Application of SOX12 inhibitor in medicine for reducing tolerance of cis-platinum to esophageal cancer cells

Abstract

The invention relates to the technical field of biological medicines, and provides application of an SOX12 inhibitor in a medicine for reducing tolerance of cis-platinum to esophageal cancer cells. The SOX12 inhibitor is applied to preparation of the medicine for reducing the tolerance of the cis-platinum to the esophageal cancer cells, the tolerance of the esophageal cancer cells to the cis-platinum is remarkably reduced by inhibiting the expression of the SOX12 in the esophageal cancer cells, and particularly, the SOX12 inhibitor can be combined with the cis-platinum to treat the esophageal cancer.

Description

Technical Field

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

[0002] Esophageal cancer (ESCA) is divided into esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Currently, the main treatments for esophageal cancer include surgical intervention, chemotherapy, and radiotherapy. Chemotherapy is an effective treatment for esophageal cancer that 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 effect of chemotherapy is significantly hindered by the emergence of drug resistance, and overcoming cisplatin resistance has become an urgent issue 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 use of a SOX12 inhibitor in a drug for reducing the tolerance of cisplatin to esophageal cancer cells, which aims to solve the problems mentioned in the background technology.

[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, playing an important role in embryonic development and cell fate determination.

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

[0006] Furthermore, the SOX12 inhibitor includes at least one RNA interference molecule or molecular compound that inhibits the expression of SOX12.

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

[0008] Furthermore, the shRNA sequence is shown in SEQ ID NO.1-2.

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

[0010] Furthermore, 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 cisplatin to esophageal cancer cells, by inhibiting the expression of SOX12 in esophageal cancer cells, thereby reducing the tolerance of cisplatin to esophageal cancer cells.

[0012] In a third aspect, the present invention provides a pharmaceutical composition for combined treatment of esophageal cancer, the pharmaceutical composition comprising a SOX12 inhibitor and cisplatin.

[0013] The present invention has the following technical effects: (1) SOX12 inhibitors are used to prepare drugs that reduce the tolerance of esophageal cancer cells to cisplatin, and 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 to overcome the chemotherapy resistance of esophageal cancer, especially SOX12 inhibitors can be combined with cisplatin to treat esophageal cancer.

[0014] (2) Inhibiting SOX12 expression in esophageal cancer cells can promote the transcription of UBR5 and TRIP12 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

[0015] A more complete understanding of exemplary embodiments of the present invention may be obtained by referring to the following drawings: Figure 1 This 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: Figure 1 A in the figure is the result of the doubling time of Eca109 cells and Eca109-CR cells measured by CCK8 (cell counting kit 8) in the CCK8 proliferation experiment, ns means p>0.05; Figure 1 B is a photograph of cultured Eca109 cells and Eca109-CR cells taken under an inverted microscope at 40 times magnification in the CCK8 proliferation experiment; Figure 1 C in the figure is the result of detecting the resistance of Eca109 cells and Eca109-CR cells to cisplatin by CCK8 cell activity assay; Figure 1 D in the figure is the comet assay results of the Eca109-CR-cisplatin group, Eca109-CR-control group, Eca109-cisplatin group and Eca109-control group. The comet assay photos taken by fluorescence microscopy were analyzed by CASP (Comet Assay Image Analysis) software. *** indicates p<0.001, **** indicates p<0.0001.

[0016] Figure 2Figure 2 is the result of flow cytometry analysis of apoptosis in Eca109 cells and Eca109-CR cells after treatment with or without cisplatin.

[0017] Figure 3 The Western blotting results of Example 2 and Example 3 of the present invention are shown in FIG. 1 , with GAPDH as the internal reference protein, wherein: Figure 3 A in the figure is the Western blotting detection result diagram of Example 2; Figure 3 B is a typical picture of SOX12 protein in Eca109 cells detected by Western blotting after the Eca109 cells in Example 3 were treated with different concentrations of cisplatin for 24 hours; Figure 3 C in the figure is a typical picture of the Eca109 cells in Example 3 treated with 3 μM cisplatin for different time periods, and the SOX12 protein in the Eca109 cells was detected by Western blotting.

[0018] Figure 4 This is a graph showing the RT-qPCR (real-time quantitative polymerase chain reaction) detection results of Example 4 of the present invention.

