LncRNA人浆细胞瘤变体易位1介导miR-148b调控脂多糖诱导巨噬细胞自噬的机制研究

Mechanism research of LncRNA PVT1 regulating LPS-induced macrophage autophagy by mediating miR-148b

目的目前LncRNA人浆细胞瘤变体易位1(LncRNA-PVT1)在脂多糖(LPS)诱导的巨噬细胞自噬的相关机制尚不清楚。文章旨在探讨LncRNA-PVT1介导miR-148b对LPS诱导的巨噬细胞自噬的相关机制。方法体外培养小鼠巨噬细胞株RAW264.7,LPS刺激并分别转染PVT1 siRNA、miR-148b mimics、PVT1,分为LPS组(细胞不进行转染)、LPS+si-NC组(转染PVT1质粒阴性对照)、LPS+PVT1 siRNA转染组(转染PVT1 siRNA质粒)、LPS+miR-NC组(转染miR-148b mimics阴性对照)、LPS+miR-148b mimics组(转染miR-148b mimics)、LPS+PVT1-NC+miR-148b mimics组(转染PVT1阴性对照和miR-148b mimics)、LPS+PVT1+miR-148b mimics组(转染PVT1和miR-148b mimics)。同时设置空白组,空白组RAW264.7细胞既不进行LPS处理也不进行转染。采用qRT-PCR检测细胞中LncRNA-PVT1、miR-148b的表达;CCK-8检测细胞活力;免疫荧光染色检测细胞中LC3的表达;ELISA检测细胞中炎症因子水平;双荧光素酶报告系统分析PVT1与miR-148b的靶向关系;蛋白免疫印迹法检测细胞中自噬蛋白表达。结果与空白组比较,LPS组PVT1 mRNA、LC3阳性表达、LC3-II/LC3-I、Beclin-1、TNF-α、IL-6水平显著升高(P<0.05),miR-148b、p62表达显著降低(P<0.05)。与LPS组、LPS+si-NC组比较,LPS+PVT1 siRNA组PVT1 mRNA、p62、TNF-α、IL-6水平显著降低(P<0.05),miR-148b、LC3阳性表达、LC3-II/LC3-I、Beclin-1表达显著升高(P<0.05)。CCK-8实验结果显示,与空白组细胞活力(100.00±0.00)比较,LPS组(73.46±3.25)显著降低(P<0.05);与LPS组细胞活力(73.46±3.25)、LPS+si-NC组(74.15±4.42)比较,LPS+PVT1 siRNA组(90.38±2.16)显著升高(P<0.05)。双荧光素酶报告基因结果表明,PVT1与miR-148b存在靶向关系。与LPS组、LPS+miR-NC组比较,LPS+miR-148b mimics组LC3阳性表达、LC3-II/LC3-I、Beclin-1表达显著上调(P<0.05),p62、TNF-α、IL-6水平显著下调(P<0.05);与LPS+miR-148b mimics组、LPS+PVT1-NC+miR-148b mimics组比较,LPS+PVT1+miR-148b mimics组细胞LC3-II/LC3-I、Beclin-1表达显著下调(P<0.05),p62、TNF-α、IL-6水平显著上调(P<0.05)。结论脓毒症中LncRNA-PVT1靶向miR-148b抑制巨噬细胞自噬,PVT1可能是脓毒症治疗的潜在研究靶点。

Objective At present,the mechanism of plasmacytoma variant translocation 1(LncRNA-PVT1)in lipopolysaccharide(LPS)-induced macrophage autophagy remains unclear.This study aims to investigate the mechanism of LncRNA PVT1 mediated miR-148b on LPS-induced macrophage autophagy.Methods Mouse macrophage strain RAW264.7 was cultured in vitro,and LPS stimulated and transfected with PVT1 siRNA,miR-148b mimics,PVT1,respectively.They were divided into LPS group(cells were not transfected),LPS+si-NC group(transfected with PVT1 plasmid negative control),LPS+PVT1 siRNA transfection group(transfected with PVT1-siRNA plasmid),and LPS+miR NC group(transfected with miR-148b mimics negative control),LPS+miR-148b mimics group(transfected with miR-miR-148b mimics),LPS+PVT1-NC+miR-148b mimics group(transfected with PVT1 negative control and miR-148b mimics),LPS+PVT1+miR-148b mimics group(transfected with PVT1 and miR-148b mimics).A blank group was set,and RAW264.7 cells in the blank group were neither treated with LPS nor transfected.The expression of LncRNA-PVT1 and miR-148b in the cells was detected by qRT-PCR;Cell viability was detected by CCK-8;The expression of LC3 in cells was detected by immunofluorescence staining.The levels of inflammatory cytokines in cells were detected by ELISA.The targeting relationship between PVT1 and miR-148b was analyzed by dual luciferase reporting system.The expression of autophagy protein in cells was detected by Western blot.Results Compared with the blank group,the positive expression of PVT1 mRNA and LC3,the levels of LC3-II/LC3-I,Beclin-1,TNF-α and IL-6 were significantly increased(P<0.05),and the expression of miR-148b and p62 were significantly decreased(P<0.05).Compared with LPS group and LPS+si-NC group,the levels of PVT1 mRNA,p62,TNF-αand IL level 6 in LPS+PVT1-siRNA group were significantly decreased(P<0.05).The positive expression of miR-148b and LC3,and the expression of LC3 II/LC3 I and Beclin 1 were significantly increased(P<0.05).The results of CCK-8 experiment showed that compared with the blank group(100.00±0.00),the cell viability of LPS group(73.46±3.25)was significantly decreased(P<0.05).Compared with LPS group(73.46±3.25)and LPS+si-NC group(74.15±4.42),cell viability in LPS+PVT1 siRNA group(90.38±2.16)was significantly increased(P<0.05).The dual luciferase reporter gene results indicated that PVT1 had a targeting relationship with miR-148b.Compared with LPS group and LPS+miR NC group,LC3 positive expression,LC3-II/LC3-I and Beclin-1 expression in LPS+miR-148b mimics group were significantly up-regulated(P<0.05),and the levels of p62,TNFα and IL 6 were significantly down-regulated(P<0.05).Compared with LPS+miR-148b mimics group and LPS+PVT1-NC+miR-148b mimics group,LC3-II/LC3-I and Beclin-1 expressions in LPS+PVT1+miR-148b mimics group were significantly down-regulated(P<0.05).The levels of p62,TNFα and IL 6 were significantly up-regulated(P<0.05).Conclusion LncRNA-PVT1 can inhibit macrophage autophagy by targeting miR-148b in sepsis,and PVT1 may be a potential research target for the treatment of sepsis.