MiR-637 Inhibits Cell Proliferation And Viability By Regulating CTSB In Cholangiocarcinoma QBC939 Cell

[objective]The inhibitory effect of miR-637 had been reported on hepatocellular carcinoma and glioma.Reportly,miR-637 expression was low in cholangiocarcinoma.But the function and mechanism of miR-637 in cholangiocarcinoma is rarely reported.CTSB was predicted to be the possible target gene of miR-637 by bioinformatics software.We found the expression of CTSB was higher in cholangiocarcinoma tissues than adjacent tissues(8.4 times)by mass spectrum analysis.CTSB was reported to play an important role in promoting progression and metastasis of variety of human malignancies.It was highly expressed in cholangiocarcinoma tissues and blood,and negatively correlated with the stage and prognosis of cholangiocarcinoma.In this study,we explored whether miR-637 regulated the viability and proliferation of cholangiocarcinoma,whether miR-637 play an effect on cholangiocarcinoma by regulating CTSB directly.[methods]MiR-637 levels in cholangiocarcinoma cell lines and normal bile duct epithelium cells were detected by qRT-PCR,as well as miR-637 levels in human cholangiocarcinoma tissues and adjacent tissues.QBC939 cells were choosed to explore the function of miR-637 in cholangiocarcinoma for its lowest relative miR-637 levers than other cell lines.After mi R-637 were infected into QBC939 by lentivirus,the infection efficiency was assessed by fluorescence rate.MTT,scratch assay,flow cytometry and Westblot were used to detect the cell proliferation,migration,cell cycle,apoptosis and the expression of apoptosis protein Caspase 3 and Caspase 9.CTSB was predicted to be a potential target gene of mi R-637 by bioinformatics software.Luciferase report assay was used to verify whether Luciferase rate of the cotransfection of miR-637 mimics and wild-type CTSB group was significantly lower than that of blank control group and mutant group.The expression of CTSB were measured by Westerblot and RT-PCR at the level of gene and protein after overexpression of miR-637,respectively.MiR-637 levels and CTSB mRNA levels in 41 cases of cholangiocarcinoma were detected by RT-PCR.Correlation analysis further verify the existence of negative correlation between levels of miR-637 and CTSB mRNA.[Results]The level of miR-637 in cholangiocarcinoma cell lines was significantly lower than in normal bile duct epithelium cells,The level of miR-637 in cholangiocarcinoma was significantly lower than that in adjacent tissues.Result of RT-PCR showed the level of mi R-637 were lowest in QBC939 than other cell lines.Result of Lentivirus infection showed the transfected cells had normal morphology and transfection efficiency(fluorescence rate)were 70-80% after 72 hours.MTT showed mi R-637 overexpression decreased the proliferation activity of QBC939 cells significantly.Scratch test showed that overexpression of miR-637 inhibited the migration of QBC939 cells.Flow cytometry analysis showed that overexpression of miR-637 promoted apoptosis while had effect on the cell cycle of QBC939.Westblot found over expression of miR-637 promoted the expression of Caspase 3 and Caspase 9.Through Targetscan database analysis,we predicted that CTSB is the potantial target gene of mi R-637.The luciferase reporter experiments showed luciferase rate was significantly decreased in cotransfection of wild-type miR-637 mimics and CTSB group than in mutant group and blank control group.Westblot and RT-PCR showed the expression levels of CTSB mRNA and protein were significantly decreased in miR-637 overexpressed cells.Correlation analysis of miR-637 and CTSB mRNA expression in 41 cases of cholangiocarcinoma tissues showed that miR-637 level was negatively correlated with the expression of CTSB mRNA.[conclusion]MiR-637 may inhibit the proliferation and viability of cholangiocarcinoma cells by targetly regulating CTSB.MiR-637 may promote the apoptosis of cholangiocarcinoma cells by upregulating apoptosis protein.In conclusion,miR-637 may play an important role in the programe of cholangiocarcinoma.Objective: To investigate the effect of Cathepsin B(CTSB)on the proliferation,migration and apoptosis of human cholangiocarcinoma(CCA)cell QBC939,and the interfering effect of sh RNA on CTSB.Materials and methods: By constructing and packaging CTSB-sh RNA lentivirus vector,the infection and interference efficiency of sh RNA on CTSB were detected,The infection efficiency and of RNAi interference,and the effect of CTSB decreased on the proliferation,migration ability,apoptosis and cell cycle of QBC939 were detected,respectively.Results: The expression of CTSB in sh RNA interfered QBC939 cell was significantly decreased compared with QBC939-NC cell(cell interfered by blank plasmid).The interference of CTSB causes the proliferation and migration ability of QBC939 cell decreased significantly.In addition,the cell in G0/1 phase,and the cell apoptosis rate significantly increased.Conclusion: CTSB plays an important role in the proliferation and migration of CCA cells.Sh RNA inhibits the expression of CTSB significantly,thus reduces the proliferation,migration and invasion ability of QBC939 cell,and promotes the apoptosis of QBC939 cell.