基于Syk/Ras/c-fos信号轴探讨miR-324-5p对HBZY-1细胞增殖的影响

Effect of miR-324-5p on proliferation of HBZY-1 cells based on Syk/Ras/c-fos signal axis

目的:探讨微小RNA-324-5p(miR-324-5p)对各组HBZY-1细胞增殖的影响及其机制。方法:采用MTT法检测各组HBZY-1细胞增殖活性,流式细胞术检测细胞凋亡情况,RT-qPCR检测HBZY-1细胞内miR-324-5p、Syk、Ras、MEK1、MEK2、ERK1、ERK2以及c-fos的表达水平,免疫荧光检测HBZY-1细胞中Syk/Ras/c-fos信号轴相关表达蛋白及对其进行亚细胞定位。结果:与LPS组相比,转染miR-324-5p-inhibitor后,miR-324-5p的表达水平显著下降,促进HBZY-1细胞增殖,Syk、Ras、MEK1、MEK2、ERK1、ERK2以及c-fos的表达水平均显著升高,Syk/Ras/c-fos信号轴被激活。p-Syk、Ras、p-MEK1/2、p-ERK1/2以及c-fos主要在细胞质中表达。结论:miR-324-5p-inhibitor可通过激活Syk/Ras/c-fos信号轴促进HBZY-1细胞的增殖,表明miR-324-5p可能参与了慢性肾小球肾炎(CGN)中肾小球系膜细胞(GMC)增殖过程,初步探讨了CGN的发病机制。

Objective:To explore the effect of miR-324-5p on the proliferation of HBZY-1 cells and its mechanism.Methods:MTT method was used to detect the proliferation of HBZY-1 cells in each group.Detection of apoptosis by flow cytometry.The expression levels of miR-324-5p,Syk,Ras,MEK1,MEK2,ERK1,ERK2 and c-fos were detected by RT-qPCR.Immunofluorescence detection of Syk/Ras/c-fos signaling axis-related expressed proteins in HBZY-1 cells and their subcellular localization.Results:Compared with LPS group,the expression of miR-324-5p decreased significantly and promoted the proliferation of HBZY-1 cells after transfection of miR-324-5p-inhibitor.The expression levels of Syk,Ras,MEK1,MEK2,ERK1,ERK2 and c-fos were significantly increased,and Syk/Ras/c-fos signal axis was activated.p-Syk,Ras,p-MEK1/2,p-ERK1/2 and c-fos were mainly expressed in the cytoplasm.Conclusion:miR-324-5p-inhibitor can promote the proliferation of HBZY-1 cells by activating Syk/Ras/c-fos signal axis,indicating that miR-324-5p may be involved in the proliferation of glomerular mesangial cells(GMC)in chronic glomerulonephritis(CGN),and preliminarily explore the pathogenesis of CGN.