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。
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.
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).
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.