CAJ | 학술논문

背景与目的：细胞周期蛋白依赖激酶4(cyclin-dependent kinase 4，CDK4)是调控细胞周期进程的重要激酶之一，有实验报道其在子宫内膜癌中呈高表达，但是其在子宫内膜癌细胞中的生物学功能及其可能机制还不十分明确。本研究旨在通过小干扰RNA(small interfering RNA，siRNA)沉默CDK4表达，并检测其对人子宫内膜癌HEC-1B细胞生物学行为的影响及其可能机制。方法：将化学合成的CDK4-siRNA转染至HEC-1B细胞；实时荧光定量PCR法检测转染前后细胞中CDK4的mRNA表达量变化；Western blot检测转染前后细胞CDK4、视母细胞瘤基因(retinoblastoma gene，Rb)及其下游p-Rb的蛋白表达量的变化；分别采用CCK-8法、流式细胞仪、Transwell肿瘤细胞侵袭实验检测细胞增殖、周期、凋亡以及侵袭能力的变化。结果：转染后HEC-1B细胞中CDK4 mRNA及蛋白表达均明显下降(P<0.01)；抑制CDK4表达后，抑制HEC-1B细胞的增殖及侵袭，转染si-CDK4组细胞发生侵袭数为(117±21)个，而转染si-control组及未处理组分别为(269±39)个和(262±35)个，差异具有统计学意义(P<0.01)；细胞转染后早期凋亡率为(21.7±3.5)%，较未处理组[(12.4±2.1)%]和si-control组[(11.8±1.9)%]明显增加(P<0.01)；细胞周期分布发生变化，G1期比例增加(P<0.01)，S期细胞比例降低(P<0.01)；进一步的Western blot结果显示，抑制CDK4表达后，细胞内p-Rb表达下降，但是总Rb表达无明显变化。结论：针对CDK4基因的特异性小RNA干扰片段能够下调CDK4基因在子宫内膜癌细胞中的表达，抑制肿瘤生物学进程。
배경여목적：세포주기단백의뢰격매4(cyclin-dependent kinase 4，CDK4)시조공세포주기진정적중요격매지일，유실험보도기재자궁내막암중정고표체，단시기재자궁내막암세포중적생물학공능급기가능궤제환불십분명학。본연구지재통과소간우RNA(small interfering RNA，siRNA)침묵CDK4표체，병검측기대인자궁내막암HEC-1B세포생물학행위적영향급기가능궤제。방법：장화학합성적CDK4-siRNA전염지HEC-1B세포；실시형광정량PCR법검측전염전후세포중CDK4적mRNA표체량변화；Western blot검측전염전후세포CDK4、시모세포류기인(retinoblastoma gene，Rb)급기하유p-Rb적단백표체량적변화；분별채용CCK-8법、류식세포의、Transwell종류세포침습실험검측세포증식、주기、조망이급침습능력적변화。결과：전염후HEC-1B세포중CDK4 mRNA급단백표체균명현하강(P<0.01)；억제CDK4표체후，억제HEC-1B세포적증식급침습，전염si-CDK4조세포발생침습수위(117±21)개，이전염si-control조급미처리조분별위(269±39)개화(262±35)개，차이구유통계학의의(P<0.01)；세포전염후조기조망솔위(21.7±3.5)%，교미처리조[(12.4±2.1)%]화si-control조[(11.8±1.9)%]명현증가(P<0.01)；세포주기분포발생변화，G1기비례증가(P<0.01)，S기세포비례강저(P<0.01)；진일보적Western blot결과현시，억제CDK4표체후，세포내p-Rb표체하강，단시총Rb표체무명현변화。결론：침대CDK4기인적특이성소RNA간우편단능구하조CDK4기인재자궁내막암세포중적표체，억제종류생물학진정。
Background and purpose: Cyclin-dependent kinase 4 (CDK4) is a kind of protein kinases regulating the cell cycle progression, which has been reported to be overexpressed in endometrial carcinoma tissues. But the role of CDK4 in endometrial carcinogenesis and relative mechanisms has not been identiifed yet. In this study, we used a small interfering RNA targeting CDK4, and explored the effects of CDK4 on endometrial cancer cells HEC-1B biological function and relative mechanisms.Methods:The chemically synthesized small interfering RNA targeting CDK4 (si-CDK4) was transiently transfected into HEC-1B cells;the quantitative real time-PCR assays and Western blot assays were performed to explore the mRNA and protein expression levels of CDK4 and its downstream genes, Rb and p-Rb, in HEC-1B cells upon transfection;Moreover, the CCK-8, lfow cytometry (FCM) and invasion assays were performed to indentify the effects of si-CDK4 on the proliferation, cell cycle distribution, apoptosis and invasion abilities of HEC-1B cells, respectively. Results:The results showed that the mRNA and protein expressions of CDK4 were suppressed in HEC-1B cells upon transfection with si-CDK4 (P<0.01);Suppression of CDK4 inhibited cell proliferation and invasion of HEC-1B cells;the number of cells migrating through the transwell membrane in si-CDK4 group was 117±21, which was much fewer than the cells in si-control (269±39) and untreated groups (262±35) (P<0.01);the early apoptosis rate of cells treated with si-CDK4 [(21.7±3.5)%] was much higher than the untreated [(12.4±2.1)%] and si-control groups [(11.8±1.9)%] (P<0.01);moreover, suppression of CDK4 increased cells in G1 phase (P<0.01) and correspondingly decreased cells in S phase (P<0.01);further Western blot results showed that suppression of CDK4 down-regulated the expression of p-Rb in cells, but did not inlfuence the expression of total Rb. Conclusion:CDK4-siRNA speciifcally and efifciently blocks the constitutively activated CDK4 in human endometrial cancer cells HEC-1B, resulting in tumor suppression.