The Study Of COMMD7 Gene Regulate The HCC Cells Growth Through ERK/MAPK Dependent Pathway

BackgroundHepatocellular carcinoma(HCC) is one of the malignancies which threaten to the human health.The morbidity is high in our country and all over the word. The main therapy is resection and chemotherapy at present, but the recurrence rate is high and the prognosis is poor. Research suggests that the occurrence of HCC is a complex progress which is regulated by multi-gene. The activation of oncogene、anti-oncogene and cancer-related genes are linked to the occurrence of HCC. Gene therapy of HCC has better prospect in recent years.COMMD7 gene is found in recent years and its function is unknown, it located in the chromosome of 20(20q11.22). Research suggests that a kind of multi-functional protein which consists of 200 amino acids is encoded by this gene. Our preliminary study found that the expression of COMMD7 is high in HCC compared with normal liver tissue, and its expression intensity is positively correlated with the extensive extent of tumor invasion. Preliminary studies indicate that after silencing COMMD7 of HepG2 cells, we detected that the expression of the downstream proteinELK4 and DUSP4 in ERK/MAPK pathway decreased by using gene chip.So we speculate that COMMD7 gene may regulate the cells growth of HepG2 through ERK/MAPK dependent pathway.Also,tumor research has proved that MAPK pathways involved in the regulation of cell proliferation、differentiation and apoptosis and is closed related to the tumorigenesis. However, how can COMMD7 gene regulate the cells growth of HepG2 through ERK/MAPK pathway, the specific mechanism is unclear.This study intends to use RNA interference technology to silence COMMD7 gene and detect the important molecule such as ERK1/2、p-ERK1/2、MEK1/2、p-MEK1/2 in ERK/MAPK pathway by using WB, at the same time,we observe the activity and apoptosis of HepG2 cells. So we can study the specific role of COMMMD7 activating ERK/MAPK pathway and clarify the molecular mechanism of HepG2 cells proliferation and lay the foundation for further gene therapy of HCC.MethodsFirstly, cell model which COMMD7 has been silenced is established and build lentivirus vectors which contain interference RNA aiming to COMMD7 and transfect into HepG2 cells. The interference effect will be verified by PCR、WB and immunofluorescence. Filtering stable cell lines and regular culture it. The experiment is divided in three groups: blank group, negative control group(NC group) and the positive group(experimental group).Secondly, we detect the activity and apoptosis of cells in each group using CCK-8 method and flow cytometry and study the effect of COMMD7 on HepG2 cells proliferation.Thirdly,we detect the important molecule such as ERK1/2、p-ERK1/2、MEK1/2、p-MEK1/2 in ERK/MAPK pathway by using WB, at the same time,we observe the activity and apoptosis of HepG2 cells. So we can study the specific role of COMMMD7 activating ERK/MAPK pathway.Results1. We build the cell model which COMMD7 gene has been silenced successfully, after the lentivirus transfected HepG2 cells for 48, the relative expression of COMMD7 decreased obviously in experimental group compared with blank group and NC group.CCK-8 results show that the cell vitality significantly decreased in experimental group compared with NC group and blank group. Flow cytometry results show that cell apoptosis rate increased obviously in experimental group compared with blank group and NC group,while there is no significant changes between NC group and blank group.2. The expression of p-ERK1/2、p-MEK1/2 which is the main protein in ERK/MAPK pathway decreased obviouslyin experimental group compared with blank group and NC group.Conclusion1. Silencing COMMD7 gene can decrease the vitality and increase the apoptosis of HepG2 cells.2. COMMD7 gene can regulate the cells growth of HepG2 through ERK/MAPK pathway, the specific mechanism may promote the expression of phosphorylated of ERK1/2 and MEK1/2.