MicroRNA-27Targets Prohibitin And Impairs Adipocyte Differentiation And Mitochondrial Function In Human Adipose-derived Stem Cells

Object:Prohibitin (PHB) has been reported to play a crucial role in adipocytedifferentiation and mitochondrial function. MicroRNAs (miRNA, miR) comprise alarge family of single-stranded RNAs that decrease gene expression by binding totarget mRNA and leading to translational repression. MiRNAs can regulate a broadspectrum of physiological and pathological progress. In this study, we designedlentivirus transduction and adipose-derived stem cell differentiation to detect thechanges of ASC mitochondria and expression of PHB and adipogenic markers. Wefocused on the effects of miR-27on adipogenesis as well as the direct target gene ofmiR-27, in order to illustrate the regulative mechanism of miR-27duringadipogenesis and provide a new therapeutic strategy for the treatment of obesity.Methods:1. Induce (Insulin, dexamethasone,3-Isobutyl-1-Methylxanthine, and indomethacin)ASC differentiate into mature adipocyte. Isolate RNA to detect the mRNA levelsof PHB and adipogenic markers (PPARγ, aP2) and the levels of miR-27a andmiR-27b at Day0,1/2,1,2,4,7,14.2. When the cell density reached30%, ASCs were transduced with Lenti/miR-Control (negative control), Lenti/miR-27a or Lenti/miR-27b. Three days later,cells were induced to adipocyte differentiation. Isolate RNA to analyze the mRNAlevels of PHB and adipogenic markers (PPARγ, aP2), and extract proteins todetect the expression of PHB protein at Day0,1,4,14. Meanwhile, observe themorphology and function of mitochondria at Day0and Day14.3. A luciferase reporter construct expressing the3’UTR of human PHB (Lenti/Luc-UTR/PHB) was created. Another construct expressing the mutated3’UTR of PHB(Lenti/Luc-UTR/PHB-mut), with a deletion of six nucleotides at the predictedmiR-27target site, was used as a control. ASCs were transduced with Lenti/miR-Control (negative control), Lenti/miR-27a or Lenti/miR-27b and incubated overnight. ASCs were then transduced with Lenti/UTR-Luc/Blank (blank control),Lenti/UTR-Luc/PHB or lenti/UTR-Luc/PHB-mut. Following an additional2daysof incubation, the luciferase activities in ASC were determined by luminometry.4. ASCs were transduced with Lenti/miR-Control (negative control), Lenti/miR-27aor Lenti/miR-27b and incubated overnight. ASCs were then transduced with ofLenti/PHB, Lenti/GFP was used as a control for Lenti/PHB. After an additional2days of incubation, ASCs were induced to differentiation. At day14of adipogenicinduction, the cells were subjected to immunoblot analysis to determine if PHBcould restore adipogenesis that inhibited by miR-27.Results:1. Compared with day0, increases in the mRNA level of PHB, PPARγ, and aP2were observed, while both miR-27a and miR-27b were down-regulated in ASCduring adipogenesis.2. In the adipogenic induction experiments, the protein levels of PHB andadipogenic markers PPARγ, and aP2were decreased upon overexpression ofmiR-27a or miR-27b.3. Lenti/Luc-UTR/Blank was used as a negative control. Results fromco-transduction experiments indicated that the relative luciferase activities inLenti/Luc-UTR/PHB-treated ASC were significantly inhibited by either miR-27aor miR-27b, whereas the luciferase activities in Lenti/Luc-UTR/PHBmut-treatedASC were unaffected. Replenishment of PHB restores the adipogenesis attenuatedby miR-27.4. Instead of normal tubular mitochondria, nearly20%of the miR-27a-ormiR-27b-overexpressing ASC consisted of fragmented mitochondria afteradipogenesis.Conclusion:1. Insulin, dexamethasone,3-Isobutyl-1-Methylxanthine and indomethacin couldinduce ASC differentiate into mature adipocyte.2. MiR-27a and miR-27b repress adipocyte differentiation. 3. Prohibitin targets miR-27in ASC and overexpression of PHB restores theadipogenesis attenuated by miR-27.4. MiR-27impairs the morphology and function of mitochondria in ASC duringadipogenesis.