MiR-34a-5p在MLN8237诱导神经母细胞瘤细胞衰老中的作用机制

目的研究小分子AURKA抑制剂MLN8237诱导神经母细胞瘤细胞衰老的作用机制。方法利用细胞转染的方法改变SK-N-SH细胞中miR-34a-5p的表达水平,通过SA-β-gal染色方法及流式细胞术检测细胞衰老及细胞周期情况。运用CCK8检测不同处理下细胞增殖的变化情况。RT-PCR及蛋白质免疫印迹(Western blot)方法用来检测细胞中相关基因及蛋白的表达情况。结果(1)MLN8237在SK-N-SH细胞中可以通过激活P53/P21通路诱导细胞衰老,同时引起miR-34a-5p表达上调(P值均<0.05)。(2)SA-β-gal染色及流式检测细胞周期显示,过表达miR-34a-5p可以引起SK-N-SH细胞衰老,靶向调控衰老相关靶基因SIRT1的表达,并导致P53、P21蛋白及mRNA表达升高(P值均<0.05),激活P53/P21衰老通路。(3)将miR-34a-5p抑制剂与MLN8237联用后,与单用MLN8237相比可以减少衰老细胞,降低P53、P21的表达(P值均<0.05),并抑制P53/P21衰老通路,且CCK8结果提示,两者联用可以显著减弱MLN8237对SK-N-SH细胞的增殖抑制作用(P值均<0.05)。结论在神经母细胞瘤SK-N-SH细胞中MLN8237是通过激活P53/miR-34a-5p/SIRT1反馈环路来诱导细胞衰老的过程。

The mechanism of miR-34a-5p in MLN8237-induced senescence of neuroblastoma cells

Objective To explore the mechanism of the senescence of neuroblastoma cells induced by the small molecule AURKA inhibitor MLN8237.Methods Cell transfection was used to change the expression level of miR-34a-5p in SK-N-SH cells.Cell senescence and cell cycle were detected by SA-β-gal staining method and flow cytometry.The changes in cell proliferation under various treatments were examined by CCK-8.RT-PCR and Western Blot were used to detect the expression of related genes and proteins in cells.Results(1)MLN8237 could induce cell senescence by activating P53/P21 pathway in SK-N-SH cells,and up-regulate the expression of miR-34a-5p(all P<0.05).(2)SA-β-gal staining and flow cytometry showed that overexpression of miR-34a-5p induced SK-N-SH cell senescence,regulated the expression of senescence-related target gene SIRT1,increased the expression of P53,P21(all P<0.05)and activated the P53/P21 senescence pathway.(3)The combination of miR-34a-5p inhibitor and MLN8237 could reduce senescent cells,down-regulate P53 and P21 expression(all P<0.05),and inhibit the P53/P21 senescence pathway compared with MLN8237 alone.The results of CCK8 also suggested that the combination could alleviate the inhibitory effect on cell proliferation of MLN8237(all P<0.05).Conclusions In neuroblastoma SK-N-SH cells,MLN8237 could induce cell senescence by activating P53/miR-34a-5p/SIRT1 feedback loop.