论文部分内容阅读
目的采用RNA干扰技术探讨人宫颈癌癌基因-2(HCCR-2)对人食管癌细胞凋亡及增殖周期的影响及其可能的分子机制。方法采用脂质体介导将pGCsi-shHCCR-2与pGCsi质粒转染人食管癌细胞株EC9706,经G418筛选获得稳定抑制HCCR-2表达的食管癌细胞模型;逆转录-聚合酶链反应(RT-PCR)及Western blot检测转染细胞HCCR-2、P21、P27 mRNA与蛋白表达;流式细胞仪检测各组细胞凋亡及增殖周期。结果成功构建稳定抑制HCCR-2表达的食管癌细胞模型。反义组、空载体组和对照组细胞HCCR-2mRNA表达量分别为0.19±0.07、0.43±0.22、0.45±0.21;相应的P21 mRNA表达量分别为0.25±0.09、0.12±0.04、0.11±0.05;相应的P27 mRNA表达量分别为0.28±0.13、0.13±0.05、0.15±0.08;相应的HCCR-2蛋白表达量分别为0.42±0.23、0.88±0.41、0.91±0.46;相应的P21蛋白表达量分别为0.78±0.34、0.36±0.15、0.35±0.17;相应的P27蛋白表达量分别为0.81±0.38、0.41±0.17、0.43±0.24,较其他组,反义组细胞HCCR-2 mRNA及蛋白表达量显著降低(P<0.01),而P21、P27 mRNA及蛋白表达量显著增加(P<0.01)。反义组、空载体组和对照组细胞凋亡率分别为(19.64±3.35)%、(6.75±0.91)%、(6.79±0.98)%,反义组较其他组细胞凋亡显著增加(P<0.01);相应的G0/G1期细胞百分数分别为(56.58±11.37)%、(41.32±8.52)%、(42.65±8.63)%,反义组较其他组处于G0/G1期细胞数显著增加(P<0.01)。结论下调EC9706细胞HCCR-2表达后,细胞凋亡增加,细胞周期出现G0/G1期阻滞,其作用机制与P21与P27表达增加有关。
Objective To investigate the effect of human cervical cancer oncogene-2 (HCCR-2) on the apoptosis and proliferation of human esophageal cancer cells by RNAi and its possible molecular mechanism. Methods The plasmid pGCsi-shHCCR-2 and pGCsi were transfected into human esophageal cancer cell line EC9706 by lipofectamine. The cells were stained with G418 to obtain the esophageal cancer cell model stably inhibiting the expression of HCCR-2. RT-PCR The expressions of HCCR-2, P21 and P27 mRNA and protein were detected by Western blot and flow cytometry. Results The esophageal carcinoma cell model stably inhibiting the expression of HCCR-2 was constructed successfully. The expression of HCCR-2 mRNA in antisense group, empty vector group and control group were 0.19 ± 0.07, 0.43 ± 0.22 and 0.45 ± 0.21, respectively. The corresponding P21 mRNA expression levels were 0.25 ± 0.09, 0.12 ± 0.04 and 0.11 ± 0.05 respectively. The corresponding P27 mRNA expression levels were 0.28 ± 0.13, 0.13 ± 0.05 and 0.15 ± 0.08, respectively. The corresponding HCCR-2 protein levels were 0.42 ± 0.23, 0.88 ± 0.41 and 0.91 ± 0.46, respectively. The corresponding P21 protein expression levels were 0.78 ± 0.34,0.36 ± 0.15,0.35 ± 0.17. The corresponding P27 protein expression levels were 0.81 ± 0.38,0.41 ± 0.17 and 0.43 ± 0.24, respectively. Compared with other groups, the expression of HCCR-2 mRNA and protein in antisense group was significantly decreased (P <0.01), but P21, P27 mRNA and protein expression increased significantly (P <0.01). The apoptotic rates in antisense group, empty vector group and control group were (19.64 ± 3.35)%, (6.75 ± 0.91)%, (6.79 ± 0.98)%, respectively. The apoptosis rate of antisense group was significantly higher than that of other groups <0.01). The percentage of cells in G0 / G1 phase was (56.58 ± 11.37)%, (41.32 ± 8.52)% and (42.65 ± 8.63)%, respectively. (P <0.01). Conclusion The down-regulation of HCCR-2 expression in EC9706 cells increases apoptosis and arrests in G0 / G1 phase of the cell cycle. The mechanism is related to the increased expression of P21 and P27.