The Effect Of Of Golgi Protein73(GP73)Knockdown Hepatocellular Cancer Cell HepG2

Backgroud/AimGP73is a newly discovered Golgi resident protein which is expressed abnormally high in the tissue and serum of hepatocellular carcinoma patients,however, its function is unclear.We just know that it is closely related with some tumors such as hepatocellular carcinoma, and presume that it may play an important role in maintaining Golgi structure and function in hepatocytes,which needs further study.Therefore, in this study we intended to explore the biological function of GP73by changing GP73expression and observing cell proliferation in hepatocellular carcinoma.Methods1.We detected GP73expression level in several cell lines using Western-blotting method and selected hepatoma cell line HepG2for this study.2.GP73-specific siRNA was designed and transfected into HepG2cell with liposome and protein of the cells was collected to analyze GP73protein expression level by Western-blotting and determine the inteference effect of the siRNA.3.Cell viability was determined by MTT assay Oh,24h,48h,72h after HepG2cells was transfected by GP73-specific siRNA to the observe the antiproliferative effect of siRNA;60h after transfection cell was blocked by nocodazole for12h plus,and then cell cycle distribution was evaluated by flow cytometry analysis; apoptosis in HepG2cells caused by siRNA was determined by flow cytometry using the Annexin V and PI double labeling72h after transfected.4. To further explore the molecular mechanism of the above phenomenons, below proteins in related signal transduction pathways were detected by Western-blotting: p-ERK, p-JNK, p-c-jun, p-Rb, p-CDK4, p-CDK6, CDK4, CDK6, cyclin D3using the specific antibodies to observe the changes of their expression.Results1.In this study,the siRNA we designed can successfully down-regulate the GP73gene expression at the protein level in HepG2cell;2.Knockdown of GP73expression induces cell proliferation inhibition in HepG2cell;3.Knockdown of GP73expression induces G1arrest in HepG2cell;4.Knockdown of GP73expression induces cell proliferation inhibition and cell cycle arrest by reducing CDK4,CDK6and cyclin D3expression and inhibiting JNK,ERK and CDK4phosphorylation in HepG2cell.ConclusionGP73affects cell proliferation and cell cycle by regulating phosphorylation of JNK,ERK and Rb.