LCMT1过表达对M2型巨噬细胞极化的影响

Effect of leucine carboxyl methyltransferase 1 overexpression on polarization of M2 macro-phages

目的:通过慢病毒载体构建亮氨酸羟基甲基转移酶1(LCMT1)过表达THP-1细胞株,探讨LCMT1对THP-1细胞和THP-1源性巨噬细胞M2型极化的调控作用。方法:利用慢病毒技术,构建LCMT1过表达载体。设置blank组、OE-control组和OE-LCMT1组。荧光显微镜观察各组细胞的绿色荧光,细胞计数试剂盒(CCK-8)法检测各组细胞增殖情况,实时荧光定量PCR和免疫印迹法检测LCMT1 mRNA和蛋白表达水平以及PP2Ac甲基化相关蛋白表达水平。将THP-1细胞经100 nmol/L佛波酯(PMA)处理48 h后诱导分化为M0型巨噬细胞;M0型巨噬细胞经20 ng/mL白细胞介素4(IL-4)刺激48 h,诱导分化为M2型巨噬细胞。实时荧光定量PCR检测M2型巨噬细胞极化标志物c型甘露糖受体1(MRC-1)、CC趋化因子配体22(CCL22)、树突状细胞特异性细胞间黏附分子结合非整合素(CD209)的mRNA表达。结果:与blank组相比,OE-control和OE-LCMT1组表达绿色荧光,OE-control组的增殖曲线下降(P<0.001),LCMT1和PP2Ac甲基化相关蛋白表达水平差异无统计学意义(P>0.05);与OE-control组相比,OE-LCMT1组增殖曲线下降(P<0.05),LCMT1和PP2Ac甲基化蛋白表达升高(P<0.05),PPME-1和PP2Ac去甲基化蛋白表达降低(P<0.01),总PP2Ac蛋白表达差异无统计学意义(P>0.05)。与Blank M0组相比,Blank M2组的M2极化标志物MRC-1、CD209、CCL22 mRNA水平升高(P<0.05);与OE-control M2组比较,OE-LCMT1 M2组的M2极化标志物MRC-1、CD209、CCL22 mRNA水平降低(P<0.05)。结论:成功构建了LCMT1过表达的THP-1细胞株,LCMT1过表达可抑制THP-1细胞的增殖;维持总PP2Ac蛋白水平,增强PP2Ac甲基化并伴随PPME-1和PP2Ac去甲基化水平降低;下调M2型巨噬细胞极化标志物的表达,抑制M2型巨噬细胞极化。

Objective:To establish the THP-1 cell lines with leucine carboxyl methyltransferase 1(LCMT1)overexpression by lentiviral vector and study the regulating effect of LCMT1 on THP-1 cells and M2 polarization of THP-1-derived macrophages.Methods:Lentiviral technology was used for the construction of LCMT1 over-expression vectors.Blank group,OE-control group,and OE-LCMT1 group were set up.The green fluorescence of each group of cells was observed by fluorescence microscope.The proliferation levels of each group of cells were detected by the cell counting kit-8(CCK-8)assay.Real-time fluorescence quantitative PCR(RT-qPCR)and western blotting were carried out to evaluate LCMT1 mRNA and protein expression level,and the expression lev-el of PP2Ac methylation-associated protein.THP-1 stable transformation cells were treated by 100 nmol/L phor-bol myristate acetate(PMA)for 48 hours and induced to polarize into M0 macrophages,and M0 macrophages were induced to polarize into M2 macrophages by 20 ng/mL interferon-4(IL-4)for 48 hours.The mRNA expression of mannose receptor C type 1(MRC-1),C-C motif chemokine ligand 22(CCL22)and dendritic cell-specific ICAM-3 grabbing non-integrin(CD209)in the polarization markers of M2 macrophages were detected by RT-qPCR.Results:Compared with the blank group,the expression of green fluorescence was expressed in the OE-control group and the OE-LCMT1 group,the proliferation curve was decreased in the OE-control group(P<0.001),and the expression levels of LCMT1 and PP2Ac methylation-associated protein differences were not statistically significant in the OE-control group(P>0.05).Compared with the OE-control group,the proliferation curve of the OE-LCMT1 group was decreased(P<0.05),the expression of LCMT1 and PP2Ac methylated proteins was increased(P<0.05),the expression of PPME-1 and PP2Ac demethylated proteins was decreased(P<0.01)and the expression of total PP2Ac protein differences was not statistically significant in the OE-LCMT1 group(P>0.05).Compared with the blank M0 group,the mRNA expression levels of MRC-1,CD209,and CCL22 of M2 polarization markers were increased in the blank M2 group(P<0.05).Compared with the OE-control M2 group,the mRNA expression levels of MRC-1,CD209,and CCL22 of M2 polarization markers were reduced in the OE-LCMT1 M2 group(P<0.05).Conclu-sion:The THP-1 cell lines with LCMT1 overexpression are successfully constructed.LCMT1 overexpression can inhibit the proliferation of THP-1 cells,maintain the total PP2Ac protein levels,enhance the PP2Ac methyla-tion with concomitant reduction in PPME-1 and PP2Ac demethylation,down-regulate the polarization markers of M2 macrophages,and inhibit the polarization of M2 macrophages.