miR-4686对A549细胞球增殖的调控作用及其机制研究

Regulation of miR-4686 on proliferation in A549 spheres

目的研究miR-4686对A549细胞球增殖的调控作用及其相关机制。方法培养A549细胞,从中分选出CD133+CD44+A549细胞并置于无血清培养基中培养,形成细胞球后进行CD133+CD44+表达检测。Western blot检测A549细胞和A549细胞球中Wnt5a、Wnt2蛋白的表达;合成miR-4686抑制物和阴性对照,分别转染A549细胞球48 h后,应用Real-time PCR检测转染前后miR-4686的表达变化后,使用Real-time PCR和Western blot分析Wnt5a、Wnt2以及β-catenin mRNA和蛋白的表达变化,利用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CD133+CD44+表达为69.800%;A549细胞球分别转染miR-4686抑制物和阴性对照物后,miR-4686的表达以及Wnt5a、Wnt2和β-catenin mRNA和蛋白的表达较对照组和miR-4686阴性对照物组显著下调(P<0.05);A549细胞球转染miR-4686抑制物后12、24、48 h,其OD值显著低于对照组和miR-4686对照物干预组(P<0.05)。结论 miR-4686介导Wnt信号通路发挥调控A549细胞球增殖的作用,其可成为治疗肺癌的潜在靶点。

Objective To study the mechanism of miR-4686 regulating the proliferation of A549 spheres.Methods CD133＋CD44＋A549 cells were sorted by FACS, and then cultured in serum-free medium. Positive rate of CD133＋CD44＋ A549 spheres were detected by FACS. Wnt5a and Wnt2 protein expression of A549 cells and A549 spheres were assayed by Western blotting. MiR-4686 inhibitors and negative control were synthesized. A549 spheres were treated with miR-4686 inhibitors and negative control separately for 48 h, and then the expression of miR-4686 were detected by real-time PCR. Wnt5a, Wnt2 and β-catenin mRNA and protein expression was assayed by RT-PCR and Western blotting, respectively. The proliferation of A549 spheres was detected by MTT assay at 12, 24 and 48 h.Results CD133＋CD44＋ A549 cells were sorted. The positive rate of CD133＋CD44＋ was 69.800% in A549 spheres. Wnt5a and Wnt2 protein expression was significant decreased in A549 cells, as compared to A549 spheres （P〈0.05）. After treatment with miR-4686 inhibitors, the expression of miR-4686 as well as the mRNA and protein expression of Wnt5a, Wnt2 and β-catenin were significant down-regulated, compared to the control group and miR-4686 negative control group （P〈0.05）. What’s more, OD values at 12, 24 and 48 h were decreased after miR-4686 inhibitor treatment （P〈0.05）.Conclusion MiR-4686 plays an important role in regulation of A549 sphere proliferation through Wnt signal pathway, which may become a novel target for treatment of non-small-cell lung cancer.