Effects And Mechanisms Of PRMT5 On Promoting Epithelial Mesenchymal Transition In Hepatocellular Carcinoma Cells

Objective: The study aimed at investigating the effects of PRMT5 on the cell proliferation, migration and invasion in hepatocellular carcinoma. The functions of PRMT5 were analyzed through controlling the cytoplasm/nucleus shuttle of snail in hepatocellular carcinoma during EMT in order to determine whether PRMT5 promotes cancer cell proliferation, migration and invasion.Methods: PRMT5 short hairpin RNA(shRNA) sequence was designed and inserted into pLKO.1-TRC vector, then packaging lentivirus to infect SMMC-7721 and BEL-7402 cells. Stable infected cells were obtained by puromycin screening. shRNA silencing efficiency was tested by Western blot and qPCR. The plasmid pHA-PRMT5 was used to rescue the expression of PRMT5 in stable cells. Cell proliferation activity was detected by CCK-8. Colony formation ability was detected by plate colony assay. Cell migration activity was detected by migration assay. Cell invasion activity was detected by invasion assay. The expression of cell invasion-related protein MMP2 and MMP9 were detected by Western blot. The expression of EMT-related protein E-cadherin, Collagen-I, β-catenin, α-SMA, Vimentin and SNAIL were detected by Western blot. The subcellular localization of SNAIL in hepatocellular carcinoma was detected by immunofluorescence and nucleocytoplasmic protein separation. Results: The specific shRNA against PRMT5 plasmid(sh- PRMT5) was successfully constructed and infected into SMMC-7721 and BEL-7402 cells. Western blot and qPCR demonstrated that the expression of PRMT5 was efficiently inhibited at both protein and mRNA levels. It revealed that PRMT5 depletion decreased cell proliferation, colony formation ability, migration, and invation. Expectedly, ectopic PRMT5 re-expression reverses these changes. It was found that knockdown of PRMT5 reduced the protein levels of cell invasion-related protein MMP2 and MMP9, whereas ectopic PRMT5 re-expression partially reverses these changes. It showed that silencing PRMT5 induces epithelial markers E-cadherin expression and downregulates expression of mesenchymal markers including Collagen-I, β-catenin, α-SMA and Vimentin, while the protein levels of SNAIL increased. Expectedly, ectopic PRMT5 re-expression downregulates epithelial markers E-cadherin, reverses expression of mesenchymal markers including Collagen-I, β-catenin, α-SMA, and Vimentin, however, the protein levels of SNAIL was still increased. It was found that knockdown of PRMT5, SNAIL translocated from nucleus into cytoplasm. However, ectopic PRMT5 re-expression, SNAIL relocated to the nucleus. Conclusion: Accordingly, it is suggested that a mechanism of PRMT5 involved in EMT through controlling the cytoplasm/nucleus shuttle of SNAIL in HCC cells.