慢病毒介导的RNA干扰ppGalNAc-T2基因表达抑制Jurkat细胞增殖和迁移(英文)

Lentivirus-mediated RNA Interference ofppGalNAc-T2 Gene Expression InhibitProliferation and Migrationof Jurkat Cell Line

在肿瘤中,黏蛋白O-糖基化有着重要的生物学功能.控制O-糖基化起始合成的是多肽∶N-乙酰氨基半乳糖转移酶家族,研究该酶家族对阐明O-糖基化在肿瘤中的作用机制有重要的意义.探讨了靶向干扰ppGalNAc-T2基因表达对白血病Jurkat细胞株增殖及迁移的影响.首先合成ppGalNAc-T2特异shRNA干扰及对照序列,将其连接至慢病毒干扰载体YH1;重组载体经双酶切、测序鉴定正确后与包装质粒共转染293T细胞,获得的病毒颗粒经过滤纯化后感染Jurkat细胞,流式细胞分选仪进行细胞分选以获得ppGalNAc-T2基因稳定干扰表达的Jurkat细胞,然后使用RT-PCR和Western blot方法对各组别细胞中ppGalNAc-T2基因表达情况进行分析,以确定ppGalNAc-T2基因表达被有效干扰;进一步利用MTT实验和Transwell实验分析ppGalNAc-T2基因干扰表达对Jurkat细胞增殖及迁移的影响.结果表明,成功构建了靶向干扰ppGalNAc-T2基因表达的慢病毒载体,感染Jurkat细胞后能稳定干扰ppGalNAc-T2基因表达.MTT和Transwell实验研究发现,下调ppGalNAc-T2基因表达对Jurkat细胞增殖和迁移有抑制作用.

Mucin O-glycosylation plays important roles in many carcinogenic events,and it is initiated by the enzymes： UDP-GalNAc∶polypeptide N-acetylgalactosaminyltransferases（ppGalNAc-Ts）.The association of ppGalNAc-T2 expression and Jurkat cells proliferation and migration was investigated.The RNAi and negative control shRNA were synthesized and then inserted into lentivirus vector YH1.After enzyme digestion and sequencing confirmation,each of the recombinant vectors and packaging vectors were cotransducted into 293T cells,and the recombinant lentivirus were packaged.Then Jurkat cells were infected by purified lentivirus,RT-PCR and Western blot showed that ppGalNAc-T2 mRNA and protein expression were remarkably decreased after RNA interference.Furthermore,the proliferation and migration abilities of Jurkat cells were detected.In conclusion,the recombinant lentivirus interfering vector was constructed targeting on ppGalNAc-T2 gene,and decreased expression of ppGalNAc-T2 gene inhibit proliferation and migration of Jurkat cells.