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目的检测干扰eIF4E基因对宫颈癌细胞中c-myc基因表达和增殖活性的影响,初步探讨e IF4E对宫颈癌细胞的作用机制。方法两种宫颈癌细胞C33A、Hela各分为未转染组(C33A-Mock、Hela-Mock),空载对照组(C33A-NC、Hela-NC)以及e IF4E干扰组(C33A-she IF4E、Hela-she IF4E),用阳离子脂质体Lip2000将上述质粒转染到宫颈癌细胞,收集细胞逆转录合成CDNA后采用qRT-PCR方法检测各组细胞中eIF4E和cmyc基因mRNA相对水平的变化,采用Western blot方法检测各组细胞中上述基因蛋白水平的改变,采用MTT法绘制细胞生长曲线检测各组细胞增殖活性的变化。结果与未转染组细胞相比,C33A-she IF4E细胞的e IF4E mRNA和蛋白水平分别下降(71±2.1)%和(69±2.4)%,c-myc mRNA和蛋白水平分别上调(5.6±0.7)倍和下降(60±2.5)%,细胞种板后1~3 d增殖无明显改变,第4、5、6、7天增殖抑制率分别为(62±1.7)%、(65±1.2)%、(69±1.4)%、(63±1.2)%;Hela-she IF4E细胞的eIF4E mRNA和蛋白水平分别下降(68±1.9)%和(61±2.1)%,c-myc mRNA和蛋白水平分别上调(3.4±0.2)倍和下降(53±1.5)%。结论 eIF4E基因敲减导致宫颈癌细胞c-myc的蛋白表达相应下调,同时显著抑制宫颈癌细胞增殖活性,提示e IF4E通过调控c-myc的蛋白表达进而影响宫颈癌细胞的生物学行为。
Objective To investigate the effect of eIF4E on c-myc gene expression and proliferation in cervical cancer cells and to explore the mechanism of eIF4E on cervical cancer cells. Methods C33A and Hela cells were divided into three groups: untransfected group (C33A-Mock, Hela-Mock), blank control group (C33A-NC, Hela-NC) and eIF4E interference group (C33A-she IF4E, Hela-she IF4E). The plasmid was transfected into cervical cancer cells with lipofectamine Lip2000. The cells were reverse transcribed into CDNA and the relative mRNA and protein levels of eIF4E and cmyc in each group were detected by qRT-PCR. Western blot was used to detect the changes of the above protein levels in each group. The cell growth curve was drawn by MTT assay to detect the changes of cell proliferation activity in each group. Results Compared with untransfected cells, e IF4E mRNA and protein levels in C33A-she IF4E cells were decreased by (71 ± 2.1)% and (69 ± 2.4)%, respectively, and c-myc mRNA and protein levels were increased by 5.6 ± 0.7) and decreased (60 ± 2.5)%, respectively. There was no significant difference in cell proliferation between 1 and 3 days after plating (62 ± 1.7%, 65 ± 1.2, ), (69 ± 1.4)% and (63 ± 1.2)%, respectively. The levels of eIF4E mRNA and protein in Hela-she IF4E cells decreased by 68 ± 1.9% and 61 ± 2.1% Levels were increased (3.4 ± 0.2) and decreased (53 ± 1.5)%, respectively. Conclusion The knockdown of eIF4E causes a down-regulation of c-myc protein expression in cervical cancer cells and significantly inhibits the proliferation of cervical cancer cells. It suggests that eIF4E may affect the biological behavior of cervical cancer cells through regulating the protein expression of c-myc.