miR-544a对非小细胞肺癌细胞系Wnt信号通路抑制因子GSK3β表达的调控作用

Expression of GSK3β,an inhibitory factor of the Wnt signaling pathway,in non-small cell lung cancer regulated by miR-544a

目的探讨微小RNA-544a(miR-544a)对肺癌干细胞的自我更新能力影响及其可能的机制。方法通过生物信息学预测miR-544a的靶向基因,用荧光素酶报告系统及western blot验证其靶向关系;构建稳定表达miR-544a的非小细胞肺癌(NSCLC)细胞系95C和95D,并用荧光定量PCR验证;用肿瘤球悬浮培养实验检测miR-544a在形成肿瘤球中的作用。结果生物信息学预测发现,miR-544a可以靶向调节Wnt信号通路的抑制子GSK3β,并通过荧光素酶报告系统得到了验证(F=201.37,P<0.01);western blot结果显示,miR-544a可以下调GSK3β的表达,而β-catenin、CD133蛋白表达水平明显上调(F分别为7.73,3.37,9.43,P均<0.01);荧光定量PCR结果显示,95C、95D细胞在转染miR-544a后,miR-544a表达水平增高,分别为20.51±0.97、15.16±1.38(F=418.05,P<0.01)。肿瘤球悬浮培养实验发现,稳定表达miR-544a的细胞能够反复形成具有肿瘤干细胞特性的肿瘤球。结论 miR-544a可以通过直接下调GSK3β的表达来激活Wnt信号通路,以维持肺癌干细胞自我更新能力。

Objective To investigate the effect and its mechanism of miR-544a on the self-renewal ability of lung cancer stem cells. Methods The target genes of miR-544a were predicted by bioinformatics, and were verified with the luciferase reporter system and western blot. In the meanwhile, the non-small cell lung cancer （NSCLC） cell lines expressing miR-544a stably, 95C and 95D, were constructed, and validated by qPCR. The tumor spheroid culture was used to assess the effect of miR-544a on the formation of lung cancer stem cells. Results Bioinformatics analysis indicated that miR-54da could regulate the expression of GSK3β, an inhibitory factor of Wnt signaling pathway, which was verified by the luciferase reporter system （ F = 201.37, P 〈 0.01 ）. western blot revealed that miR-544a could inhibit the expression of GSK3i3, while increase the levels of β-catenin and CD133 proteins in 95C and 95D cells （ F = 7.73, 3.37 and 9.43, respectively, P 〈 0.01 ）. qPCR showed that the expressions of miR-544a increased to 20.51 ± 0.97 and 15.16 ± 1.38 （P 〈0.01 ）, respectively, after miR-544a was transfected into 95C or 95D cells. The tumor spheroid culture revealed that the cells expressing miR-544a stably could form the spheroids with the features of tumor stem cells repeatedly. Conclusion miR- 544a was able to activate the Wnt signaling pathway by reducing the expression of GSK3β, and subsequently maintain the self-renewal ability of lung caner stem ceils.