Construction Of Glycosyltransferase β 3GnT2 ,β3GnT8 Eukaryotic Expression Vector And β 3GnT8 RNA Interference Cell Lines SGC-7901 And Effect Of β 3GnT8 On The MRNA Expression Of Related Genes

Objective: Construction ofβ3GnT8 RNA Interference Cell Lines to probe the effect ofβ3GnT8 on the mRNA expression level ofβ3GnT2,MMP2,TIMP2 , detection of 3GnT2,β3GnT8 mRNA expression level in gastric,glioma and breast cancer cell lines and Construction of their eukaryotic expression vectors.Methods: RT-PCR was used to detectβ3GnT2,β3GnT8 mRNA expression level in three types of cancer cell lines above.β3GnT8 interference vector pSilenCircle-T8Si and control vector were transfected into SGC-7901 cells, stably transfected cells were selected by G418, RT-PCR was used to detectβ3GnT2,β3GnT8,MMP2,TIMP2 m RNA expression level ,β3GnT8 expression on protein level in different transfected SGC-7901 cells was detected by Western blot ;the whole encoding fragments ofβ3GnT2,β3GnT8 with restriction endonuclease recognition sites were got by PCR amplification and their eukaryotic expression vectors p EGFP-C1-β3GnT2,pEGFP-C1-β3GnT8 were constructed.Results:β3GnT2,β3GnT8 were expressed in all cell lines shown above ,a little higher in gastric cancer cell lines. The stable SGC-7901β3GnT8 knockdown cell lines was established by fluorescence microscope observation,RT-PCR and Western blot detection but there was no significant change whenβ3GnT2 mRNA expression level were detected . The whole encoding fragments ofβ3GnT2,β3GnT8 with restriction endonuclease recognition sites were constructed into pEGFP-C1 vector.Conclusion:β3GnT2,β3GnT8 were both expressed in all cell lines detected, the mRNA expression of MMP2 was affected byβ3GnT8, the whole encoding fragments ofβ3GnT2,β3GnT8 were cloned and their eukaryotic expression vectors were constructed successfully ,andβ3GnT8 RNA Interference Cell Lines SGC-7901 was also constructed successfully, so foundation for more extensive functional research was established.