| Purpose The 5-year survival rate of pancreatic cancer is about 8%.Existing cytotoxic treatments,such as gemcitabine,a first-line nucleoside mimic used to treat this type of cancer and other cancers,have a high failure rate and do not significantly improve the overall survival rate of patients with pancreatic cancer.Therefore,it is very necessary to find and develop alternative therapy,which can be used alone or combined with existing treatments to better treat pancreatic cancer.More than 95 per cent of 20000 to25000 transcribed human genes undergo alternative splicing,increasing proteome diversity.Homotypes derived from the same gene can have different and,in some cases,opposite functions.There is increasing evidence that abnormal RNA splicing is a common and driving event in the development and progression of cancer.In addition,abnormal splicing events that lead to cancer drug / therapeutic resistance are more common than previously thought.The study of alternative splicing regulatory factors related to pancreatic cancer can help us to find new ways to fight pancreatic cancer.Chemotherapy is related to the chemotherapy resistance of tumor cells,and some studies have found that alternative splicing plays an important role in the chemotherapy resistance of tumors.CLK1(cell division cycle kinase 1)is one of the newly discovered splicing regulators.At present,CLK1 is increasingly found to be related to the pathobiology of some diseases,including neurological diseases,metabolic disorders and cancer.It was found that there were extensive and highly variable alternative splicing of CLK1 in 12 carcinoid lines.At the same time,CLK1 is a gene highly expressed in pancreatic cancer.This paper intends to explore the relationship between the expression of CLK1 in tumor cells and the repair of DNA double strand breaks and chemotherapy resistance,to further improve the theory of CLK1 regulating chemotherapy-induced DNA damage repair and chemotherapy resistance,and to determine the mechanism of this protein may lead to new therapeutic strategies.Method In this study,lentiviral vectors overexpressing and knocking down CLK1 were transfected into Panc1 cells by lentivirus packaging,and the mechanism of chemotherapy resistance was observed under the action of gemcitabine.Specifically,we first detected the viability of Panc1 cells by MTT method,calculated their IC50(semi-inhibitory concentration),and observed the relationship between the changes of IC50 after CLK1 knockout / overexpression and the expression of CLK1,and then we detected the relationship between the expression of CLK1 and the drug resistance of gemcitabine and the degree of cell injury induced by gemcitabine.At the same time,we detected the expression of several proteins involved in the repair of DNA double strand breaks induced by gemcitabine and the expression of m RNA(messenger ribonucleic acid): DNA-PKcs(deoxyribonucleic acid dependent protein kinase catalytic subunit),Ku70 and Ku80 in the experimental group and the control group.Result In Panc1 cells with CLK1 gene knockdown,the IC50 of gemcitabine decreased compared with the control group.After the use of gemcitabine,the expression of γ H2 AX increased compared with the control group,and the protein and m RNA expression of DNA-PKcs,Ku70 and Ku80 increased.Compared with the control group,the IC50 of panc1 cells overexpressing CLK1 gene increased compared with the control group.After the use of gemcitabine,the expression of γ H2 AX decreased compared with the control group,and the protein and m RNA expression of DNA-PKcs,Ku70 and Ku80 decreased.Conclusions The expression of CLK1 gene was positively correlated with gemcitabine resistance of pancreatic cancer cells,negatively correlated with DNA damage related protein γ H2 AX,and positively correlated with DNA damage repair related genes such as Ku70/Ku80/DNA-PKcs.It can be inferred that CLK1 gene can enhance the ability of DNA damage repair of pancreatic cancer cells to improve their chemotherapy resistance. |
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