CAJ | 학술논문

目的明确miR-216b是否通过靶向调控蛋白激酶Cα(PKCα)基因的表达来抑制鼻咽癌细胞的增殖和侵袭.方法构建PKCα 3'非编码区(UTR)-荧光素酶报告载体,通过荧光素酶报告系统检测miR-216b对PKCα 3'UTR-荧光素酶活性的影响.将miR-216b模拟物转染鼻咽癌细胞CNE2,采用实时荧光定量PCR和Western blot法检测PKCα mRNA和蛋白的表达水平.将PKCαsiRNA转染CNE2细胞,通过MTS细胞增殖实验和Transwell侵袭实验观察PKCα基因表达下调对CNE2细胞增殖和侵袭的影响.将PKCα重组质粒与miR-216b模拟物共转染CNE2细胞,通过MTS细胞增殖实验检测CNE2细胞的增殖能力.结果双荧光素酶报告检测结果显示,miR-216b能特异性地与PKCα mRNA的3'UTR结合,下调荧光素酶活性,其活性约为转染对照模拟物细胞的62.4％.过表达miR-216b的CNE2细胞中PKCα mRNA和蛋白的表达水平均降低,与对照细胞比较,分别降低49.1％和55.7％.siRNA干扰PKCα表达能抑制CNE2细胞的增殖和侵袭能力,能部分模拟miR-216b的抑瘤功能.过表达PKCα能部分逆转miR-216b对CNE2细胞的增殖抑制作用.结论 miR-216b通过靶向调控PKCα基因的表达来抑制鼻咽癌细胞的增殖和侵袭.
목적 명학miR-216b시부통과파향조공단백격매Cα(PKCα)기인적표체래억제비인암세포적증식화침습.방법 구건PKCα 3'비편마구(UTR)-형광소매보고재체,통과형광소매보고계통검측miR-216b대PKCα 3'UTR-형광소매활성적영향.장miR-216b모의물전염비인암세포CNE2,채용실시형광정량PCR화Western blot법검측PKCα mRNA화단백적표체수평.장PKCαsiRNA전염CNE2세포,통과MTS세포증식실험화Transwell침습실험관찰PKCα기인표체하조대CNE2세포증식화침습적영향.장PKCα중조질립여miR-216b모의물공전염CNE2세포,통과MTS세포증식실험검측CNE2세포적증식능력.결과 쌍형광소매보고검측결과현시,miR-216b능특이성지여PKCα mRNA적3'UTR결합,하조형광소매활성,기활성약위전염대조모의물세포적62.4％.과표체miR-216b적CNE2세포중PKCα mRNA화단백적표체수평균강저,여대조세포비교,분별강저49.1％화55.7％.siRNA간우PKCα표체능억제CNE2세포적증식화침습능력,능부분모의miR-216b적억류공능.과표체PKCα능부분역전miR-216b대CNE2세포적증식억제작용.결론 miR-216b통과파향조공PKCα기인적표체래억제비인암세포적증식화침습.
Objective To elucidate whether miR-216b suppresses cell proliferation and invasion by targeting PKCα,thus to reveal the molecular mechanism that miR-216b functions as a tumor suppressor in nasopharyngeal carcinoma (NPC).Methods PKCα 3' UTR-luciferase vector was constructed and dualluciferase reporter gene assay was employed to examine the effect of miR-216b on luciferase activity.Nasopharyngeal cancer CNE2 cells were transfected with miR-216b mimics,and then qRT-PCR and Western blotting were performed to detect the expressions of PKCa mRNA and protein.The effects of PKCα downregulation on cell proliferation and invasion were assessed after PKCα siRNA were transfected into CNE2 cells.CNE2 cells were cotransfected with miR-216b mimics and PKCα plasmid,and the proliferation of CNE2 cells was assayed using a MTS cell proliferation assay kit.Results The results of dual-luciferase reporter gene assay demonstrated that miR-216b could bind to the 3'-untranslated region (UTR) of PKCα and inhibited the luciferase activity to 62.4％ of that of the mimics control cells.The expressions of PKCα mRNA and protein were significantly down-regulated by 49.1％ and 55.7％,respectively,in comparison with that of the control cells.siRNA-mediated downregulation of PKCα suppressed the proliferation and invasion ability of CNE2 cells,and could partially mimic the tumor-inhibiting effect of miR-216b.Moreover,the overexpressed PKCα may partially reverse the inhibitory effect of miR-216b on proliferation of CNE2 cells.Conclusion miR-216b suppresses cell proliferation and invasion by targeting PKCα in NPC cells.