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- 2017
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
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Abstract:
摘要 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1.2×109 TU/mL和1.0×109 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Naa10 shRNA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
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