Wang Ping, Cui Zhu-mei, Luo Bing, Lin Ping
Department of Obstetrics and Gynecology, Afflicated Hospital of Medical College, Qingdao University, Qingdao 266003, China.
Zhonghua Fu Chan Ke Za Zhi. 2006 Mar;41(3):190-3.
To study the effect of small interference RNA on E6, E7 mRNA of human papillomavirus type-18 (HPV18) in cervical cancer cells.
The specific HPV18E6 and E7 siRNA synthesized by in virtro transcription. The cell activity was detected by methyl thiazolyl tetrazolium (MTT) assay to determine the most suitable concentration, then cells were transfected into HPV18 cervical cancer cells by oligofectamine. HPV18E6, E7 mRNA level was examined by semi-quantitative RT-PCR, and the cell cycles were analyzed by flow cytometry.
The activity of the HeLa cells had an obvious suppression after transfection to 0.57 +/- 0.05 and 0.62 +/- 0.04 compared with the contrast of 0.87 +/- 0.05. The expression of HPV18E6 mRNA level was 0.63 +/- 0.04 before transfection, decreased to 0.53 +/- 0.04, 0.46 +/- 0.02, 0.56 +/- 0.03 evidently after transfection at 24, 48, 72 hours. The expression of HPV18E7 mRNA level was 0.66 +/- 0.03 before transfection and decreased to 0.60 +/- 0.05, 0.52 +/- 0.04, 0.59 +/- 0.02 after transfection at 24, 48, 72 hours. The number of cells in G(2) phase was increased after HPV18E6 siRNA transfection from (1.4 +/- 1.2)% to (66.9 +/- 3.5)%, S phase was declined from (39.4 +/- 0.4)% to (0 +/- 5.5)%, and they were (47.2 +/- 0.5)% and (5.6 +/- 4.2)% in E7 group.
RNAi exists in HeLa cells, and its effect has specificity.
研究小干扰RNA对子宫颈癌细胞中人乳头瘤病毒18型（HPV18）E6、E7 mRNA的影响。
通过体外转录合成特异性HPV18E6和E7 siRNA。采用甲基噻唑基四氮唑（MTT）法检测细胞活性以确定最适宜浓度，然后用脂质体转染试剂将其转染至HPV18子宫颈癌细胞中。采用半定量逆转录聚合酶链反应（RT-PCR）检测HPV18E6、E7 mRNA水平，并用流式细胞术分析细胞周期。
与对照组（0.87±0.05）相比，转染后HeLa细胞活性明显受到抑制，分别为0.57±0.05和0.62±0.04。转染前HPV18E6 mRNA水平为0.63±0.04，转染后24、48、72小时明显下降至0.53±0.04、0.46±0.02、0.56±0.03。转染前HPV18E7 mRNA水平为0.66±0.03，转染后24、48、72小时下降至0.60±0.05、0.52±0.04、0.59±0.02。HPV18E6 siRNA转染后G(2)期细胞数量从(1.4±1.2)%增加至(66.9±3.5)%，S期细胞数量从(39.4±0.4)%下降至(0±5.5)%，E7组分别为(47.2±0.5)%和(5.6±4.2)%。
RNA干扰存在于HeLa细胞中，且其作用具有特异性。
