Naa10基因RNA干扰慢病毒载体的构建及其对口腔鳞癌细胞生长的影响

Construction of Lentiviral Vector of Naa10 Small-interfering RNA and Its Effect on Growth of Oral Squamous Cell Carcinoma

目的:构建人N-α-乙酰基转移酶10(N-α-acetyltransferase 10,Naa10)基因有效的慢病毒RNAi载体并转染人口腔鳞癌细胞SCC-15,研究其对口腔鳞癌细胞生长的影响。方法:根据人Naa10基因mRNA序列设计合成3个siRNA片段,转染人口腔鳞癌SCC-15细胞株并进行验证,将最有效的干扰序列克隆至pLV-shRNA载体上,测序正确后进行包装,测定病毒颗粒滴度后,将慢病毒感染SCC-15细胞以测定Naa10的表达情况,并通过生长曲线研究干扰Naa10对SCC-15细胞生长的影响。结果:3个靶点的siNaa10均能有效抑制Naa10基因的表达,其中2号靶点最为有效(P<0.005);重组RNAi质粒pLV-shNaa10经测序证实构建成功,pLV-shNaa10可在293T细胞中成功包装;测定病毒颗粒LV-shNaa10、LV-NC滴度分别为1.2×10~9 TU/mL和1.0×10~9 TU/mL;SCC-15细胞感染LV-shNaa10后,Naa10蛋白表达明显降低(P<0.005);干扰Naa10可促进人口腔鳞癌细胞SCC-15的生长。结论:成功构建了Naa10shRNA慢病毒表达载体,感染人口腔鳞癌细胞SCC-15后,有效抑制了内源性Naa10基因的表达,干扰Naa10可促进SCC-15细胞的生长。

Objective：To construct the effective lentiviral vector of Naa10small-interfering RNA,and transfect it into oral squamous cell carcinoma SCC-15 cells so as to investigate its effect on the growth of SCC-15 cells.Methods：Three pairs of siRNA sequences against different parts of Naa10 mRNA were separately transfected into SCC-15 cells.The transfection efficiency was verified,and then the most effective shNaa10 sequence was cloned into pLV-shRNA.pLV-shNaa10 was verified by sequencing and co-transfected into 293 Tcells to obtain packaged lentivirus particles LV-shNaa10.After viral titer determination,SCC-15 cells were infected with LV-shNaa10 and the expression of Naa10 p was detected by Western Blot.Growth curves were used to study the effect of Naa10 knockdown on the growth of SCC-15 cells.Results：Naa10siRNAs could suppress Naa10 expression and the second siRNA was the most effective（P〈0.005）.LV-shNaa10 and LV-NC harvested from 293 Tcells had a titer of 1.2×10-9 TU/mL and 1.0×10-9 TU/mL,respectively.After infection with LV-shNaa10,the expression of Naa10protein in SCC-15cells was down-regulated（P〈0.005）,which could in turn markedly facilitate the growth of SCC-15cells.Conclusion：The lentivirus-mediated Naa10shRNA was successfully constructed,and knocking down of Naa10could promote the growth of SCC-15cells.