Effect Of MIA2 On Growth And AKT Signaling Of Human Hepatocellular Carcinoma HepG2 Cellline

PART ONE The construction of p LVX-IRES-Zs Green1-MIA2 recombinant plasmid and establishment of its high efficient tranfection and expression systemObjective To clone human peroxisome proliferator-activated receptor ? gene(h PPAR?) and construct a eukaryotic expression vector of p LVX-IRES-Zs Green1-MIA2 carrying MIA2 gene. Transfect the Hep G2 cellls with the vector and detect the expression of MIA2 gene in transfected Hep G2 cells in order to further study the function and molecular signaling pathways MIA2 provide research foundationMethods Primer pairs which contained cutting sites of Bam HI and Sal I endonucleases were designed and used to amplify the MIA2 gene from total RNA of Hep G2 cells by RT-PCR. Both the PCR product of MIA2 gene and plasmids were excised by Bam HI and Sal I endonucleases. The products of enzymes digestion were recovered from agarose gel and then ligated with each other. The recombinant plasmids were transformed into DH5? competent cells prepared by Ca Cl2 method, Doubly digesting of Bam HI and Sal I endonucleases and sequencing were used to identify the integrity and fidelity of MIA2 gene contained in the recombinant plasmid from the positive clones. The correct recombinant plasmid was transfected into Hep G2 cells for the purpose of expressing cloned MIA2 gene. Real-time quantitative PCR, immunocytochemistry, and western blot assays were used to analyze the expression of MIA2 at m RNA and protein levels in the transfected Hep G2 cellls.Results MIA2 gene sequence contained in recombinant plasmid p LVX-IRES-Zs Green1-MIA2 was verified by enzyme digestion as well as sequence analysis. The sequence of inserted MIA2 gene was in accordance with the corresponding sequence found in Gene Bank database, High expression transfected Hep G2 cells under fluorescence microscope and the transfection efficiency of p LVX-IRES-Zs Green1-MIA2 was 60%-70%, high efficient expression of m RNA and protein which could be detected by real-time quantitative PCR, immunocytochemistry, and western blot,respectively.Conclusion The recombinant plasmid p LVX-IRES-Zs Green1-MIA2 has been constructed successfully with highly efficient expression in transfected Hep G2 cellsPART TWO Effect of MIA2 on growth and Akt signaling of human Hepatocellular carcinoma Hep G2 celllineObjective To investigate the MIA2 inhibited the growth of Hep G2 cells, and to explore the molecular mechanism is involved in AKT signaling pathway through upregulation.Method Culture Hep G2 cells in vitro, the transfected p LVX-IRES-Zs Green1 group(NC group) and p LVX-IRES-Zs Green1-MIA2 group(MIA2 group). The activity of cell was determined by MTT assay, the cell apoptosis was detected by flow cytometry, the level of the m RNA was analyed by RT-PCR and the level of the protein was analyed by Western blotting.Results Compared with the control group, MIA2 group was significantly higher than the NC group,AKT1 gene expression level increases.Conclusion MIA2 can susppress the occurrence of liver tumor by up regulating the expression of AKT1,which maybe one mechanism of its anti Antitumor effects.