[0019] Figure 5 This is a graph showing the results of detecting the ubiquitination of SOX12 in Eca109 cells by immunoblotting in Example 5 of the present invention.

[0020] Figure 6 This is a graph showing the results of detecting cell viability using the CCK8 detection method according to Example 6 of the present invention, *** indicates p<0.001, and **** indicates p<0.0001.

[0021] Figure 7 This is a graph showing the results of detecting the viability of Eca109 cells using the CCK8 detection method of Example 7 of the present invention, *** indicates p<0.001, and **** indicates p<0.0001.

[0022] Figure 8 This is a diagram of the comet assay result of Example 8 of the present invention. The comet assay photographs taken by a fluorescence microscope were analyzed by CASP (Comet Assay Image Analysis) software. **** indicates p<0.0001.

[0023] Fig. 9 : is a result diagram of the effect of increased SOX12 protein level on DNA repair efficiency in Example 9 of the present invention; wherein: Fig. 9 A in the figure is the result of detecting the expression of GFP (green fluorescent protein) and m-Cherry (red fluorescent protein) in cells by flow cytometry; Fig. 9 B in the figure is the statistical analysis result of DNA DSBs repair efficiency, *** indicates p<0.001, **** indicates p<0.0001.

[0024] Fig.10 This is a result diagram showing the effect of the SOX12 protein on the NER repair efficiency in Example 10 of the present invention; Fig.10 A in the figure is the result of detecting the expression of GFP (green fluorescent protein) and RFP (red fluorescent protein) in cells by flow cytometry; Fig.10 B in the figure is the result of statistical analysis of DNA NER repair efficiency, ns means p>0.05.

[0025] Fig.11 This is a graph showing the results of Western blotting to detect the expression of proteins related to DNA damage response in Example 11 of the present invention, with GAPDH as the internal reference protein.

[0026] Fig.12 This is a graph showing the result of immunofluorescence detection of expression in Example 12 of the present invention, and the image is taken using a Leica laser confocal microscope, including DAPI (4',6-diamidino-2-phenylindole), γ-H2AX protein, and a result graph combining the two.

[0027] Fig.13 This is a graph showing the RT-qPCR detection results of Example 13 of the present invention.

[0028] Fig.14 This is a graph showing the results of Western blotting in Example 14 of the present invention, with GAPDH as the internal reference protein.

[0029] Fig.15 This is a graph showing the results of Western blotting in Example 15 of the present invention, with GAPDH as the internal reference protein.

[0030] Fig.16 This is a graph showing the results of Western blotting in Example 16 of the present invention, with GAPDH as the internal reference protein.

[0031] Fig.17 This is a graph showing the results of Western blotting in Example 17 of the present invention, with GAPDH as the internal reference protein.

[0032] Fig.18 This is a graph of the RT-qPCR detection results of Example 18 of the present invention.

[0033] Fig.19 This is a graph showing the results of Western blotting in Example 19 of the present invention, with β-actin as the internal reference protein.

[0034] Fig. 20 This is a graph showing the results of Western blotting in Example 20 of the present invention, with β-actin as the internal reference protein.

[0035] Fig.21 This is a graph showing the results of Western blotting in Example 21 of the present invention, with β-actin as the internal reference protein.

[0036] Fig. 22 This is a graph of the Western blotting detection results of Example 22 of the present invention, with β-actin as the internal reference protein. DETAILED DESCRIPTION

[0037] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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 intended to limit the present invention.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments rather than limiting the present invention.

[0039] In some embodiments, the present invention provides use of a SOX12 inhibitor in the preparation of a medicament for reducing the tolerance of cisplatin to esophageal cancer cells.

[0040] Specifically, the SOX12 inhibitor includes at least one RNA interference molecule or molecular compound that inhibits the expression of SOX12.

[0041] Specifically, the RNA interference molecule includes at least one of shRNA, siRNA, dsRNA or miRNA.

[0042] Specifically, the shRNA sequence is shown as SEQ ID NO.1-2.

[0043] Specifically, the esophageal cancer cells are esophageal squamous cell carcinoma cells.

[0044] Specifically, the esophageal squamous cell carcinoma cells are Eca109 cells.

