CAJ | 학술논문

目的：研究miR-21抑制物在A549细胞球增殖的作用及其相关机制。方法在无血清培养基中培养A549细胞球，形成细胞球后进行CD133＋CD44＋表达检测。实验分为A549细胞球组：未经处理；IHN组：miR-21抑制物转染A549细胞球组；IHN-NC组：miR-21抑制物阴性对照物转染A549细胞球组。 Western印迹检测A549细胞和A549细胞球中DKK2、β-catenin蛋白以及QPCR检测miR-21在两种细胞中的表达；合成miR-21抑制物和阴性对照，分别转染A549细胞球48 h后，应用QPCR检测转染前后 miR-21的表达变化，以及使用 Western印迹分析 DKK2、β-catenin蛋白的表达变化，利用噻唑蓝（ MTT）检测12、24、48 h细胞的增殖能力。结果流式细胞术检测 A549细胞球中CD133＋CD44＋阳性率为65.800％； IHN组miR-21的表达以及β-catenin蛋白的表达较A549细胞球组和IHN-NC组显著下调（ P＜0.05），而DKK2蛋白表达显著升高（ P＜0.05）；MTT结果示IHN组和IHN-NC组在12、24、48 h时抑制率显著高于A549 sphere组（P＜0.05），IHN组在24、48 h时相点抑制率明显高于IHN-NC组（P＜0.05）。结论 miR-21抑制物可有效抑制A549 sphere的增殖，其可能成为治疗肺癌的潜在靶点。
목적：연구miR-21억제물재A549세포구증식적작용급기상관궤제。방법재무혈청배양기중배양A549세포구，형성세포구후진행CD133＋CD44＋표체검측。실험분위A549세포구조：미경처리；IHN조：miR-21억제물전염A549세포구조；IHN-NC조：miR-21억제물음성대조물전염A549세포구조。 Western인적검측A549세포화A549세포구중DKK2、β-catenin단백이급QPCR검측miR-21재량충세포중적표체；합성miR-21억제물화음성대조，분별전염A549세포구48 h후，응용QPCR검측전염전후 miR-21적표체변화，이급사용 Western인적분석 DKK2、β-catenin단백적표체변화，이용새서람（ MTT）검측12、24、48 h세포적증식능력。결과류식세포술검측 A549세포구중CD133＋CD44＋양성솔위65.800％； IHN조miR-21적표체이급β-catenin단백적표체교A549세포구조화IHN-NC조현저하조（ P＜0.05），이DKK2단백표체현저승고（ P＜0.05）；MTT결과시IHN조화IHN-NC조재12、24、48 h시억제솔현저고우A549 sphere조（P＜0.05），IHN조재24、48 h시상점억제솔명현고우IHN-NC조（P＜0.05）。결론 miR-21억제물가유효억제A549 sphere적증식，기가능성위치료폐암적잠재파점。
Objective To study the mechanism and role of miR-21 inhibitors on proliferation of A549 sphere.Methods A549 spheres were cultured in serum-free medium and positive rate of CD 133+CD44+in A549 spheres were detected by FACS .There were A549 sphere, IHN and IHN-NC groups.The expressions of DKK2 andβ-catenin protein and miR-21 were assayed by Western blot and QPCR re-spectively between A549 cells and A549 spheres.miR-21 inhibitiors and negative control were synthetized .A549 spheres were treated with miR-21 inhibitors and negative control respectively for 48 h, and then the expression of miR-21 was detected by Real-time PCR, and the DKK2 andβ-catenin protein expressions were assayed by Western-blot.The inhibition rate of A549 spheres was detected by MTT in 12, 24, 48 h timepoint.Results CD133+CD44+A549 cells were sorted.Positive rate of CD133+CD44+was 65.800%in A549 spheres.Compared with those of A549 sphere and IHN-NC groups , the expressions of miR-21 and β-catenin protein in IHN group were significantly decreased and DKK2 protein expression was higher (P<0.05).Compared with that of A549 sphere group, the proliferation inhibition rate of IHN and IHN-NC group in 12, 24, 48 h were higher (P<0.05 respectively), and the proliferation inhibition rate of IHN was significant higher than that of IHN-NC group(P<0.05).Conclusions miR-21 inhibitors could effectively inhibit the A549 sphere proliferation, and it might be-come a novel target for treatment non small cell lung cancer .