Effects Of Silencing SFRP5 To The Cellular Property In Human Cholangiocarcinoma RBE Cells

Objective: To investigate the silence efficiency of lentivirus-mediated shRNA(short hairpin RNA)silencing SFRP5(secreted frizzled-related proteins-5)on proliferation, migration and apoptosis of cholangiocarcinoma RBE cells.Method:In our research,The human cholangiocarcinoma RBE cell line were cultured in vitro,In the present work, We designed the interference sequence to amplify the coding region of SFRP5,The plasmid was verification by DNAsequencing, after transfected the sFRP-5 knockdown vector(pLVX-shRNA2) into RBE cells by using the FuGENE? HD Transfection Reagentas the cell conversion agent, we obtained a RBE cell line with stable SFRP5 knockdown, which was validated by reverse transcription quantitative polymerase chain reaction and western blotting. The CCK-8 assay was used to measure the capability of cells proliferation, The wound healing assay and transwell migration assay was used to exploring the effects of SFRP5 knockdown on cell motility, the cell cycle and apoptosis was measured by using flow cytometry.Results: The results of Immunocytochemistry shows SFRP5 expression is positive in RBE cells,so we selected the RBE cell line for Subsequent experiments, after transfected the recombinant plasmid into 293 T cells,we observed the successful transfected cells with green fluorescence under the inverted fluorescence microscope, the sequencing results were positive with Lentivirus titer of1×109 TU/ml,Western blotting and RT-PCR assays were performed to validatethat SFRP5 was down-regulated in RBE-shRNA- SFRP5 cells in compare with RBE-NC-cells. The protein level of SFRP5 transfected group was signifcantly lower than the NC group, while the mRNA level is also reduced with p=0.0087, The CCK-8 assay results showed that RBE cells with the transfection of SFRP5 shRNA grew significantly faster than that of the NC one While the transwell migration assay was shown that the number of migrated cells of SFRP5 knockdown group[(310±15.63)]was significantly higher comparing to the NC group[(75±8.6)], The results of flow cytometric analyses showed the cell apoptosis rate(2.3%)was significantly lower in RBE-shRNA- SFRP5cells(12.3%).Conclusion: SFRP5 may play an important regulatory role in the development of intrahepatic cholangiocarcinoma, low expression of SFRP5 activate wnt signaling pathway, and promote tumor development. After sucessfully constructed a RBE Cell Line with stable knockdown ofSFRP5, our results indicate that SFRP5 knockdown enhances proliferation and migration potential of RBE cells in vitro, and can decrease cell apoptosis at the same time. Our results suggest SFRP5 might act as tumor suppressor in cholangiocarcinoma RBE cells, and the present study lay the foundation for the future experiments in vitro.