【摘要】 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。更多还原

【Abstract】 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 CD133+CD44+in A549 spheres were detected by FACS. There were A549 sphere,IHN and IHN-NC groups. The expressions of DKK2 and β-catenin protein and miR-21 were assayed by Western blot and QPCR respectively 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 and β-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 become a novel target for treatment non small cell lung cancer.更多还原