Studies Of The Correlation Between DNA Mismatch Repair Gene Expression And Cisplatin Sensitivity In Endometrial Carcinoma

Endometrial carcinoma is a common female genital malignant tumor in China,and its incidence rate is about 22.7/100,000,and it is increasing year by year.However,the pathogenesis of endometrial carcinoma is still not clear.It is currently believed that the occurrence and development of this disease is a multi-factorial and multi-gene involved process.Cisplatin is a first-line anticarcinoma drug widely used in clinical practice,and it can achieve good curative effect in the treatment of various types of malignant tumors.Cisplatin can bind to DNA,cause cross-linking,and change the function of DNA structure to destroy DNA.In women with Hereditary Non-polyposis Colorectal Cancer(HNPCC)syndrome,the cumulative lifetime risk of endometrial carcinoma is 40%to 60%,Endometrium carcinoma is often one of the most common extraintestinal tumors in women with Lynch syndrome,also known as Lynch syndrome related endometrial carcinoma(LS-EC).These diseases are genetically susceptible and mainly favor the loss or inactivation of the DNA mismatch repair(MMR)system.Mismatch Repair(MMR)is a hereditary non-polyposis colorectal cancer(Hereditary Non).-polyposis Colorectal Cancer,a group of genetic susceptibility genes isolated from HNPCC.It is not an oncogene or a tumor suppressor gene.It is a novel molecular mechanism of tumor pathogenesis discovered after oncogenes and tumor suppressor genes.With the in-depth study of the hereditary tendency of hereditary nonpolyposis colorectal cancer(HNPCC)syndrome and Lynch syndrome,we found that mismatch repair gene mutations are closely related to Lynch-related endometrial carcinoma,Detection of mismatch repair gene mutations is one of the effective methods for screening patients with Lynch syndrome-associated endometrial carcinoma.Early MMR gene screening is very important for early diagnosis and postoperative adjuvant therapy of Lynch syndrome patients and its close relatives.Cisplatin is a non-specific anti-tumor drug that inhibits cell mitosis.However,in recent years,with the development of tumor cell resistance,the clinical application and efficacy of cisplatin have been greatly restricted.The emergence of drug resistance is multifaceted,and it is currently believed to be mainly related to the enhancement of DNA repair ability,the mutation of P53 gene and the lack of DNA mismatch repair ability.Mismatch Repair(MMR)is a group of genetic susceptibility genes isolated from Hereditary Non-polyposis Colorectal Cancer(HNPCC),which plays a key role in maintaining the integrity of DNA replication.Any mutation in one of these genes can lead to defects in gene mismatch repair,manifested as replication errors or microsatellite instability,leading to tumor resistance.However,the specific mechanism of action of mismatch repair genes in endometrial cancer has not been fully investigated.Therefore,this study used real-time quantitative PCR and Western blot to detect the expression levels of mismatch repair genes in different endometrial cancer cell lines Ishikawa and RL95-2.The chemosensitivity of Ishikawa and RL95-2 endometrial cancer cell lines to cisplatin was measured by CCK8 cell proliferation assays.In addition,in this study,the expression and knockdown of the mismatch repair gene MLH1 was conducted in endometrial cancer cells.We observed the changes in the chemosensitivity of different treated endometrial cancer cells to cisplatin,and further studied the potential mechanism of cisplatin resistance.Meanwhile,the nude mouse model experiment of endometrial cancer was established to further validate our research.This study suggests that MLH1 may be a new target for the treatment of endometrial cancer,providing a theoretical basis for individualized treatment and gene therapy of endometrial carcinoma.Part 1:The correlation between the expression of mismatch repair genes in endometrial cancer cells and cisplatin sensitivityPurpose:1.Real-time quantitative PCR and Western blot were used to detect the expression levels of MLH1,PMS2,and MSH6 in human endometrial carcinoma cell lines Ishikawa and RL95-2.2.Sensitivity of Ishikawa and RL95-2 cells to cisplatin was detected by CCK8 cell proliferation assay,flow cytometry analysis.Methods:1.Real-time quantitative PCR was used to detect the gene expression levels of mismatch repair genes MLH1,PMS2 and MSH6 in different endometrial carcinoma cell lines Ishikawa and RL95-2.2.The protein expression levels of mismatch repair genes MLH1,PMS2 and MSH6 in Ishikawa and RL95-2 cell lines were detected by Western blot.3.The cell viability of Ishikawa and RL95-2 cell