The Study On The Role Of MicroRNA-218 Through Survivin In The Pathogenesis Of Cholangiocarcinoma

Objectives: To observe the expression of mi R-218 in cholangiocarcinoma,and to explore the target gene Survivin of miRNA-218,and to study its effect on the biological behaviors of cholangiocarcinoma cells in cell lines,and to verify the connection between them.Therefore,the study of the pathogenesis of cholangiocarcinoma provides a new direction for the early diagnosis and treatment of cholangiocarcinoma.Methods:Two kinds of cholangiocarcinoma cell lines(CCLP1 and QBC939)and non tumor bile duct cell lines(H69)were cultured.Real-time PCR was used to detect the mi R-218 expression and compared the difference between cholangiocarcinoma cell line and non tumor bile duct cell line.The miRNA-218 minics and negative control were transfected into CCLP1 and QBC939 cells,and establish miRNA-218 group,negative control group and blank control group.Apoptotic cells were detected by cell apoptosis detection kit,BD Martigel with Matrigel invasion chamber was invasive.Chemical synthesis of Survivin3'UTR fragments containing miRNA-218 predictive binding sites and cloned into pMIR-Luc plasmids.In order to prove that it is miRNA-218,the binding site leads to the change of luciferase activity.The mutant Survivin3'UTR fragment designed at miRNA-218 binding site is cloned into pMIR Luc plasmid.The CCLP1 and QBC939 cells were co transfected to miRNA-218 mimic and pMIR-Survivin,miRNA-218 mimic and pMIR-Survivin-mut,respectively.The activity of luciferase was detected and the correlation between miRNA-218 and Survivin was determined.Results:The expression in the cholangiocarcinoma cell line(CCLP1 and QBC939)was significantly lower than the non tumor cholangiocarcinoma cell line(H69)(P<0.01).In the mi RNA-218 group,the apoptosis rate of the cells in the negative control group and the blank control group increased significantly,P<0.05.This indicates that miRNA-218 plays a negative regulatory role in the development of cholangiocarcinoma.Compared with the negative control group and the NC group,the transfection of miRNA-218 mimics could significantly inhibit the invasiveness of bile duct cancer cells,with a statistically significant P<0.01.The results of luciferase activity assay: miRNA-218 mimic and pMIR-Survivin,miRNA-218 mimic and pMIR-Survivin-mut were co transfected in CCLP1 and QBC939 cells.The results showed that wild type 3 'end of report gene vector non encoding region of the luciferase activity and the negative control group and blank control group significantly decreased compared with statistical significance P<0.05 mutant 3' non encoding region of reporter gene luciferase activity and no significant changes compared to negative control group and blank control group,no the statistical significance of P>0.05.The results indicate that Survivin is a direct target of miRNA-218.MiRNA-218 may negatively regulate the occurrence and development of cholangiocarcinoma through binding withSurvivin3'UTR.Conclusions:1.The expression of miRNA-218 in cholangiocarcinoma is decreased,and it can increase the apoptosis of cholangiocarcinoma cells,inhibit the invasion of cholangiocarcinoma cells,and play a negative role in the occurrence and development of cholangiocarcinoma.2.MiRNA-218 can interact with Survivin3'UTR directly.The negative regulatory effect of miRNA218 on cholangiocarcinoma is likely to play a role directly through the Survivin.