Construction Of ORF/antisense Expression Plasmid Of Human UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase10and Primary Study On Its Function In LoVo

Background&ObjectHuman 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 GalNAc a-0Thr/Ser glycosidic linkage. Preliminary study prompted the abnormal expression of the enzyme may be related to the uncontrolled growth of the tumor and malignant transformation. ppGalNAc-T10ppGalNAc-T10is a few studies in the 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-T10in cellular proliferation, migration and metastasis of Colorectal Cancer.MethodsPCR synthesis full length of ppGalNAc-T10cDNA containing restriction sites (Xba I,EcoR Ⅰ). pMD18T-GalNAcT10-ORF and pMD18T-GalNAcT10-antisense were build, and the sequence and size of ppGalNAc-T10cDNA fragment were confimed to be correct. After objectibe vector pcDNA3.1was digested, it was separately connected with GalNAcT10-ORF and GalNAcT10-antisense. Sense and antisense eukaryotic expression vectors pcDNA3.1-GalNAcT10-ORF and pcDNA3.1-GalNAc T10-antisense were separately transfected into293T cells. WesternBlot was used to identify the expressed product. pcDNA3.1-T10-ORF、pcDNA3.1-T10-antisense and vacuity carrier pcDNA3.1was separately transfected LoVo. Western blot was used to detect express status of ppGalNAc-T10.Detect the proliferation of LoVo affected by ppGalNAc-T10through CCK8; the change of capability of motility by Wound Healing experiment and invation by transwell experiment. Studied the variational characteristics of TGF-β1and TGF-βR1expression on mRNA and protein level with RT-PCR and Western blot.ResultsThe results of digestion confirmed the right length of inserted DNA, which was the same as the ppGalNAc-T10cDNA, and ppGalNAc-T10protein was highly expressed in293T cells which were transfected with pcDNA3.1-ppGalNAcT10-ORF, and low expressed in293T cells which were transfected with pcDNA3.1-ppGalNAcT10-antis-ense. The protein express status detected by Western blot method in LoVo showed difference. With the increase of expression of ppGalNAc-T10, the cell growth was depressed and the ability of motility and invation decline, TGF-βⅠ and of TGF-βRⅡ on mRNA and protein level increased. however, the LoVo transfected pcDNA-T10-antisense,its cell growth was increased, the ability of motility and invation was also increase, TGF-βⅠ and of TGF-PRⅡ on mRNA and protein level reduce.ConclusionpcDNA3.1-ppGalNAcT10-ORF and pcDNA3.1-ppGalNAcT10-antisense have been successfully constructed and laied the foundation for further in-depth study of the biological function of ppGalNAc-T10. It is possible that the tune-up and tune-down of pp GalNAc-T10have influence on biological proporties of colon tumor. Due to the expression of TGF-βⅠ and TGF-βRⅡ which are associated with tumor proliferation, metastasis and invasion, We speculate that inhibition of the expression of ppGalNAc-T10decreased ability of the overall survival of the malignant cells, while inhibiting the outward migration of malignant cells.