shRNA抑制宫颈癌Hela细胞株HPV18 E6、E7基因表达的研究

Gene Expression of Human Papillomavirus 18 E6,E7 in Hela Cells by shRNA Inhibition

应用短发夹RNA(Short hairpin RNA,shRNA)表达载体抑制宫颈癌Hela细胞株HPV18 E6、E7基因的表达。应用已鉴定的shRNA表达载体pHPV1、pHPV2转染Hela细胞,G418筛选阳性细胞,建立稳定转染细胞株;倒置荧光显微镜检测转染情况;提取细胞内总RNA,RT-PCR方法检测HPV18 E6、E7 mRNA;WesternBlot检测HPV18 E6、E7蛋白表达的变化;采用灰度分析软件对PCR扩增条带与蛋白质条带进行灰度分析。pHPV1实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的31%、38%,E6、E7蛋白分别为阴性对照组的37%、31%;pHPV2实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的54%、77%,E6、E7蛋白分别为阴性对照组的52%、83%。pHPV1、pHPV2表达载体能抑制Hela细胞HPV18 E6、E7的表达,针对外显子区434-452的pHPV1抑制作用更强。

Gene expression vector of short hairpin RNA （shRNA） was used to inhibit HPV （human papillomavirus） 18 type E6, E7 gene in Hela cells to express. The identified shRNA expression vectors pHPV1 and pHPV2 were transfected into Hela cells. The positive cells were screened by G418 and established stable transfected cells. The transfection results were examined by inverted fluorescent microscope. Total RNA inside the cells was extracted and HPV 18 E6, E7 mRNA were detected by RT-PCR. The corresponding protein expression variation was detected by Western Blot. Gray scale analysis software was applied for the gray scale of the PCR amplified bands and protein bands. The results showed that the content of HPV18 E6, E7 mRNA inside cell of pHPV1 tested group were respectively 31%, 38% of negative control group, and E6, E7 protein were respectively 37%, 31% of negative control group. The content of HPV18 E6, E7 mRNA inside cell of pHPV2 tested group were respectively 54% , 77% of negative control group, and HPV E6, E7 protein were 52% , 83% of negative control group. Therefore, shRNA expression vectors pHPV1, pHPV2 could inhibit the expression of Hela cell HPV E6 and E7 gene. pHPV1 inhibition of HPVI aiming at extron 434-452 was even stronger.