[0045] In some embodiments, the present invention provides a method for reducing the tolerance of cisplatin to esophageal cancer cells by inhibiting the expression of SOX12 in esophageal cancer cells to reduce the tolerance of cisplatin to esophageal cancer cells.

[0046] In some embodiments, the present invention provides a pharmaceutical composition for the combined treatment of esophageal cancer, the pharmaceutical composition comprising a SOX12 inhibitor and cisplatin.

[0047] Experimental Materials: Human esophageal epithelial cell line HEEC cells were purchased from Genentech; HEK293T cells were purchased from the American Type Culture Collection (ATCC); esophageal squamous cell carcinoma cell lines (Eca109, KYSE150, KYSE410) were purchased from Fenghui Biotechnology. 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.

[0048] The shRNA targeting SOX12 was designed and purchased by Suzhou Jima Biotechnology. The SOX12 shRNA sequence used is as follows: SOX12 shRNA1: GGCUGCGGCUCAAGCACAUTT (SEQ ID NO.1); SOX12 shRNA2: GCCCGUCUAGCAUCGCAGATT (SEQ ID NO. 2).

[0049] Embodiment 1: (1) Eca109-CR (a cisplatin-resistant cell line derived from Eca109 cells) was established from Eca109 cells, and CCK8 (cell counting kit 8) proliferation assay was performed on Eca109 cells and Eca109-CR cells, respectively.

[0050] The results of CCK8 proliferation assay are as follows Figure 1 As shown in A and B in Figure 1, it can be seen that the doubling time of Eca109-CR cells is 29.75 hours, which is slightly longer than the doubling time of Eca109 cells: 27.65 hours (Figure 1 Figure 1 There is no significant difference in morphology between Eca109 cells and Eca109-CR cells except that Eca109-CR cells are slightly smaller (as shown in A in FIG). Figure 1 (as shown in B in the figure).

[0051] (2) The resistance of Eca109 cells and Eca109-CR cells to cisplatin was detected by CCK8 cell activity assay.

[0052] The results of CCK8 cell activity assay were as follows Figure 1As shown in C, it can be seen that the resistance of Eca109-CR cells to cisplatin is significantly higher than that of Eca109 cells. The IC50 (half inhibitory concentration) of Eca109 cells is 1.478 μM, and the IC50 of Eca109-CR cells is 16.74 μM.

[0053] (3) The 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 h; Eca109-CR-control group: Eca109 cells were not treated with cisplatin; Eca109-cisplatin group: Eca109 cells were treated with 6 μM cisplatin for 12 h; Eca109-control group: Eca109 cells were not treated with cisplatin. After that, each group was subjected to comet assay.

[0054] Comet assay results Figure 1 As shown in D, it can be seen that after 12 hours of cisplatin treatment, the degree of DNA damage in Eca109-CR cells was lower than that in Eca109 cells.

[0055] (4) Eca109 cells and Eca109-CR cells were treated with or without 3 μM cisplatin for 24 hours, and then cell apoptosis was analyzed by flow cytometry.

[0056] The results of cell apoptosis analysis were as follows Figure 2 As shown, it can be seen that after 24 hours of cisplatin treatment, 21% of Eca109 cells showed early apoptosis and 62.9% showed late apoptosis and cell death; only 8.74% of Eca109-CR cells showed early apoptosis and 4.77% showed late apoptosis and cell death.

[0057] In summary, Eca109-CR cells have become resistant to cisplatin.

[0058] Example 2: The expression of SOX12 protein in Eca109 cells and Eca109-CR cells was detected by Western blotting.

[0059] Western blot results Figure 3 As shown in A, it can be seen that the expression level of SOX12 protein in Eca109-CR cells is significantly higher than that in Eca109 cells.

[0060] Embodiment 3: (1) After Eca109 cells were treated with different concentrations of cisplatin (0 μM, 1.5 μM, 3 μM, 6 μM, 12 μM, and 24 μM) for 24 hours, the effect of cisplatin concentration on SOX12 protein expression in Eca109 cells was analyzed by Western blotting.

[0061] Western blot results Figure 3 As shown in B, it can be seen that within a certain concentration range, cisplatin treatment significantly increased the expression of SOX12 protein in Eca109 cells.

