Effects of FTO on m6A and proliferation of mouse mesangial cells
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摘要:
目的：构建表达脂肪与肥胖相关（fat mass and obesity associated，FTO）基因的重组质粒，并检测其对小鼠肾小球系膜细胞（mice mesangial cell，MMC）N6-甲基腺嘌呤（m6A）修饰及增殖能力的影响。方法：PCR法扩增FTO基因片段，并将其插入表达载体 pCMV-MCS-EGFP质粒中以构建重组质粒pCMV-FTO。将目的质粒pCMV-FTO及对照质粒pCMV分别转染MMC，采用qRT-PCR法检测FTO mRNA表达水平，蛋白质印迹法检测细胞FTO蛋白和相关增殖标志物的表达，CCK8法检测细胞增殖，m6A RNA甲基化定量试剂盒检测m6A含量。结果：菌落PCR鉴定以及测序结果证实重组质粒pCMV-FTO构建成功。RT-qPCR及蛋白印迹结果显示转染目的质粒pCMV-FTO后FTO表达明显增加。过表达FTO后，m6A含量、细胞增殖水平及细胞周期蛋白D1（Cyclin D1）、增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）蛋白水平均显著下降。结论：FTO可以降低 MMC中m6A修饰水平及抑制细胞增殖。
Abstract:
Objective：This study aims to construct a recombinant plasmid expressing fat mass and obesity associated（FTO）gene and to detect its effects on m6A modification and proliferation of mouse mesangial cells（MMCs）. Methods：The FTO gene fragment was amplified by PCR and inserted into the expression vector pCMV-MCS-EGFP plasmid to construct the recombinant plasmid pCMV-FTO. The target plasmid pCMV-FTO and the control plasmid pCMV were transfected into MMCs，respectively，and the mRNA expression of FTO was measured with real-time quantitative PCR（RT-qPCR）. The total protein was extracted，and FTO，EGFP，proliferation markers of Cyclin D1 and PCNA were detected by Western blotting. The proliferation of MMCs were studied with CCK8 method. The m6A content was measured using the m6A RNA methylation quantification kit. Results：Colony PCR identification and sequencing confirmed that the recombinant plasmid pCMV-FTO was successfully constructed. The results showed that the mRNA and protein level of FTO was significantly increased after transfection of the target plasmid pCMV-FTO. After overexpression of FTO，m6A content，the proliferation of MMCs and Cyclin D1，PCNA protein levels decreased significantly. Conclusion：FTO can reduce the level of m6A modification and inhibit cell proliferation in MMCs.