lines under different concentrations of cisplatin was detected by CCK8 cell proliferation assay.The apoptosis rate of Ishikawa and RL95-2 cell lines under different concentrations of cisplatin was detected by flow cytometry.To investigate the relationship between the sensitivity of Ishikawa and RL95-2 endometrial cancer cell lines to different concentrations of cisplatin drugs and the expression of mismatch repair genes.and investigate the relationship between the sensitivity of Ishikawa and RL95-2 endometrial cancer cell lines to different concentrations of cisplatin and the expression level of mismatch repair genes.4.Statistical analysis was performed using GraphPad Prism version 6.01.Differences between the two or three groups were analyzed using Student’s t test.And P value<0.05 was considered to indicate a statistically significant difference.Results:1.The mismatch repair genes MLH1,PMS2 and MSH6 were normally expressed in endometrial carcinoma RL95-2 cell line,but in Ishikawa cell line,the expression of MLH1,PMS2 and MSH6 was significantly decreased.MLH1 and PMS2 proteins were not expressed in Ishikawa cell line.the differences were statistically significant(P<0.05).2.the CCK8 cell proliferation-toxicity assays was used to detect the IC50 values of Ishikawa and RL95-2 cells against cisplatin,at the same 0,5,10,15,20,25umol cisplatin chemotherapeutic drug concentration gradient,the IC50 of RL95-2 cells were 2.5umol and the IC50 of Ishikawa cells were 82.6umol/ml,respectively.The difference between the two was statistically significant,and RL95-2 cells were more sensitive to cisplatin than Ishikawa.3.Flow cytometry was used to detect the apoptosis rate of Ishikawa and RL95-2 cells cultured for 72h without cisplatin.The apoptosis rate of RL95-2 cells was 15.61%and Ishikawa was 13.32%without cisplatin.The difference was not significant(P>0.05).After 72 hours of 5 umol of cisplatin,the apoptosis rate of RL95-2 cells was 51.18%and the apoptosis rate of Ishikawa cells was 18.79%(P<0.05).the difference was statistically significant.The results showed that RL95-2 cells with high mismatch repair gene were more sensitive to cisplatin and higher apoptosis rate than Ishikawa cells under the same cisplatin concentration..Conclusions:1.Mismatch repair genes MLH1,PMS2,and MSH6 are highly expressed in genes and protein levels in endometrial carcinoma RL95-2 cells,In Ishikawa cells,MLH1,PMS2,and MSH6 exhibited low expression at the gene level,in which MLH1 and PMS2 were deleted at the protein level.2.The endometrial carcinoma cells Ishikawa and RL95-2 showed significant differences in cell viability and apoptotic rate under the same cisplatin concentration gradient.RL95-2 cells with high expression of mismatch repair genes MLH1,PMS2,and MSH6 are more sensitive to cisplatin drugs.This suggests that the expression levels of mismatch repair genes MLH1,PMS2,MSH6 may affect the sensitivity of endometrial carcinoma cells to cisplatin.Part 2:MLH1 enhanced the chemosensitivity of human endometrial carcinoma cells to cisplatin by activating the MLHl/c-Abl signaling pathwayPurpose:1.The sensitivity of the treated Ishikawa and RL95-2 cells to cisplatin was detected by specific overexpression or silencing of gene expression of MLH1 in endometrial carcinoma cells Ishikawa and RL95-2.2.Western blot analysis was used to investigate the specific mechanisms of cisplatin chemotherapy resistance3..CCK8 cell proliferation assay,cell apoptosis,cell cycle detection and animal model experiments were used to further verify the relationship between the expression level of mismatch repair gene MLH1 and cisplatin sensitivity of endometrial carcinoma ce ls.Metheods:1.Different MLH1 siRNA fragments were designed and synthesized according to the gene sequence of MLH1 and transfected into RL95-2 cells.The interference effect of siRNA was verified by RT-PCR and Western blot,and the optimal siRNA fragment was selected to specifically knock down the expression of MLHI in RL95-1 cell s.2.The adenoviral vector,ADV-MLH1,was constructed and transfected into Ishikawa cells to specifically up-regulate MLH1.3.CCK8 cell proliferation assay,flow cytometry and Honest immunofluorescence staining were used to detect the sensitivity of Ishikawa and RL95-2 cells to cisplatin before and after treated.4.Western blot was used to detect the expression of c-Abl,bcl-2,caspase-9,caspase-3 and PARP in MLH1/c-Abl apoptosis signaling pathway in cisplatin-induced endometrial carcinoma cells.5.The nude mouse model experiment of endometrial cancer was established,ADV-MLH1 was used to specifically up-regulate the expression of MLH1 in Ishikawa cells,the relationship between the expression level