特异siRNA抑制宫颈癌细胞中人乳头状瘤病毒16型E6表达的研究

目的 优化HPV-16 E6癌基因特异的U6质粒表达的siRNA，抑制HPV癌基因表达及其对子宫颈癌细胞生长繁殖的影响。方法 选择4个分别针对HPV-16 E6 mRNA外显子和内含子序列为靶序列，合成DNA链，构建表达HPV-16 E6短发卡样dsRNA的重组pSilencer1．0-U6载体，导入HPV-16DNA阳性的宫颈癌细胞株CaSki中，观察该细胞中HPV-16 E6、E7基因表达水平及其蛋白含量的变化，并观察细胞生长被抑制的情况。结果 4种HPV-16 E6 siRNA均能降低宫颈癌细胞CaSki的生长速率。通过细胞生长曲线观察到HPV-16 E6 shRNA表达质粒导入细胞0-96h内，可降低细胞生长速度。荧光定量RT-PCR检测HPV-16 E6 siRNA可使宫颈癌细胞株CaSki中HPV-16 E6、E7基因转录的mRNA水平降低，其中针对E6 mRNA内含子的重组shRNA只抑制E6基因的表达水平。Western blot分析表明，4个HPV-16 E6 siRNA作用72h后，未能检测到宫颈癌细胞中HPV-16 E6蛋白。结论 HPV-16 E6 siRNA能使宫颈癌细胞CaSki生长缓慢；选择针对E6内含子的siRNA作用位点，特异性抑制E6表达；而针对E6外显子的siRNA作用位点，可抑制E6和E7基因的表达，是用于治疗HPV阳性宫颈癌细胞的理想靶位。

Inhibition of human papillomavirus type 16 E6 gene expression with HPV16 E6 specific siRNA in cervical carcinoma cell line

Objective To study the HPV-16 E6 specific shRNA expressed by U6 plasmid to inhibit the expression of HPV oncogene and the growth of cervical cancer cell. Methods We selected 4 target mRNA sequences to the exon and interon of HPV-16 E6 mRNA, and synthesized dsDNA to construct the recombinant pSi-lencer1.0-U6 vector which expresses HPV-16 E6 short hairpin dsRNA. After transfected into HPV-16 DNA positive cervical cancer cell line CaSki, then observe the expression level of HPV-16 E6,E7 genes and their proteins as well as the inhibition effects of cell growth. Results The cellular growth velocity was down-regulated both by 4 HPV-16 E6 specific siRNA in 0-96 h after recombinant pSilencer1.0-U6 vector transfected into CaSki cell. The abundance of HPV-16 E6 E7 mRNA inside CaSki cells were also reduced by the 4 HPV-16 E6 specific siRNA respectively, while 1 HPV-16 E6 specific siRNA targetting to the interon sequence of E6 gene inhibited the HPV-16 E6 mRNA only. HPV-16 E6 protein was undetectable with Western blot assay 72 h later after 4 HPV-16 E6 specific siRNA transfected into CaSki cell. Conclusion It suggested that the growth of cervical cancer cells was inhibited by HPV-16 E6 specific siRNA. The HPV16 E6 specific siRNA targeting to the interon of E6 mRNA can inhibit the expression of HPV-16 E6 gene only, while the HPV-16 E6 specific siRNAs targeting to the exon of E6 mRNA can inhibit the expression of both E6 and E7 genes, which can be used as a good candidates of cancer therapeutical siRNA drug.