The Role And Molecular Mechanism Of Mismatch Repair Gene MLH1 In Pancreatic Cancer Invasion And Metastasis

BackgroundPancreatic cancer is one of the most lethal malignant neoplasms.Highly invasive phenotype,rapid disease progression,and early metastasis are the main factors contributing to its extremely poor prognosis.However,the molecular mechanism of its invasion and metastasis remains further elucidated.MLH1(Mut L homologue 1),as a key gene of DNA mismatch repair,has been widely studied in colorectal cancer,endometrial cancer and other tumors.However,the molecular mechanism of MLH1 regulating the invasion and metastasis of pancreatic cancer is still insufficient.Our previous study found that the expression of mismatch repair genes was positively correlated with good prognosis of pancreatic cancer patients,and significantly negatively correlated with tumor differentiation and lymph node metastasis,suggesting that MLH1 may play an inhibitory role in the invasion and migration of pancreatic cancer.Therefore,this study aims to clarify the role of MLH1 in invasion and metastasis of pancreatic cancer,and provide potential therapeutic targets for precisely targeted therapy of pancreatic cancer.ObjectiveThis study aims to explore the possible role of mismatch repair genes in pancreatic cancer based on the TCGA database,the effects of MLH1 on the malignant biological behaviors of pancreatic cancer and to preliminarily reveal the molecular mechanism of MLH1 regulation on invasion and metastasis of pancreatic cancer.Methods1.The expression,functional enrichment and prognosis of mismatch repair genes MLH1,MSH2 and MSH6 in pancreatic cancer were analyzed by bioinformatics methods,and the possible role of mismatch repair genes in pancreatic cancer was preliminarily explored by using transcriptome data from the TCGA database.2.The expression of MLH1 in different pancreatic cancer cell lines was investigated using real-time quantitative(qRT-PCR)and western blot.And lentivirus transfection was used to screen and establish stable MLH1 knockdown,overexpression and corresponding control regular transfected pancreatic cancer cell lines.3.CCK-8 cell proliferation assays and colony formation assays were used to detect the effect of MLH1 on the proliferation of pancreatic cancer cells in vitro.The effects of MLH1 on migration and invasion of pancreatic cancer cells in vitro and in vivo were investigated by using transwell assays and wound healing assays and orthotopic transplantation of nude mouse tumor model.4.Transcriptome sequencing,rescue experiment,and immunohistochemistry of tissue samples were used to explore the possible molecular mechanism of MLH1 regulation of pancreatic cancer invasion and metastasis.Results1.The possible role of mismatch repair genes in pancreatic cancer based on TCGA In the TCGA database,the expression levels of mismatch repair genes MLH1,MSH2 and MSH6 were not considerably different between pancreatic and normal pancreatic tissues.The expression level of MLH1 in pancreatic cancer tissues was lower than that in normal pancreatic tissues on the trend.The expression of MSH2 and MSH6 was significantly positively correlated.The expression of MLH1 was significantly associated with multiple invasion and metastasis genes,and the differentially expressed MLH1 genes were enriched in multiple invasion and metastasis-related pathways.The expression of MLH1 in stage I patients was significantly higher than that in stage Ⅱpatients,and tended to increase in subgroups of T1/T2 and N0.The overall survival,progression-free survival and disease-specific survival of patients with high expression of MSH2 and MSH6 were significantly shortened(P<0.05),and MSH2 had statistical significance in multivariate Cox regression analysis of prognosis(P<0.05).2.The regulation of MLH1 on malignant biological behaviors of pancreatic cancer In vitro experiments showed that the proliferation,migration and invasion abilities of BxPC-3 cells were significantly enhanced after MLH1 knockdown compared with the control group,and the proliferation,migration and invasion abilities of MIA PaCa-2 cells were significantly decreased after overexpression of MLH1 compared with the control group(P<0.05).Compared with the control group,BxPC-3 MLH1 knockdown cell lines had significantly enhanced liver metastasis ability in vivo,while MLH1 overexpression cell lines had the opposite(P<0.05).3.The mechanisms of MLH1 regulation on invasion and metastasis of pancreatic cancerTranscriptome sequencing showed that G protein-coupled Receptor C family 5C(GPRC5C)was significantly negatively correlated with MLH1(P<0.05).Pearson correlation analysis,Spearman correlation analysis and Kendall correlation analysis with the GEPIA2 online tool showed that GPRC5C was negatively correlated with MLH1 expression in pancreatic cancer(PAAD-TCGA)tissues(P<0.05).In addition,MLH1 was down-regulated and GPRC5C up-regulated at protein level and transcription level in BxPC-3 MLH1 knockdown cells.On the contrary,MLH1 was negatively correlated with GPRC5C in MIA PaCa-2 MLH1 overexpression cells.In nude mouse orthotopic transplanted tumor,the expression of GPRC5C increased in BxPC-3 MLH1 knockdown cells,and decreased in overexpression cells.Immunohistochemical staining results showed that GPRC5C expression was low in patients with high MLH1 expression,and GPRC5C expression was increased in patients with low MLH1 expression(P<0.05).Further rescue experiments showed that the proliferation,migration and invasion abilities of BxPC-3 MLH1 knockdown cells were significantly reduced after GPRC5C knockdown(P<0.05),which reversed the promoting effect of MLH1 deletion on migration and invasion of pancreatic cancer cells.Conclusion1.In the TCGA database,the expression level of MLH1 in pancreatic cancer tissues was lower than that in normal pancreatic tissues on the trend.MLH1 may be involved in regulating invasion and metastasis of pancreatic cancer.MSH2 is an independent risk factor for the poor prognosis of pancreatic cancer and can be used as a potential prognostic marker of pancreatic cancer.2.MLH1 inhibited proliferation,migration and invasion of pancreatic cancer cells in vitro,and inhibited liver metastasis of pancreatic cancer cells in vivo.3.MLH1 negatively regulated GPRC5C at the transcriptional level to inhibit pancreatic cancer invasion and metastasis.