of MLH1 and the sensitivity of Ishikawa cells to cisplatin was further verified.6.Statistical methods same as the first part.Results:1.In this study,four loci were selected to design MLH1-siRNA,which was successfully transfected into endometrial cancer cell line RL95-2.The interference effects of each group were verified by RT-PCR and Western blot,and the interference of siRNA-1832 was confirmed.The effect is more than 80%.2.The MLH1 adenoviral vector(ADV-MLH1)was successfully constructed and successfully transfected into the Ishikawa cells.The overexpression of MLH1 was verified by RT-PCR and Western blot,confirming that the expression level of MLH1 was confirmed to be up-regulated by 80%-90%.3.When MLH1-siRNA-1832 was transfected into RL95-2 cells,the results of CCK8 cell proliferation assay showed that after specific knockdown of MLH1 expression,the IC50 of the cisplatin drug in the experimental group increased from 2.5 umol/mL to 15 umol/mL compared with the control group,and the cell survival rate was significantly increased.Cytotoxicity Significantly decreased(P<0.05).The results of cell cycle assay showed that the cells in the G1/G0 phase decreased and the proportion of cells in the S phase increased in the experimental group.cell apoptosis results:the apoptosis rate of the experimental group with MLH1 knockdown was 12.9%,and the apoptosis rate was significantly lower than that of the normal RL95-2 cells in the control group(36.9%in the negative control group;48.3%in the blank control group).(P<0.05).The results of hoechst 33258 immunofluorescence staining showed that the apoptotic bodies in the RL95-2 cells of the experimental group were significantly reduced compared with the control group under the fluorescence microscope.4.ADV-MLH1 was successfully infected Ishikawa cells.The results of CCK8 cell proliferation assay showed that the IC50 of the experimental group decreased from 82.6umol/mL to 12.5umol/mL after 72 hours of cisplatin treatment after specific overexpression of MLH1.After MLH1 up-regulation,cell viability decreased significantly and cell cytotoxicity of cisplatin increased significantly(P<0.05).The cell cycle detected a slight increase in the G1/G0 phase cells in the experimental group,and the proportion of cells in the S phase decreased.The results of apoptosis experiment showed that the apoptosis rate of MLH1 overexpression group was 28.3%,and the apoptosis rate was significantly higher than that of Ishikawa cells in the control group(negative control group:15.4%;blank control group:18.8%).(P<0.05).hoechst 33258 immunofluorescence staining showed that the apoptotic bodies in Ishikawa cells of the experimental group were significantly increased compared with the control cells5.We detected an increase in the expression of c-Abl protein in Ishikawa cells with upregulation of MLH1,the Ishikawa cells in the experimental group showed a 1.6-fold and 2.0-fold increase in c-Abl protein at 48 and 72 hours after cisplatin treatment,respectively(P<0.05).Cleaved caspase-3 protein was increased by 2.3-fold and 2.5-fold(P<0.05).Cleaved PARP increased 1.68-fold(P<0.05)in 48 hours of cisplatin induction and 2.2-fold in 72 hours of cisplatin induction(P<0.05).The Bcl-2 protein showed a tendency to gradually decrease as the induction time of cisplatin increased.When the expression of MLH1 in RL95-2 cells was specifically knocked down,the expression levels of the above pathway proteins showed an opposite trend6.A mouse xenograft model was employed to investigate the effect of MLH1 on tumor growth upon treatment with cisplatin,Tumors were much smaller in the ADV-MLH1 group than in the ADV-NC group.On day 30,tumor size in the ADV-NC group increased to an average of 763±11.2.0 mm3,compared with 203.6±8.7 mm3 in the ADV-MLH1 group,the differences were statistically significant(P<0.05)Conclusions:1.Overexpression of MLH1 in endometrial carcinoma cells can significantly enhance the chemosensitivity of endometrial cancer cells to cisplatin and increase the apoptosis of endometrial cancer cells.Conversely,knockdown of MLH1 expression significantly reduced the sensitivity of endometrial carcinoma cells to cisplatin.2.The overexpression of MLH1 enhances the sensitivity of endometrial carcinoma cells to cisplatin,and its mechanism may be through activation of the MLH1/c-Abl apoptotic signaling pathway.3.This study demonstrates that MLH1 may plays an important role in the apoptosis of endometrial cancer cells.The discovery of this mechanism of action is more conducive to our understanding of the occurrence of endometrial caicinoma,MLH1 may be a new target for the treatment of endometrial cancer,and requires more further research.