The Role And Mechanism Of CLK1 In The Development And Progression Of Pancreatic Cancer

Objective:Pancreatic cancer is known as the"king of cancers".With the continuous innovation of medical technology,the mortality rate of most malignant tumors has been decreasing year by year,but the five-year survival rate of pancreatic cancer still hovers around 5%.Due to the lack of early specific symptoms,the pathogenesis of pancreatic cancer is very insidious and highly aggressive.A large number of patients are already in the advanced stage at the first diagnosis and miss the opportunity of surgery.Meanwhile,pancreatic cancer is not sensitive to radiotherapy and chemotherapy,and treatment means are extremely scarce.At present,the pathogenesis and development mechanism of pancreatic cancer are still unclear.It is of great clinical significance and social value to further explore epigenetic events in the occurrence and development of pancreatic cancer,as well as explore new molecular markers and therapeutic targets of pancreatic cancer.CLK1 is an important functional molecule that mediates alternative splicing,and has been reported to be closely related to the occurrence and development of a variety of malignant tumors,but its expression and role in pancreatic cancer have not been reported.This study is the first to confirm that CLK1 is highly expressed in pancreatic cancer and is closely related to the prognosis of pancreatic cancer patients.CLK1 is closely related to cell cycle disturbance and proliferation of pancreatic cancer in vivo and in vitro.We screened and verified the functions of splicing factors that may be regulated downstream by CLK1,and completely observed differential splicing events mediated downstream by CLK1.This study aims to clarify the role of CLK1 in pancreatic cancer cell cycle disturbance and its mechanism,and to fully explore and discuss the specific mechanism of CLK1 in pancreatic cancer,in an effort to provide a new scientific basis and theoretical basis for early diagnosis and targeted therapy of pancreatic cancer.Methods:1.TCGA and GEO public databases,immunohistochemistry and western blotting were used to detect the expression levels of CLK1 mRNA and protein in patients with pancreatic cancer.At the same time,the correlation between CLK1 expression and clinicopathological characteristics and prognosis of patients with pancreatic cancer was further explored by combining the clinical data of patients with pancreatic cancer.2.Lentivirus packaging technology stably overexpressed or interfered with CLK1expression in pancreatic cancer cell lines,and the transfection effect was verified by western blotting.3.The effect of CLK1 on the proliferation of pancreatic cancer cells was detected by CCK8 test,cell counting method and plate colony formation test.4.Flow cytometry（PI staining）was used to evaluate the effect of CLK1 on the cell cycle of pancreatic cancer.Cell cycle regulatory proteins p21 and p53 were detected by Western blotting.5.The effect of CLK1 on the proliferation of pancreatic cancer cells was verified in vivo by subcutaneous tumor-forming experiment in nude mice;6.Phosphorylated protein profiling technology was used to screen splicing factors that might affect CLK downstream,and Western blotting was used to verify the screening results.The splicing factors were verified by plasmids with different phosphorylated states packaged by lentivirus.7.Variable splicing events regulated by CLK1 and splicing factors were completed by high-throughput sequencingResults:1.Both mRNA and protein expression of CLK1 are increased in pancreatic cancer.2.CLK1 expression is closely related to the clinical,clinicopathological characteristics and survival prognosis of pancreatic cancer patients.3.High expression of CLK1 promotes proliferation of pancreatic cancer cells,and interferes with CLK1expression to inhibit proliferation of pancreatic cancer cells.4.CLK1 mediates the cell cycle disturbance of pancreatic cancer,and regulates the expression of P21 and P53 in cell cycle.5.CLK1 mediates SRSF5^Ser-250（serine 250）phosphorylation.6.The CLK1-SRSF5 axis is mainly involved in the cycle-dependent alternative splicing of pancreatic cancer cells.7.The CLK1-SRSF5 axis is mainly involved in the exon jumping of the alternative splicing type in pancreatic cancer.8.Clk1-SRSF5 axis mediates Cyclin L2Exon6.3 hopping,Cyclin L2 splicing mode changes are involved in the regulation of pancreatic cancer malignant behavior,Cyclin L2 long chain promotes pancreatic cancer cell proliferation,Cyclin L2 short chain inhibit pancreatic cancer cell proliferation.Conclusion:1.CLK1 is closely related to pancreatic cancer and is expected to be developed as a new target for the diagnosis and treatment of pancreatic cancer.2.CLK1mediates pancreatic cancer cell cycle disturbance and promotes pancreatic cancer cell proliferation in vitro and in vivo.3.CLK1 phosphorylates Sr SF5250Ser and is involved in the regulation of malignant behavior in pancreatic cancer cells.4.The CLK1-SRSF5axis mainly mediates the cycle-related alterable splicing changes in pancreatic cancer cells5.CLK1-Sr SF5 axis mediates Cyclin L2Exon6.3 hopping and participates in the regulation of malignant behavior of pancreatic cancer,which can be used as a potential target for the treatment of pancreatic cancer in clinical practice.