CAJ | 학술논문

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-2 mRNA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。
목적：채용RNA간우기술탐토인궁경암암기인-2（HCCR-2）대인식관암세포조망급증식주기적영향급기가능적분자궤제。방법채용지질체개도장pGCsi-shHCCR-2여pGCsi질립전염인식관암세포주EC9706，경G418사선획득은정억제HCCR-2표체적식관암세포모형；역전록-취합매련반응（RT-PCR）급Western blot검측전염세포HCCR-2、P21、P27 mRNA여단백표체；류식세포의검측각조세포조망급증식주기。결과성공구건은정억제 HCCR-2표체적식관암세포모형。반의조、공재체조화대조조세포 HCCR-2 mRNA표체량분별위0.19±0.07、0.43±0.22、0.45±0.21；상응적P21 mRNA표체량분별위0.25±0.09、0.12±0.04、0.11±0.05；상응적P27 mRNA표체량분별위0.28±0.13、0.13±0.05、0.15±0.08；상응적HCCR-2단백표체량분별위0.42±0.23、0.88±0.41、0.91±0.46；상응적P21단백표체량분별위0.78±0.34、0.36±0.15、0.35±0.17；상응적P27단백표체량분별위0.81±0.38、0.41±0.17、0.43±0.24，교기타조，반의조세포HCCR-2 mRNA급단백표체량현저강저（P＜0.01），이P21、P27 mRNA급단백표체량현저증가（P＜0.01）。반의조、공재체조화대조조세포조망솔분별위（19.64±3.35）%、（6.75±0.91）%、（6.79±0.98）%，반의조교기타조세포조망현저증가（P＜0.01）；상응적G0/G1기세포백분수분별위（56.58±11.37）%、（41.32±8.52）%、（42.65±8.63）%，반의조교기타조처우G0/G1기세포수현저증가（P＜0.01）。결론하조EC9706세포HCCR-2표체후，세포조망증가，세포주기출현G0/G1기조체，기작용궤제여P21여P27표체증가유관。
ObjectiveTo investigate the effect of human cervical cancer oncogene-2 (HCCR-2) silencing by RNA interference on the apoptosis and cell cycle of esophageal cancer cell line EC9706. MethodspGCsi- shHCCR-2 and pGCsi plasmids were transfected into EC9706 cells. The expression of HCCR-2, P21 and P27 in EC9706 cells were confirmed by RT-PCR and Western blot. The apoptosis and cell cycle arrest were analyzed using flow cytometry. Results The levels of HCCR-2, P21 and P27 mRNA in pGCsi-shHCCR-2, pGCsi and control groups were: HCCR-2 0.19±0.07, 0.43±0.22 and 0.45±0.21, respectively; P21 0.25±0.09, 0.12±0.04 and 0.11±0.05, respectively; P27 0.28±0.13, 0.13±0.05 and 0.15±0.08, respectively. The protein levels in pGCsi-shHCCR-2, pGCsi and control groups were: HCCR-2 0.42±0.23, 0.88±0.41 and 0.91±0.46, respectively; P21 0.78±0.34, 0.36±0.15 and 0.35±0.17, respectively; P27 0.81±0.38, 0.41±0.17 and 0.43±0.24, respectively. The mRNA and protein expression levels of HCCR-2 were significantly reduced, whereas the mRNA and protein expression levels of P21 and P27 were significantly increased in pGCsi-shHCCR-2 transfected group compared to those in the other two groups (P<0.01). The apoptosis rate in pGCsi-shHCCR-2, pGCsi and control groups were(19.64±3.35)%, (6.75±0.91)% and (6.79±0.98)%, respectively. The apoptosis of EC9706 cells wassignificantly increased in pGCsi-shHCCR-2 transfected group compared to those in the other two groups (P<0.01). The percentage of G0/G1phase cells in pGCsi-shHCCR-2, pGCsi and control groups were (56.58±11.37)%, (41.32±8.52)% and (42.65±8.63)%, respectively. The number of cells in the G0/G1phase was significantly increased in pGCsi-shHCCR-2 transfected group compared to the other two groups (P<0.01). Conclusion Down- regulation of HCCR-2 can blocks the cell cycle in the G0-G1 phase and induce cell apoptosis in EC9706 cells by the upregulation of the P21 and P27 expression.