Construction Of RNA Interference Expression Plasmid Of Human UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase 2 And Primary Study On Its Function

Object:Human UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase is a glycosyl- transferase involved in the first step of O-glycan synthesis. It catalyses the transference of GalNAc group from UDP-GalNAc to the threonine (Thr)or serine (Ser) residue within a definite sequence on protein polypeptide chain to form a GalNAcα-O Thr/Ser glycosidic linkage. Previous research revealed that ppGalNAcT maybe concerned with the growth and transfusion of tumor. ppGalNAc-T2 has a widely distribution and substrates spectrum in its family, so it is perhaps the rate-limiting enzyme of O-glycosylation ,have important biological significance so this article chose it to study .This thesis aims to approach the function of ppGalNAc-T2 in infiltration, transfusion and growth of tumor. Methods:To follow the principle of selecting RNA interference target sequence, to utilize www resource, design five potential small interference RNA(siRNA)of ppGalNAc-T2 mRNA. With PCR amplified siRNA endogeny express system with RNA pol III promoter, and transfected in to SGC7901cells. Use RT-PCR to detect the mRNA expression of ppGalNAc-T2 in transfected cells, and select the siRNA with the highest ppGalNAc-T2 inhibit efficacy. Chemical synthesize this fluorescently-labeled RNA Oligo, transfected SGC7901 cells and observe with fluorescence microscope. Use RT-PCR to detect the expression of ppGalNAc-T2, accredited the depressant effect of siRNA to ppGalNAc-T2. Subcloned this siRNA to the RNAi eukaryotic expression plasmid pSilenCircle. Transfected the recombinant plasmid into SGC7901 cells . A series of subcellular clones aiming at the blocking of ppGalNAcT2 gene expression of SGC7901 cells were established by means of G418 selection . Studied the variational characteristics of MMP2, MMP14 and TGF-β1 expression on mRNA and protein level with RT-PCR and Western blot. Detect the cell proliferation by MTT. Examined the cell cycle by flow cytometry.