GPC6基因RNA干扰病毒载体的构建及在鼻咽癌传代细胞株中的表达

Construction of a Lentivirus interfering vector carrying GPC6 Gene with RNA interfering and its expression in nasopharyngeal carcinoma cells CNE-2Z

目的构建磷脂酰基醇蛋白聚糖-6(glypican-6,GPC6)的RNA干扰(RNA interference,RNAi)慢病毒载体,转染人鼻咽癌细胞株CNE-2Z细胞,鉴定沉默效应。方法设计GPC6基因特异性RNA干扰靶序列,与经Agel和EcorI酶切后的GV115载体连接,产生重组慢病毒质粒GV115,筛选阳性克隆,测序鉴定,与慢病毒包装载体共转染293T细胞,荧光/绝对定量(qPCR)法测定滴度。慢病毒感染鼻咽癌细胞株CNE-2Z细胞,RT-PCR检测靶基因的沉默效率。结果双酶切鉴定GPC6基因干扰病毒载体,证实短发夹RNA正确插入慢病毒载体,DNA测序证实插入序列正确,293T细胞包装慢病毒后,其GPC6基因慢病毒载体的病毒滴度为2.0×108 TU/mL,干扰效率为80%。shRNA干扰鼻咽癌CNE-2Z细胞GPC6基因后,RT-PCR、结果显示感染后GPC6基因的mRNA水平显著下降,证明重组慢病毒对CNE-2Z细胞中GPC6基因有明显敲减作用。结论成功构建针对鼻咽癌细胞株CNE-2Z细胞株的GPC6基因RNA干扰慢病毒载体,并获得GPC6基因稳定干扰的鼻咽癌细胞株,为进一步研究GPC6基因在鼻咽癌发生过程中的作用奠定基础。

Objective To construct a lentiviral vector carrying GPC6（glypican-6）gene with RNA interfering（RNAi）and transfect to human nasopharyngeal carcinoma cell line CNE-2 Zcells,and to identify the silencing effect.Methods Specific RNA interference target sequence forGPC6 was designedand connected with GV115 vector digestedby Agel and EcorI enzyme.Recombinant lentivirus plasmid GV115 was establishedand positive clones and sequences were screened.Lentiviral packaging vector were transfected into 293 Tcellsto product and amplify lentivirus.The titer of recombinant lentiviral was determined by fluorescence/absolute quantitative（qPCR）method.After the infection oflentivirus mediated siRNA-GPC6 of CNE-2 Zcell lines,the efficiency of silence of GPC6 were observed by RT-PCR.Results Double enzymeidentification siRNA-GPC6 showed that the shRNA sequence was successfully inserted into the lentiviral vectors.The titer of RNAi virus was 2.0×10^8 TU/mL and the efficiency of infection virus of GPC6 was approximately 80%after the infection of lentivirus mediated siRNA-GPC6.The results of RT-PCR showed that mRNA level of GPC6 genewas significantly decreased,which proved that the recombinant lentivirus had obvious silence GPC6 gene expression in human nasopharyngeal carcinoma cell line CNE 2 Zcells.Conclusion A lentiviral vector targeting human GPC6 gene,capable of stable GPC6 gene knockdown in CNE-2 Zcells,has been successfully constructed,which is foundationto further study the role of GPC6 gene in the pathogenesis of nasopharyngeal carcinoma.