 proteins.

[0100] Example 12: Eca109 cells were transfected with HA-SOX12 plasmid for overexpression of SOX12. After setting up the control and treating with 3 μM cisplatin for 24 hours, the expression level of γ-H2AX protein was detected by immunofluorescence.

[0101] The results of immunofluorescence detection are as Figure 12 shown. It can be seen that the overexpression of SOX12 significantly enhanced the expression of γ-H2AX protein.

[0102] Example 13: SOX12 was knocked down using SOX12 shRNA1 and SOX12 shRNA2 in Eca109 cells. After setting up the control, the mRNA expression levels of SOX12, RNF168, RNF8, CHK2, CHK1, H2AX, BRCA1, and PARP1 in Eca109 cells were detected by RT-qPCR.

[0103] The results of RT-qPCR detection are as Figure 13 shown. It can be seen that the changes in the mRNA expression levels of H2AX and CHK1 were inconsistent with the changes in protein expression when SOX12 expression was absent, and only the mRNA expression level of CHK2 was significantly decreased.

[0104] Example 14: MG132 rescue experiment: SOX12 was knocked down using SOX12 shRNA1 and SOX12 shRNA2 in Eca109 cells. After setting up the control, the cells were treated with or without 20 μM MG132 (proteasome inhibitor), and then the expression levels of CHK2, H2AX, and SOX12 in Eca109 cells were detected by Western blotting.

[0105] The results of Western blotting detection are as Figure 14As shown, it can be seen that MG132 significantly restores the expression levels of CHK2 and H2AX proteins in Eca109 cells.

[0106] According to the detection results of Examples 9 - 14, inhibiting the expression of SOX12 in Eca109 cells significantly inhibits the activation of the DNA damage repair signaling pathway, and SOX12 may be involved in DNA damage repair by affecting certain ubiquitin-related proteins.

[0107] Example 15: Ubiquitination modification plays a key role in DNA damage repair. RNF168 is an important E3 ubiquitin ligase involved in DNA damage repair. Therefore, SOX12 was knocked down in Eca109 cells using SOX12 shRNA1 and SOX12 shRNA2, and a control was set. After treatment with 3 μM cisplatin and without cisplatin for 24 hours respectively, the protein levels of SOX12 and RNF168 in Eca109 cells were detected by Western blotting.

[0108] The detection results by Western blotting are as Figure 15 shown. It can be seen that after knocking down SOX12 in Eca109 cells, the expression of RNF168 protein is significantly reduced.

[0109] Example 16: Eca109 cells and KYSE150 cells were transfected with HA - SOX12 plasmid for overexpression of SOX12, and a control was set. Then, the protein levels of RNF168 in Eca109 cells and KYSE150 cells were detected by Western blotting.

[0110] The detection results by Western blotting are as Figure 16 shown. It can be seen that after overexpressing SOX12 in Eca109 cells and KYSE150 cells, the expression of RNF168 protein increases, and according to Example 13, knocking down SOX12 does not reduce the mRNA expression of RNF168 (as Figure 13 shown).

[0111] Example 17: MG132 rescue experiment: SOX12 was knocked down in Eca109 cells using SOX12 shRNA1 and SOX12 shRNA2, and a control was set. After treatment with or without 20 μM MG132 for 6 hours respectively, the protein levels of RNF168 and SOX12 were detected by Western blotting.

[0112] The detection results by Western blotting are as Figure 17 shown. It can be seen that after treating Eca109 cells with MG132, the reduction of RNF168 protein caused by SOX12 deficiency can be significantly restored.

[0113] According to the detection results of Examples 15 - 17, SOX12 protein regulates the expression of RNF168 protein through the ubiquitin - proteasome system.

[0114] Example 18: SOX12 was knocked down in Eca109 cells using SOX12 shRNA1 and SOX12 shRNA2, and after setting a control, the mRNA levels of SOX12, TRIP12, HERC2, UBR5, and CUL4A in Eca109 cells were detected by RT - qPCR.

[0115] The results of RT - qPCR detection are as Figure 18 shown. It can be seen that the knockdown of SOX12 in Eca109 cells significantly increased the mRNA expression of TRIP12 and UBR5, but had no effect on the mRNA expression of HERC2 and CUL4A.

