The Changes Of Subcellular Localization For P21 On Cell Proliferation And Apoptosis Of HepG2

Part IConstruction of the wild and mutated P21 encoded by a eukaryotic expression plasmidObjective:To construct the wild and mutated P21 for Nuclear Localizational signal encoded by a eukaryotic expression plasmid so that they will make a firm base for investigating the changes of subcellular localization on cell cycle and apoptosis of HepG2.Method:The coding genes of the wild and mutated P21 for Nuclear Localizational signal were amplified from the plasmid pCEP-WAF1 which contains human P21cDNA by mega primer PCR and then they were cloned into pMD18-T vector. The recombinational plasmids were digested with BamH I and EcoR I and were correctly inserted into BamH I/ EcoR I restriction sites of the pDsRedl-C1 expression vector so that they could encodes fusions protein emitting red fluorescence. The recombinant pDsRedl-C1-P21WT (wild type of Nuclear Localizational signal) and pDsRed1-C1-P21MT (mutant type of Nuclear Localizational signal) were identified by PCR, enzyme digestion, and were confirmed by DNA sequencing at last.Results:The wild and mutant P21 encoded by a eukaryotic expression plasmid pDsRedl-Cl were successfully constructed. Restriction analysis indicated that they were correctly inserted into the pDsRed1-C1 vector.Conclusion:The wild and mutant P21 encoded by a eukaryotic expression plasmid were successfully constructed. Part IIThe effects of the wild and mutant P21 encoded by a eukaryotic expression plasmid on proliferation and apoptosis of HepG2Objective:To analyze the intracellular localization of the wild and mutant P21; to detect the expression level of p21 mRNA;to explore the effects of the wild and mutant P21 encoded by a eukaryotic expression plasmid pDsRedl-C1 on cell cycle and apoptosis of HepG2.Methods:The wild and mutant P21 of Nuclear Localizational signal encoded by a eukaryotic expression plasmid pDsRedl-C1 were transfected into HepG2 hepatoma cells using lipofectamine transfection reagent respectively. The plasmid pDsRedl-C1 was transfected as blank controls. The expression levels of p21 mRNA were detected by RT-PCR, and intracellular localization of the p21 were analyzed by fluorescence microscope, the cell proliferation was assayed by MTT. The cell cycle of HepG2 stained by AnnexinⅤ/FITC were detected by flow cytometry. HepG2 of each group was induced to apoptosis by actinomycin-D for 24 hours, and the rate of apoptosis was measured by flow cytometry with double staining of Annexin V-FITC/PI.Result:After transfection with the wild and mutant P21, the expression of red fluorescent protein were observed by inverted fluorescence microscope in the empty vector group, the wild-type group, mutant-type group. Red fluorescence distribute diffusely in the cells of empty vector group, while in the wild-type group, mutant Group, Red fluorescence concentrated together into a massive corporation; pDsRed1-C1-p21WT groups, the fluorescence was mainly localized in the nucleus of HepG2-P21WT, HepG2-P21NLS" groups mainly located in the cytoplasm. The specific mRNA bands extracted from the stably screened cells of HepG2-P21WT, HepG2-P21NLS" expresses with a high-abundance scale. Cells of empty vector group also have a small number of specific bands which belongs to endogenous expression in HepG2 cells. The mutant P21 plasmid accelerates cell proliferation while wild P21 plasmid inhibits cell proliferation by MTT assays, as the curve moved to the left and become steepening in the mutant group (P<0.01) while right and flattening in the wild group (P＜0.01) cell-cycle analysis shows that:the wild-type group of HepG2 cells compared with the mutant, cell ratio of G0/G1 increased significantly (P<0.01);cell ratio of apoptosis for each group were 2.21 (P＜0.01);12.30 (P＜0.01);4.26 (P>0.05);4.34 (P>0.05) respectively by flow cytometry.Conclusion:the wild type of P21 is mainly localized in the nucleus; while the mutant type of P21 is mainly localized in the cytoplasm. The mutant type of P21 accelerates cell proliferation, promotes cell-cycle and enhances its capacity of anti-apoptosis while wild type of P21 inhibits cell proliferation, cell-cycle and promotes apoptosis.