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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-16 DNA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0-96 h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。West- ern blot分析表明,4个HPV-16 E6 siRNA作用72 h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。
Objective To optimize the expression of siRNA targeting U6 plasmid of HPV16 E6 oncogene, inhibit the expression of HPV oncogene and its effect on the growth and proliferation of cervical cancer cells. Methods Four pairs of exon and intron sequences of HPV-16 E6 mRNA were selected as target sequences respectively. DNA strands were synthesized and a recombinant pSilencer1.0-U6 vector encoding HPV-16 E6 short hairpin dsRNA was constructed. HPV-16 DNA positive cervical cancer cell line CaSki, observe the changes of HPV-16 E6, E7 gene expression levels and protein content in the cells, and observe the cell growth is inhibited. Results All four HPV-16 E6 siRNAs could reduce the growth rate of cervical cancer cells CaSki. The cell growth curve was observed HPV-16 E6 shRNA expression plasmid into cells within 0-96 h, can reduce cell growth rate. Fluorescent quantitative RT-PCR detection of HPV-16 E6 siRNA in cervical cancer cell line CaSki HPV-16 E6, E7 gene transcription level decreased, in which E6 mRNA intron-containing recombinant shRNA inhibition of E6 gene expression levels. West-ern blot analysis showed that HPV-16 E6 protein was not detected in cervical cancer cells after four HPV-16 E6 siRNAs were treated for 72 h. Conclusions HPV-16 E6 siRNA can slow down the growth of cervical cancer cell CaSki. The siRNA targeting E6 intron can specifically inhibit the expression of E6. However, siRNA targeting E6 exon can inhibit E6 and E7 Gene expression is the ideal target for the treatment of HPV-positive cervical cancer cells.