| Hepatocellular Carcinoma(HCC) is one of the most common carcinomas,contributes to the higher morbility and mortality among cancer patients because of its highest malignancy.HCC endangers the lives and health of Chinese people because each year the newly developed hepatic cancer patients in the Chinese mainland accout for 42.5 percent of the the cases in the world.In perspective,surgical therapy and liver transplantation are effective,but very few patients are indicative of surgical operations due to high malignancy,fast growth,susceptibility for relapse and metastasis and a great portion of them are tentatively treated with chemotherapy,radiotherapy as well as other alleviative treatments.Therefore, a thorough alternative understanding of the pathogenesis of HCC thus holds the promise of finding an effective chemoprevention and treatment for this cancer.The formation of HCC is a multi-factor, multi-step and multi-gene process.The studies on the pathogenesis of HCC shows that p21 has involved in the genesis and development of HCC. p21(p21WAF1/CIP1)belongs to the Cip/Kip family of CDK inhibitors. The p21 gene encodes a 21kDa protein which binds and inhibits cyclin/CDK complex , predominantly blocks G1/S phase cell cycle transition. p21 has also been shown to inhibit PCNA binding to DNA polymerase delta (polδ)and hereby block DNA replication. polδis a cell cycle-dependent protein with important roles in DNA replication,repair, recombination and cell cycle regulation. DNA polymerase delta is encoded by the POLD1 gene, the transcription of which is strictly cell cycle-dependent, but the mechanisms has not yet well known.The replication of eukaryotic chromosomes is a complex but highly regulated process. If DNA replication is perturbed, abnormally replicated DNA may lead to tumorigenesis.As the profound role of p21 in cell cycle regulation and DNA replication ,our research studied the behavior effects on HCC cell line SMMC-7721 through regulating the expression of p21.We confirmed the role of p21 in cell cycle arrest ,the probable influence on the invasion and metastases of HCC,and the association with POLD1gene.Objective: To study the behavior effects on SMMC-7721 cells through regulating the expression of p21.To confirm the role of p21 in cell cycle arrest ,the probable influence on the invasion and metastasis of HCC,and the association with POLD1gene.Methods:1. Transient transfection:SMMC-7721 cells were transient transfected by p21 eukaryotic expression vector named pXJ41-p21 and negative control vector named pXJ41-neo. The chemically synthesized small interfering RNA(siRNA) targeted on p21were transient transfected into cells. 2. Construction of stable cell lines: the stable cell line with p21 overexpression was obtained after G418 selection. The stable cell line with p21 knocked-down was obtained by limiting dilution assay after infected by lentivirus-mediated target silenced p21. 3.The expression of p21 and POLD1 mRNA was detected by RT-PCR. The expression of p21 and POLD1 protein was detected by Western-blot. 4.The behavior effects of cells were explored by cell growth curve, cell clones formation, transwell migration and invasion assay,flow cell cytometry. Results:1.When cells were transient transfected with p21,the levels of p21 mRNA were up-regulated at 48h,and the levels of POLD1 mRNA were down-regulated.Cell proliferation was significantly inhibited, the number of cells blocked in G0/G1 increased (P<0.05).The levels of p21 mRNA and protein were higher in the cells stably transfected with p21, accompanied with down-regulation of POLD1 mRNA and protein expression.The abilities about cell proliferation ,invasion and migration were inhibited, cell cycle progression was blocked in G0/G1 phase and apoptosis rate was lower in comparison with control groups.2. When cells were transient transfected with siRNA targeted on p21,the levels of p21 mRNA were down-regulated at 48h,and the levels of POLD1 mRNA were up-regulated.Cell proliferation was significantly increased,cell cycle arrest in G0/G1 phase was inhibited and apoptosis rate was higher.The levels of p21 mRNA and protein were down-regulated in the stable cell line knocked down p21, accompanied with up-regulation of POLD1 mRNA and protein expression.The abilities about cell proliferation ,invasion and migration were significantly increased, cell cycle arrest in G0/G1 phase was inhibited and apoptosis rate was higher.Conclusion:The results demonstrate that p21 gene can induce G0/G1 phase arrest and inhibit the cell cycle progression in SMMC-7721 cells.p21 also can protect cells from apoptosis,and involve in the invasion and metastasis of HCC probably.The expression of POLD1 correlates negatively with p21 expression,and p21 probably involves in regulation of POLD1. |