[0062] (2) Eca109 cells were treated with 3 μM cisplatin for different time periods (0 h, 8 h, 12 h, 24 h, 36 h, and 48 h), and the effect of cisplatin treatment time on SOX12 protein expression in Eca109 cells was analyzed by Western blotting.

[0063] Western blot results Figure 3 As shown in C, it can be seen that after cisplatin treatment, the expression level of SOX12 protein in Eca109 cells is time-dependent, and the expression level of SOX12 protein in Eca109 cells reaches a peak after 24 hours of cisplatin treatment.

[0064] Example 4: After Eca109 cells were treated 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).

[0065] RT-qPCR test results are as follows Figure 4 As shown, it can be seen that cisplatin treatment did not significantly upregulate the mRNA expression of SOX12 in Eca109 cells.

[0066] Example 5: Eca109 cells were co-transfected with Flag-SOX12 and HA-ub plasmids. After 24 hours, they 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.

[0067] The results of immunoblotting were as follows Figure 5 As shown, it can be seen that cisplatin treatment significantly reduced the ubiquitination of SOX12 protein in Eca109 cells.

[0068] According to the detection results of Example 2 to Example 5, the increased protein expression of SOX12 in Eca109 cells induced by cisplatin may be achieved through the ubiquitin-proteasome system.

[0069] Example 6: Eca109 cells were set as: 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 being treated 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 CCK8 detection method.

[0070] The results of CCK8 assay are as follows Figure 6 As shown, it can be seen that knockdown of SOX12 in Eca109 cells significantly reduced the resistance of Eca109 cells to cisplatin compared with the control group.

[0071] Example 7: Eca109 cells were set as: a control group; a SOX12 overexpression group; and treated with different concentrations (0 μM, 3 μM, 6 μM and 12 μM) of cisplatin for 24 hours, and then the viability of Eca109 cells was detected by CCK8 detection method.

[0072] CCK8 assay results Figure 7 As shown, it can be seen that overexpression of SOX12 in Eca109 cells enhances the resistance of Eca109 cells to cisplatin compared with the control group.

[0073] Example 8: The Eca109 cells were set as follows: Eca109-SOX12-cisplatin group: after transfection with SOX12 plasmid, the cells were treated with 12 μM cisplatin for 4 hours; Eca109-SOX12-control group: after transfection with SOX12 plasmid, the cells were not treated with cisplatin; Eca109-empty vector-cisplatin group: after transfection with empty vector, the cells were treated with 12 μM cisplatin for 4 hours; Eca109-empty vector-control group: after transfection with SOX12 plasmid, the cells were not treated with cisplatin; and comet assays were performed on each group.

[0074] Comet assay results Figure 8 As shown, it can be seen that Eca109 cells overexpressing SOX12 exhibited significantly less cisplatin-induced DNA damage in the comet assay compared with the control group.

[0075] According to the detection results of Example 6 to Example 8, inhibiting the expression of SOX12 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.

[0076] Example 9: One of the main mechanisms by which cancer cells develop resistance to cisplatin is the enhanced activation of the DNA repair pathway. Therefore, the HR / NHEJ (homologous recombination / non-homologous end joining) repair efficiency detection system was used to detect the effect of increased SOX12 protein levels on DNA repair efficiency: HEK293T cells were co-transfected with HR / NHEJ repair detection plasmid and HA-SOX12 plasmid or HA-SOX12 (1-280) plasmid, and a control was set up. After 72 hours, the expression of GFP (green fluorescent protein) and m-Cherry (red fluorescent protein) in the cells was detected by flow cytometry.

[0077] The effect of increased SOX12 protein levels on DNA repair efficiency Fig. 9 As shown in A and B in Figure 1, it can be seen that the increase in SOX12 protein levels significantly improves the efficiency of both HR and NHEJ DNA repair mechanisms; exogenous expression of a SOX12 (1-280) protein fragment lacking the transcriptional activation domain (TAD) does not affect the HR / NHEJ repair efficiency. This result suggests that SOX12 protein may affect HR / NHEJ repair efficiency through transcriptional activity.

[0078] Example 10: Cisplatin-induced DNA damage is mainly repaired through the NER (nucleotide excision repair) pathway, and the NER pathway is closely related to the tumor resistance to cisplatin. Therefore, the effect of SOX12 protein on NER repair efficiency was detected: HEK293T cells were co-transfected with NER repair detection plasmid and HA-SOX12 or 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.

