抑制HPV18型E7蛋白的表达对Hela细胞生物学特性及miR-204表达的影响

Effects of inhibiting expression of human papillomavirus type18 E7 protein on biological properties of Hela cells and expression of miR-204

目的通过小干扰RNA(siRNA)抑制宫颈癌Hela细胞中人乳头瘤病毒(HPV)18型E7的表达,研究E7蛋白对Hela细胞的生物学特性和miR-204表达的影响和关系,探索HPV致癌的分子机制。方法设计合成靶向HPV18型E7基因的siRNA,转染Hela细胞,抑制HPV18-E7的表达;采用RT-PCR和Western blot分别检测Hela细胞中HPV18-E7的mRNA和蛋白水平,用流式细胞术检测Hela细胞的细胞周期和凋亡,通过荧光实时定量PCR检测miR-204的表达水平。结果 pRNAT-HPVE7与两个对照组比较,明显抑制了Hela细胞HPV18-E7的mRNA(t值分别为2.69、2.46,均P<0.05)和蛋白(t值分别为3.93、4.20,均P<0.05)的表达,细胞周期趋于正常,凋亡率增高(t值分别为-6.87、-6.92,均P<0.05),miR-204表达水平也明显升高(t值分别为0.26、0.22,均P<0.05)。结论抑制HPV18-E7基因的表达能改变Hela细胞的生物学特性和上调miR-204表达。

Objective To study on the effects of E7 protein on biological properties of Hela cells and expression of miR-204 through inhibiting the expression of human papillomavirus type 18 E7（ HPV18-E7） protein in cervical cancer Hela cells with small interfering RNA（ siRNA） so as to explore the molecular mechanism of HPV carcinogenesis. Methods SiRNA targeting HPV18-E7 was designed and synthesized,and siRNA was transfected into Hela cells to inhibit the expression of HPV18-E7. Reverse transcription-polymerase chain reaction（ RT-PCR） and Western blot were used to assess the level of mRNA and protein of HPV18-E7 in Hela cells. In addition,flow cytometry was used to detect cell cycle and apoptosis of Hela cells. Florescent real-time quantitative PCR was used to measure the level of miR-204 expression. Results Compared with two control groups,results showed that pRNAT-HPVE7 could significantly inhibit the expression of mRNA（ t value was 2. 69 and 2. 46,respectively,both P〈0.05） and protein（ t value was 3. 93 and 4. 20,respectively,both P〈0.05） of HPV18-E7 in Hela cells. Besides,cell cycle showed a tendency to be normal while an increase was witnessed in apoptosis rate（ t value was- 6. 87 and- 6. 92,respectively,both P〈0.05）. In addition,a significant increase in miR-204 expression was also seen（ t value was 0. 26 and 0. 22,respectively,both P〈0.05）. Conclusion Inhibited expression of HPV18-E7 genes can lead to changes in the biological properties of Hela cells and up-regulation of miR-204 expression.