METTL3-m6A途径抑制肺动脉平滑肌细胞增殖的实验研究

Role of METTL3-dependent m6A in inhibiting proliferation of pulmonary artery smooth muscle cells

目的探索甲基转移样酶3(METTL3)依赖的6-甲基腺嘌呤(m6A)修饰在体外调控肺动脉平滑肌细胞增殖中的功能。方法培养SD大鼠肺动脉平滑肌细胞(PASMCs),采用siRNA敲低METTL1的表达。根据转染siRNA的不同分为siNC组(对照siRNA转染组)与siMETTL1组(METTL3 siRNA转染组)。两组PASMCs分别经过siNC及siMETTL1转染后加入含20%FBS的DMEM完全培养液诱导细胞发生增殖。通过试剂盒比色法检测两组PASMCs的m6A总体修饰差异、通过CCK-8细胞活性检测及EdU细胞增殖实验检测两组PASMCs增殖能力改变。结果与siNC组比较,siMETTL3组METTL1的基因表达[(1.0±0.1)vs(0.4±0.1)]及蛋白表达[(1.81±0.05)vs(1.35±0.09)]发生显著下调,差异均有统计学意义(P<0.05);且与siNC组比较,siMETTL3组PASMCs的m6A修饰发生显著下调[(0.10±0.01)vs(0.03±0.01)]、细胞活力显著下降[(0.35±0.01)vs(0.11±0.01)]、Ed U+细胞数显著降低[(48.22±2.92)vs(30.29±1.78)],差异均有统计学意义(P<0.05)。结论敲低METTL3的表达可下调m6A修饰并抑制PASMCs的增殖功能。

Objective To investigate the role of methyltransferase-like 3(METTL3)dependent N6-methyladenine(m6 A)modification regulates the proliferation of pulmonary artery smooth muscle cells.Methods SD rat pulmonary artery smooth muscle cells(PASMCs)were cultured,and siRNAs were used to knock down METTL1 expression.The PASMCs were divided into siNC group(control siRNA transfected group)and siMETTL1 group(METTL3 siRNA transfected group)according to the different siRNAs.After transfection,the DMEM cultured medium containing 20%FBS were added to induce PASMCs proliferation.The differences in the global m6 A levels of PASMCs in the two groups were detected by the colorimetric assay kit,and the changes in the proliferation ability of PASMCs in the two groups were detected by CCK-8 assay and Ed U assay.Results Compared with the siNC group,significant down-regulation of METTL1 gene expression and protein expression were observed in the siMETTL3 group:(1.0±0.1)vs(0.4±0.1),(1.81±0.05)vs(1.35±0.09).Significant downregulation of global m6 A level with(0.10±0.01)vs(0.03±0.01),decreased cell viability with(0.35±0.01)vs(0.11±0.01),and decreased Ed U+cells with(48.22±2.92)vs(30.29±1.78)were observed in the siMETTL3 group.Conclusion Knockdown of METTL3 can downregulate m6 A and inhibit the proliferation of PASMCs.