Studies On The Regulation Of CLK1 On Apoptosis-related Gene In Pancreatic Cancer Cells

Background CLK1 is an important protein kinase that regulates the alternative splicing of RNA and in a variety of cancers it is observed to influence the development and progression of the cancers.Pancreatic cancer is difficult to treat,but induction of apoptosis of pancreatic cancer cells is an effective treatment at present.This study aims to explore the effect of CLK1 on apoptosis of pancreatic cancer cells and the possible molecular mechanism,so as to provide theoretical support for seeking the best therapeutic agents for pancreatic cancer.Methods Firstly,we used flow cytometry to analyze the apoptosis level of pancreatic cancer cell lines after the change of CLK1 expression level.And the change of apoptosis rate in human primary pancreatic cancer cells by TUNEL assay after TG003 was used to inhibite the CLK1 expression.Seconedly,I carried out the protein profiling to compare the expression of proteome in pancreatic cancer cells between pancreatic cancer cells and pancreatic cancer cells with CLK1 was inhibited to find out the differences.Because the results of protein profiling was the m RNA splicing related proteins changed mostly,i analyzed the change of occurrence of alternative splicing events by RNA-seq technology.Finally,I detected the expression of apoptosis-related genes by quantitative PCR,and find out the apoptosis-related genes with expression changed after CLK1 inhibition.Western blot was executed to verify the result.Results(1)The results of Flow cytometry showed that apoptosis was increased after CLK1 knockdown in Panc1 cell lines that express high level of CLK1 at first,while apoptosis was inhibited after over expression of CLK1 in BXPC3 cell lines that express low level of CLK1.The apoptosis rate was increased significant in human pancreatic cancer cells after TG003 was used to inhibit the expression of CLK1.(2)The results of Proteome mass spectrometry of pancreatic cancer cells showed that the phosphorylation of about 220 proteins in pancreatic cancer cells was changed after CLK1 inhibition,among which the m RNA splicing related proteins changed the most significantly,and the RNA-seq results showed that the alternative splicing events in cells were inhibited.(3)The expression of CLK1 was inhibited in Panc1 cells.Compared with the control group,the gene and protein expression levels of Bax were significantly increased,while the expression level of Bcl2 was decreased.The expression level of Caspase3 gene increased,and the change of protein expression level was not obvious.The results of Casepase3 activity detection showed that the expression level of c-casepase3 was increased and the activity was enhanced.Conclusion Inhibition of CLK1 expression can increase apoptosis of pancreatic cancer cell lines and human primary pancreatic cancer cells,suggesting that CLK1 is involved in the regulation of pancreatic cancer cell apoptosis.The potential mechanism may be that CLK1 regulates the expression levels of Bax and Bcl2 by affecting alternative splicing,thereby altering the activation of Caspase3 and affecting cell apoptosis.