siRNA靶向抑制RPL6基因表达对人宫颈癌HeLa细胞增殖及凋亡的影响

目的 探讨小干扰RNA（siRNA）靶向抑制核糖体蛋白L6（RPL6）基因表达对人宫颈癌HeLa细胞增殖及凋亡的影响。方法 优化siRNA转染条件，将2条靶向抑制RPL6基因的siRNA载体片段（siRPL6-1、siRPL6-2）分别高效转染人宫颈癌HeLa细胞（抑制组），同时设转染无义序列（siControl）的空转染组及不进行任何处理的对照组。分别于转染24、48、72、96 h后采用实时定量PCR（qRT-PCR）检测RPL6表达水平以筛选抑制率高的siRPL6载体用于后续实验。噻唑蓝比色（MTT）法检测各组转染24、48、72、96 h后的增殖抑制率，流式细胞术检测各组转染48、96 h后的细胞凋亡和细胞周期情况， Western blotting检测各组转染96 h后的cyclin A、CDK2和p21CIP1表达情况。结果 抑制组的RPL6 mRNA水平均低于空转染组和对照组（P＜0.05），siRPL6-2片段的干扰效率高于siRPL6-1，故后续实验选择siRPL6-2片段；与其余两组相比，抑制组转染后的增殖抑制率、凋亡率及caspase-3活化率均升高，且G0／G1期细胞比例升高，但S期和G2／M期细胞比例均降低，以上差异均有统计学意义（P＜0.05）；抑制组转染后的p21CIP1水平升高，cyclin A和CDK2 水平降低，与空转染组和对照组的差异有统计学意义（P＜0.05）。空转染组和对照组以上指标的差异均无统计学意义（P＞0.05）。结论 通过siRNA抑制RPL6基因表达可抑制增殖并诱导凋亡和细胞周期阻滞，对宫颈癌防治有一定价值。

Effect of siRNA targeting RPL6 gene on the proliferation and apoptosis of human cervical cancer HeLa cells

Objective To explore the effect of siRNA targeting RPL6 gene on the proliferation and apoptosis of human cervical cancer HeLa cells. Methods After the optimization of the transfection conditions， 2 siRNA vector fragments （siRPL6-1， siRPL6-2） targeting RPL6 gene were effectively transfected into HeLa cells of human cervical cancer（inhibition group）， respectively. Meanwhile， there were also empty transfection group whose cells were transfected with the antisense sequence （siControl） and control group without any treatment. The RPL6 expression levels were detected by real time quantitative PCR （qRT-PCR） at 24， 48， 72 and 96 h after transfection， and the siRPL6 vector with the higher inhibition rate was used for the following experiments. Thiazolyl blue （MTT） was used to detect the proliferation inhibition rate at 24， 48， 72， 96 h after transfection. The apoptosis and cell cycle of the cells were detected by flow cytometry after 48 and 96 h. The expressions of CDK2， cyclin A and p21CIP1 after 96 h were detected by Western blotting. Results The level of mRNA RPL6 in the inhibition group was lower than that in the empty transfection group and the control group （P＜0.05）. Since the interference efficiency of the siRPL6 2 fragment was higher than that of the siRPL6-1， so the subsequent trials chose the siRPL6-2 fragment. Compared with other two groups， the proliferation inhibitory rate， apoptosis rate and caspase 3 activation rate together with the proportion of cells in G0／G1 phase increased， but the proportion of cells in S phase and G2／M phase decreased in inhibition group with statistically significant difference （P＜0.05）； After transfection， the levels of p21CIP1 increased but the levels of CDK2 and cyclin A were decreased in inhibition group versus other groups （P＜0.05）. Conclusion The inhibition of RPL6 gene expression by siRNA can inhibit the proliferation and induce apoptosis and cell cycle arrest， and it can be valuable for the prevention and treatment of cervical cancer.