[0079] The results of the effect of SOX12 protein on NER repair efficiency are as follows Fig.10 As shown in A and B, it can be seen that overexpression of SOX12 protein or SOX12 truncated protein has no significant effect on NER repair efficiency.

[0080] Example 11: SOX12 was knocked down in Eca109 cells using SOX12 shRNA1 and SOX12 shRNA2, and after setting up a control, the cells were treated with or without 3 μM cisplatin for 24 hours, and then the expression of DNA damage response-related proteins (CHK1 / p-CHK1, CHK2 / p-CHK2 and H2AX / γH2AX) was detected by Western blotting.

[0081] Western blot results Fig.11As shown, it can be seen that knockdown of SOX12 resulted in a significant decrease in the expression of H2AX and γ-H2AX proteins, as well as a significant decrease in the expression of CHK1 / p-CHK1 and CHK2 / p-CHK2 proteins, regardless of whether cisplatin was treated or not.

[0082] Example 12: Eca109 cells were transfected with HA-SOX12 plasmid to overexpress SOX12, and a control was set. After being treated with 3 μM cisplatin for 24 hours, the expression of γ-H2AX protein was detected by immunofluorescence.

[0083] Immunofluorescence test results Fig.12 As shown, it can be seen that overexpression of SOX12 significantly enhanced the expression of γ-H2AX protein.

[0084] Example 13: SOX12 was knocked down in Eca109 cells using SOX12 shRNA1 and SOX12 shRNA2, and after setting controls, the mRNA expressions of SOX12, RNF168, RNF8, CHK2, CHK1, H2AX, BRCA1 and PARP1 in Eca109 cells were detected by RT-qPCR.

[0085] RT-qPCR test results are as follows Fig.13 As shown, it can be seen that the changes in mRNA expression of H2AX and CHK1 are inconsistent with the changes in protein expression when SOX12 expression is lost, and only the mRNA expression of CHK2 is significantly reduced.

[0086] Example 14: MG132 rescue experiment: SOX12 was knocked down in Eca109 cells using SOX12 shRNA1 and SOX12 shRNA2, and after setting up controls, the cells were treated with or without 20 μM MG132 (proteasome inhibitor), and the expression of CHK2, H2AX and SOX12 in Eca109 cells was detected by Western blotting.

[0087] Western blot results Fig.14 As shown, it can be seen that MG132 significantly restored the expression levels of CHK2 and H2AX proteins in Eca109 cells.

[0088] According to the detection results of Example 9 to Example 14, inhibiting the expression of SOX12 in Eca109 cells significantly inhibits the activation of the DNA damage repair signaling pathway, and SOX12 may participate in DNA damage repair by affecting certain ubiquitin-related proteins.

[0089] 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 up. After treatment with 3 μM cisplatin and without cisplatin for 24 hours, the SOX12 and RNF168 proteins in Eca109 cells were detected by western blotting.

[0090] Western blot results Fig.15 As shown, it can be seen that after SOX12 knockdown in Eca109 cells, the expression of RNF168 protein was significantly reduced.

[0091] Example 16: Eca109 cells and KYSE150 cells were transfected with HA-SOX12 plasmids to overexpress SOX12, and after setting controls, the RNF168 protein in Eca109 cells and KYSE150 cells was detected by Western blotting.

[0092] Western blot results Fig.16 As shown, it can be seen that after overexpression of SOX12 in Eca109 cells and KYSE150 cells, RNF168 protein expression increased, and according to Example 13, knockdown of SOX12 did not reduce RNF168 mRNA expression (such as Fig.13 as shown).

[0093] Example 17: MG132 rescue experiment: SOX12 was knocked down in Eca109 cells using SOX12 shRNA1 and SOX12 shRNA2. After setting up controls, the cells were treated with or without 20 μM MG132 for 6 hours, and the RNF168 and SOX12 proteins were detected by Western blotting.

[0094] Western blot results Fig.17 As shown, it can be seen that MG132 treatment of Eca109 cells can significantly restore the reduction of RNF168 protein caused by SOX12 deficiency.

[0095] According to the detection results of Example 15 to Example 17, SOX12 protein regulates the expression of RNF168 protein through the ubiquitin-proteasome system.

