CAJ | 학술논문

目的在肺腺癌细胞株A549中探讨微小RNA(miRNA)-145调控肺腺癌起始细胞发生及其分子机制.方法采用无血清培养方法富集A549起始细胞微球,脂质体转染miR-145阻遏物及模拟物,进行细胞微球定量分析,流式细胞术检测CD133+细胞比例,Western blot检测上皮细胞间质化(EMT)相关转录因子Snail、Slug、上皮细胞标志E-钙黏蛋白(E-cadherin)、角蛋白18(Cytokeratin 18)及间充质细胞标志波形蛋白(Vimentin)、N-钙黏蛋白(N-cadherin)的蛋白水平.结果与对照组比较,过表达miR-145可显著下调A549起始细胞微球中CD133+细胞比例[(25.39±12.37)％比(43.70±17.58)％,P＜0.05]；Snail、Slug、Vimentin、N-cadherin的蛋白水平明显下调,E-cadherin、Cytokeratin 18的蛋白水平明显上调.抑制miR-145可显著增加A549起始细胞微球CD133+细胞比例[(58.89±23.54)％比(45.60±18.58)％,P＜0.05]；上调Snail、Slug、Vimentin、N-cadherin的蛋白水平,下调E-cadherin、Cytokeratin 18的蛋白水平.结论 miR-145可抑制肺腺癌起始细胞的发生,其机制与调控EMT通路相关.
목적 재폐선암세포주A549중탐토미소RNA(miRNA)-145조공폐선암기시세포발생급기분자궤제.방법 채용무혈청배양방법부집A549기시세포미구,지질체전염miR-145조알물급모의물,진행세포미구정량분석,류식세포술검측CD133+세포비례,Western blot검측상피세포간질화(EMT)상관전록인자Snail、Slug、상피세포표지E-개점단백(E-cadherin)、각단백18(Cytokeratin 18)급간충질세포표지파형단백(Vimentin)、N-개점단백(N-cadherin)적단백수평.결과 여대조조비교,과표체miR-145가현저하조A549기시세포미구중CD133+세포비례[(25.39±12.37)％비(43.70±17.58)％,P＜0.05]；Snail、Slug、Vimentin、N-cadherin적단백수평명현하조,E-cadherin、Cytokeratin 18적단백수평명현상조.억제miR-145가현저증가A549기시세포미구CD133+세포비례[(58.89±23.54)％비(45.60±18.58)％,P＜0.05]；상조Snail、Slug、Vimentin、N-cadherin적단백수평,하조E-cadherin、Cytokeratin 18적단백수평.결론 miR-145가억제폐선암기시세포적발생,기궤제여조공EMT통로상관.
Objective To investigate whether microRNA-145 (miR-145) could regulate the lung adenocarcinoma initiating cells genesis by using A549 cell line,and the possible molecular mechanisms.Methods The initiating cells microspheres were derived from the serum-free-cultured A549 celld.Pre-miR-145 mimics and anti-miR-145 inhibitor were transfected into A549 initiating cells microspheres by Lipofectamine 2000.The number of A549 initiating cell microspheres was counted by light microscopy,and the percent of CD133 + cells wsd measured by flow cytometry.Western blotting was applied for determining the protein levels of epithelial-mesenchymal transition (EMT) related proteins:Snail,Slug (EMT-related transcription factor),Cytokeratin 18,E-cadherin (epithelial markers) and N-cadherin,Vimentin (mesenchymal markers).Results As compared with the control group,the percent of CD133 + cells was remarkably decreased in Pre-miR-145 mimics groups [(25.39 ± 12.37) ％ vs (43.70 ± 17.58) ％,(P ＜0.05)],whereas significantly increased in the anti-miR-145 inhibitor groups [(58.89 ± 23.54) ％ vs (45.60 ± 18.58)％,(P ＜ 0.05)].The overexpression of miR-145 led to the downregulation of Snail,Slug,Vimentin and N-cadherin,but the upregulation of E-cadherin and Cytokeratin 18.However,repressing miR-145 apparently up-regulated the Snail,Slug,Vimentin and N-cadherin,and down-regulated the Ecadherin and Cytokeratin 18.Conclusion miR-145 can inhibit the A549 initiating cells genesis,which might be controbuted to the regulation of EMT pathway.