敲减长链非编码RNA MIR4697HG抑制骨髓间充质干细胞成脂向分化

Knockdown of long non-coding RNA MIR4697 host gene inhibits adipogenic differentiation in bone marrow mesenchymal stem cells

目的:初步探究长链非编码RNA(long non-coding RNA,lncRNA)MIR4697宿主基因(MIR4697 host gene,MIR4697HG)对骨髓间充质干细胞(bone marrow stem cells,BMSCs)的成脂向分化调控作用。方法:将BMSCs进行成脂诱导,在不同的时间点(0、1、2、3、5、7、10 d)收集RNA,通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)技术检测成脂分化调控相关的过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)、CCAAT增强子结合蛋白α(CCAAT/enhanced binding proteinα,CEBP/α)、脂联素(adiponectin,ADIPQ)编码基因的mRNA以及lncRNA MIR4697HG的表达水平。为了防止脱靶效应,本研究构建了两条不同序列的MIR4697HG shRNA(shMIR4697HG-1,shMIR4697HG-1)并通过慢病毒感染BMSCs,建立MIR4697HG稳定敲减的BMSCs细胞系。采用油红O染色、蛋白质印迹实验和qRT-PCR等方法检测敲减MIR4697HG对BMSCs成脂分化能力的影响。结果:体外诱导BMSCs成脂向分化时,成脂标志基因PPARγ、CEBP/α和ADIPQ表达量均显著升高,在此过程中lncRNA MIR4697HG表达也明显增加(P<0.01)。慢病毒感染BMSCs 72 h后,荧光显微镜下可以观察到90%以上细胞成功表达绿色荧光蛋白,qRT-PCR结果显示MIR4697HG敲减效率高于60%;BMSCs敲减MIR4697HG后,在BMSCs成脂诱导7 d时,成脂基因PPARγ、CEBP/α和ADIPQ的转录物(mRNA)水平显著下降(P<0.01),同时PPARγ和CEBP/α的蛋白质水平也显著降低(P<0.01)。敲减MIR4697HG的BMSCs成脂向分化能力减弱。结论:lncRNA MIR4697HG对BMSCs成脂向分化有调控作用,敲减MIR4697HG可抑制BMSCs的成脂向分化,提示lncRNA MIR4697HG可能成为治疗骨质疏松症等成脂肪异常疾病的潜在靶点。

Objective:To preliminarily investigate the role of long non-coding RNA(lncRNA)MIR4697 host gene(MIR4697HG)in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods:For adipogenic differentiation,BMSCs were induced in adipogenic media for 10 days.The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptorγ(PPARγ),CCAAT/enhanced binding proteinα(CEBP/α)and adiponectin(ADIPQ)were detected by quantitative real-time polymerase chain reaction(qRT-PCR)at different time points(0,1,2,3,5,7,10 days).The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses.To avoid off-target effect,two target sequences(shMIR4697HG-1,shMIR4697HG-2)were designed.And then cells were induced to differentiate in adipogenic medium.Oil red O staining,Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs.Results:The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation(P<0.01),and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ,CEBP/αand ADIPQ.Observed by fluorescence microscopy,more than 90%transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group,shMIR4697HG-2 group and shNC group transfection for 72 h.And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%.Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ,CEBP/αand ADIPQ were significantly decreased in the MIR4697HG knockdown group(P<0.01),while the expression levels of PPARγand CEBP/αproteins were decreased remarkably as well(P<0.01).Consistently,MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs,which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs.Conclusion:lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs,and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs.These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.