Hu Xumin, Tang Jianhua, Hu Xuyun, Bao Peng, Pan Jinxin, Chen Zhipeng, Xian Jiaqi
Department of Orthopedics, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.
Department of Orthopedics, People's Hospital of Gaozhou, Gaozhou, China.
Cell Physiol Biochem. 2018;47(2):545-555. doi: 10.1159/000489988. Epub 2018 May 22.
BACKGROUND/AIMS: In this study, the molecular mechanisms of miR-27b and lipoprotein lipase (LPL) that regulate human adipose-derived mesenchymal stem cells (hASCs) adipogenic differentiation were detected.
Microarray analysis was applied to screen for differentially expressed miRNAs and mRNA during hASCs adipocyte differentiation induction. MiR-27b and LPL were found to have abnormal expression. Then, a dual luciferase reporter assay was employed to validate the targeting relationship between miR-27b and LPL. We also utilized qRT-PCR, western blot, cellular immunofluorescence and an oil red O staining assay to analyze the regulation of miR-27b and LPL during adipogenic differentiation.
The microarray analysis demonstrated that, during adipogenic differentiation, miR-27b was down-regulated, while LPL was up-regulated but tended to become stable 14 days after induction. A dual luciferase reporter assay confirmed the negative targeting regulatory relationship between miR-27b and LPL. After overexpressing and silencing miR-27b, LPL was found to be reversely regulated by miR-27b according to qRT-PCR and western blot. The fat-formation-related biomarkers CCAAT-enhancer binding protein α (c/EBPα) and peroxisome proliferator-activated receptors γ (PPARγ) had decreasing levels after over-expressing miR-27b or knockdown of LPL followed by adipogenic differentiation. Meanwhile, the oil red O staining assay revealed that the accumulation of lipid droplets decreased. There was no change in the expression of c/EBPα, PPARγ, or lipid droplet accumulation when overexpressing miR-27b and LPL.
During the adipogenic differentiation of hASCs, miR-27b expression decreased, and LPL expression increased. The abnormal expression of miR-27b and LPL effectively regulated the adipogenic differentiation of hASCs.
背景/目的：本研究检测了miR-27b和脂蛋白脂肪酶（LPL）调控人脂肪间充质干细胞（hASCs）成脂分化的分子机制。
应用基因芯片分析筛选hASCs诱导分化为脂肪细胞过程中差异表达的miRNAs和mRNA。发现miR-27b和LPL表达异常。随后，采用双荧光素酶报告基因检测法验证miR-27b与LPL之间的靶向关系。我们还利用qRT-PCR、蛋白质免疫印迹法、细胞免疫荧光法和油红O染色法分析miR-27b和LPL在成脂分化过程中的调控作用。
基因芯片分析表明，在成脂分化过程中，miR-27b表达下调，而LPL表达上调，但诱导14天后趋于稳定。双荧光素酶报告基因检测法证实了miR-27b与LPL之间的负向靶向调控关系。过表达和沉默miR-27b后，根据qRT-PCR和蛋白质免疫印迹法发现LPL受miR-27b反向调控。过表达miR-27b或敲低LPL后再进行成脂分化，脂肪形成相关生物标志物CCAAT增强子结合蛋白α（c/EBPα）和过氧化物酶体增殖物激活受体γ（PPARγ）水平降低。同时，油红O染色显示脂滴积累减少。过表达miR-27b和LPL时，c/EBPα、PPARγ的表达及脂滴积累均无变化。
在hASCs成脂分化过程中，miR-27b表达降低，LPL表达升高。miR-27b和LPL的异常表达有效调控了hASCs的成脂分化。