[0096] Example 18: SOX12 was knocked down in Eca109 cells using SOX12 shRNA1 and SOX12 shRNA2, and after setting controls, the mRNA levels of SOX12, TRIP12, HERC2, UBR5 and CUL4A in Eca109 cells were detected by RT-qPCR.

[0097] RT-qPCR test results are as follows Fig.18 As shown, it can be seen that 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.

[0098] Example 19: HEEC cells and KYSE410 cells were transfected with HA-SOX12 plasmids to overexpress SOX12, and after setting controls, the TRIP12 and UBR5 proteins in HEEC cells and KYSE410 cells were detected by Western blotting.

[0099] Western blot results Fig.19 As shown, it can be seen that overexpression of SOX12 in HEEC cells and KYSE410 cells reduced TRIP12 and UBR5 protein expressions.

[0100] Example 20: Eca109 cells were transfected with HA-SOX12 to overexpress SOX12; HA-SOX12 (1-280) plasmid was transfected to express SOX12 (1-280), and after setting controls, the TRIP12 and UBR5 proteins in Eca109 cells were detected by Western blotting.

[0101] Western blot results Fig. 20 As shown, it can be seen that overexpression of SOX12 in Eca109 cells reduced the expression of TRIP12 and UBR5 proteins, while SOX12 (1-280) expression had no significant effect on the expression of TRIP12 and UBR5 proteins.

[0102] Example 21: SOX12 was knocked down in Eca109 cells using SOX12 shRNA1 and SOX12 shRNA2, and after setting controls, the TRIP12 and UBR5 proteins in Eca109 cells were detected by Western blotting.

[0103] Western blot results Fig.21 As shown, it can be seen that knockdown of SOX12 in Eca109 cells significantly increased the expression of TRIP12 and UBR5 proteins.

[0104] 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 expression of SOX12, TRIP12 and UBR5 proteins in Eca109 cells was detected by Western blotting.

[0105] Western blot results Fig. 22 As shown, it can be seen that cisplatin inhibited the expression of UBR5 and TRIP12 proteins in a dose-dependent manner.

[0106] According to the detection results of Example 18 to Example 22, SOX12 in Eca109 cells negatively regulates the expression of TRIP12 and UBR5 through a transcriptional mechanism.

[0107] From the analysis of the results of Examples 1 to 22, the present invention draws the following conclusions: SOX12 inhibitors are used to prepare drugs that reduce the tolerance of esophageal cancer cells to cisplatin, and 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 to overcome the chemotherapy resistance of esophageal cancer, and in particular, SOX12 inhibitors can be combined with cisplatin to treat esophageal cancer.

[0108] Prior art studies have found that DNA damage repair is an important reason for esophageal cancer cells to develop tolerance to cisplatin, and RNF168 plays a key role in the DNA damage repair pathway by ubiquitinating histone H2A. The present invention confirms that inhibiting SOX12 expression in esophageal cancer cells can promote the transcription of UBR5 and TRIP12 to inhibit the stability of RNF168 protein, thereby inhibiting the activation of the DNA damage repair pathway, thereby achieving the purpose of reducing the tolerance of esophageal cancer cells to cisplatin.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of SOX12 inhibitors in the preparation of drugs that reduce the tolerance of cisplatin to esophageal cancer cells.

2. The use according to claim 1, characterized in that: The SOX12 inhibitor includes at least one RNA interference molecule or molecular compound that inhibits the expression of SOX12.

3. The use according to claim 2, characterized in that: The RNA interference molecule includes at least one of shRNA, siRNA, dsRNA or miRNA.

4. The use according to claim 3, characterized in that: The shRNA sequence is shown as SEQ ID NO.1-2.

5. The use according to claim 4, characterized in that: The esophageal cancer cells are esophageal squamous cell cancer cells.

6. The use according to claim 5, characterized in that: The esophageal squamous cell carcinoma cells are Eca109 cells.

7. A method for reducing the tolerance of cisplatin to esophageal cancer cells, characterized in that: By inhibiting the expression of SOX12 in esophageal cancer cells, the resistance of cisplatin to esophageal cancer cells is reduced.

8. A pharmaceutical composition for combined treatment of esophageal cancer, characterized in that: The pharmaceutical composition comprises a SOX12 inhibitor and cisplatin.

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