[0116] Example 19: HEEC cells and KYSE410 cells were transfected with HA - SOX12 plasmid respectively for overexpression of SOX12, and after setting a control, the protein levels of TRIP12 and UBR5 in HEEC cells and KYSE410 cells were detected by Western blotting.

[0117] The results of Western blotting detection are as Figure 19 shown. It can be seen that the overexpression of SOX12 in HEEC cells and KYSE410 cells decreased the protein expression of TRIP12 and UBR5.

[0118] Example 20: Eca109 cells were transfected with HA - SOX12 respectively for overexpression of SOX12; transfected with HA - SOX12(1 - 280) plasmid for expression of SOX12(1 - 280), and after setting a control, the protein levels of TRIP12 and UBR5 in Eca109 cells were detected by Western blotting.

[0119] The results of Western blotting detection are as Figure 20 shown. It can be seen that the overexpression of SOX12 in Eca109 cells decreased the protein expression of TRIP12 and UBR5, while the expression of SOX12(1 - 280) had no significant effect on the protein expression of TRIP12 and UBR5.

[0120] Example 21: SOX12 was knocked down in Eca109 cells using SOX12 shRNA1 and SOX12 shRNA2, and after setting a control, the protein levels of TRIP12 and UBR5 in Eca109 cells were detected by Western blotting.

[0121] The results of Western blotting detection are asFigure 21 As shown, it can be seen that the knockdown of SOX12 in Eca109 cells significantly increases the protein expression of TRIP12 and UBR5.

[0122] Example 22: After Eca109 cells were treated with different concentrations (0 μM, 1 μM, 3 μM, 6 μM, 9 μM, and 12 μM) of cisplatin for 12 hours, the protein levels of SOX12, TRIP12, and UBR5 in Eca109 cells were detected by Western blotting.

[0123] The results of Western blotting detection are as Figure 22 shown. It can be seen that cisplatin inhibits the protein expression of UBR5 and TRIP12 in a dose-dependent manner.

[0124] According to the detection results of Examples 18 - 22, SOX12 in Eca109 cells plays a negative regulatory role in the expression of TRIP12 and UBR5 through a transcriptional mechanism.

[0125] From the result analysis of Examples 1 - 22, the following conclusions are obtained in the present invention: The application of SOX12 inhibitors in the preparation of drugs for reducing the tolerance of esophageal cancer cells to cisplatin can reduce the tolerance of esophageal cancer cells to cisplatin by inhibiting the expression of SOX12 in esophageal cancer cells. SOX12 can be used as a therapeutic target for overcoming chemotherapy resistance in esophageal cancer. In particular, SOX12 inhibitors can be combined with cisplatin for the treatment of esophageal cancer.

[0126] It has been found in prior art research that DNA damage repair is an important reason for the tolerance of esophageal cancer cells to cisplatin, and RNF168 plays a key role in the DNA damage repair pathway by ubiquitinating histone H2A. The present invention confirms that inhibiting the expression of SOX12 in esophageal cancer cells can promote the transcription of UBR5 and TRIP12, inhibit the stability of RNF168 protein, thereby inhibiting the activation of the DNA damage repair pathway, and achieving the purpose of reducing the tolerance of esophageal cancer cells to cisplatin.

[0127] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of an SOX12 inhibitor in the preparation of a drug for reducing the tolerance of esophageal cancer cells to cisplatin, characterized in that: The SOX12 inhibitor inhibits the activation of the DNA damage repair pathway and reduces the cisplatin tolerance of esophageal cancer cells by promoting the transcription of UBR5 and TRIP12 in esophageal cancer cells, thereby inhibiting the stability of the RNF168 protein. The SOX12 inhibitor is an shRNA that inhibits SOX12 expression, and the shRNA sequences are as shown in SEQ ID NO.1-2.

2. The application according to claim 1, characterized in that: The esophageal cancer cells are esophageal squamous cell carcinoma cells.

3. The application according to claim 2, characterized in that: The esophageal squamous cell carcinoma cells are Eca109 cells.

4. A pharmaceutical composition for the combined treatment of esophageal cancer, characterized in that: The pharmaceutical composition comprises a SOX12 inhibitor and cisplatin. The SOX12 inhibitor is an shRNA that inhibits SOX12 expression, and the shRNA sequences are as shown in SEQ ID NO.1-2.

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