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 receptor γ (PPARγ), CCAAT/enhanced binding protein α (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/α and 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/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P < 0.01), while the expression levels of PPARγ and CEBP/α proteins 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.
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 protein α，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。
Keywords: Adipogenic differentiation; Bone marrow mesenchymal stem cells; Long non-coding RNA; MIR4697HG.