Effect On Invation And Proliferation Of Human Hepatoma Cell By ShRNA Inhibiting EVL Gene Expression

Background:The primary hepatocellular cancer is the sixth most common cancer and have the third highest mortality rate in all kind of cancers. Alone with the growing of global population and aged tendency of population, the development of liver cancer reveal a growing trend all over the world. It’s worth noting that more than half of the liver cancer occurred in china, that’s serious harm to people’s life and health in our country. EVL (Ena/VASP like protein) is a member of Ena/VASP family proteins and is implicated in processes that require dynamic actin remodeling such as cell adhesion, axon guidance and the motility of cell. Ena/VASP family proteins have a highly conserved structure that consist of EVH1, Pro-rich and EVH2 domain.In fibroblasts, Ena/VASP family protein regulate the motility of fibroblasts by controling the reassemble of actin in lamellipodia. Ena/VASP family proteins show high expression in several types of cancer. The research using real time PCR revealed that EVL was highly expressioned in the most of breast cancer patients’serum. In this work, we found that the expression levels of EVL in the hepatoma cell was higher than normal hepatocyte. The research of EVL’s function in hepatoma cell will help us learn the mechanism for the development of HCC better and provide powerful basis for the treatment and searching the marker of hepatocellular cancer.Method:Comparing the expression levels of EVL between the hepatoma cell line SMMC-7721 and hepatocyte line L02 using western bloting. To study the function of EVL in hepatoma cells, stable EVL-knockdown transgenic cell lines were generated by transfecting human hepatoma cells with shRNA plasmids. The hepatoma cells were transfected by lipofectamine 2000 and transfection efficiency was observed by fluorescence microscope.The mRNA and protein expression levels of EVL was detected by real time PCR and western blot. Then, We using MTT assay, Colony-forming assay, Transwell invasion assay and Flow cytometry to study the influence on SMMC-7721 cells proliferation, invasion and cell cycle after transfected. The function of EVL in hepatoma cells was analysed by comparing the experimental group with the control group.Result:Western blot showed that the level of EVL was increased in hepatoma cells. The plasmid transfected SMMC-7721 cells by lipofecamine 2000 showed green fluorescence, it indicate that the plasmid had been successful transfected in hepatoma cells. Real time PCR and western blot indicated that the mRNA and protein levels of EVL in the hepatoma cells were lower than hepatocyte cells. The cell growth curve made by MTT assay show the EVL defected hepatoma cells growed slower than wild type. In colony-forming assay, the colony-forming rate of the shRNA-EVL transfected cells was lower than control. The numbers of cell permeating septum of treatment group was lower than control suggested the defected of EVL result in the decrease of hepatoma cell’s invasion ability. The cell cycle was detected by flow cytometry, the EVL defected hepatoma cells in G0/G1 phase was increased. It suggested that the hepatoma cell cycle will be blocked in G0/G1 phase.Conclusion:It suggest that EVL has an important role in the hepatocellular carcinoma malignant proliferation and invasion, because the proliferation and invasion ability of hepatoma cells was decreased when EVL expression was inhibited.