Molecular Mechanism Of MiR - 148a On Adipogenic Differentiation Of Human Adipose - Derived Mesenchymal Stem Cells

Obesity is a chronic metabolic disease which excess body fat has accumulated or adipose tissue distribution of anomalies. Obesity is a major risk factor for noncommunicable diseases such as:cardiovascular diseases (CVD), hypertension, dyslipidemia, type2diabetes, fatty liver disease and some cancers, which are the fifth leading risk for global deaths. In China, the prevalence of obesity in children, adolescents and adults has dramatically increased over the past2decades, and the popularity is as much as in the developed countries, which represents a major public health problem.At the cellular level, increased adipose tissue mass is due to the proliferation and hypertrophy of adipocytes, with the degree of hypertrophy relative to hyperplasia influencing the percentage of body fat and the metabolic consequences of obesity. Additionally, adipose-derived mesenchymal stem cells or preadipocytes in response to stimulation by multiple factors, such as genetic factor, environmental factor or dietary factor, which may change the potential of proliferation and differentiation that are resulted in the development of obese. Human adipose-derived mesenchymal stem cells (HMSC-Ad) represent a population of self-renewing and multipotent cells that differentiate into adipocytes and play an important role in adipose tissue hyperplasia. Since the molecular pathways in this process are not fully elucidated, investigating the mechanism of adipocyte differentiation in HMSC-Ad may provide a better understanding of the pathogenesis of obesity and perhaps provide a novel therapeutic target for the management of metabolic diseases, such as obesity and diabetes.In recent years, with the rapid development of life science and technology, recent genome-wide studies have shown that the human genome is pervasively transcribed and produces many thousands of regulatory non-protein-coding RNAs (ncRNAs), which regarded as ’junk’ and fulfilled critical roles as transcriptional and post-transcriptional regulators in normal development and physiology and when dysfunctional, underpin disease; the interpretations of the mechanism of development and disease have become increasingly diverse, the ncRNA directly challenged the traditional view of RNA as simply an intermediary between DNA and protein. MicroRNAs (miRNAs) are a class of small, endogenous RNAs of21-25nucleotides (nts) in length, which functions in transcriptional and post-transcriptional regulation of gene expression. Aberrant expression of miRNAs has been implicated in numerous disease states. And more than20miRNA-based therapies are under research. Several preclinical and clinical trials have been initiated for miRNA-based therapeutics. Since miRNA is complementary to a part of one or more messenger RNAs (mRNAs), which will provide a new view in the development of disease. Although miRNA has been part of the research in obesity and adipocyte differentiation, it is minimal compared to the total number of miRNA, and therefore we continue to explore miRNA function and mechanism of obesity-related research, which will bring the great promising future in prevention and treatment of obesity and related metabolic diseases.Previously we used the miRNA array to identify the differential expression of adipocyte specific miRNA in HMSC-Ad and adipocytes. We found that miR-148a was significantly differential expression in adipogenesis. As we know, Dicer is a cellular enzyme required for the processing of pre-miRNA molecules into mature miRNA. Reseachers utilized primary cells isolated from mice bearing Dicer-conditional alleles to study adipogenesis in the presence or absence of miRNA biogenesis; their results reveal that Dicer is required for adipogenic differentiation of mouse embryonic fibroblasts and primary cultures of pre-adipocytes. Thus, to investigate the relationship between miR-148a and the development of obesity, we first measured the expression level of miR-148a in the different obesity model, such as adipocytes, high fat diet induced mouse and obesity subjects. In addition, candidate target genes of miR-148a were examined by bioinformatics analysis, which lays the foundations for the further study in the development of obesity. Additionally, we determined whether miR-148a directly affected adipocyte differentiation and further understood the mechanism underlying the regulation of adipogenesis by miR-148a. Furthermore, promoter analysis of the miR-148a locus indicated that it is an intergenic miRNA with putative multiple adipogenesis-related transcriptional factor binding sites in the promoter region. Thus, our findings provide a basis for the role of miR-148a in adipogenesis that may underlie the development of obesity and associated metabolic disorders.Part Ⅰ:The relationship between miR-148a and obesityObjective:Examination the expression profile of miR-148a in adipocytes, obesity subjects and HFD-induced mice and bioinformatics analysis of miR-148a, so as to investigate the relationship between miR-148a and obesity and to lay foundation and provide theoretical basis for the further studies the role of miR-148a in the development of obesity.Methods:HMSC-Ad, Pre-Ad (human preadipocytes) and3T3-L1preadipocytes were incubated with insulin, dexamethasone,3-isobutyl-l-methylxanthine (MIX) and rosiglitazone, and then were differentiated into mature adipocytes. Abdomen fat biopsies were prospectively collected from patients undergoing surgery, which were divided into two groups based on BMI:lean subjects (17to23.9kg/m2, n=18) and obese or overweight or subjects (BMI≥24kg/m2, n=12). We examined the expression profile of miR-148a in adipocytes, adipose tissue of obesity subject and HFD-induced mice by Realtime PCR; we analyzed the species conservation of miR-148a by miRBase; predicted target genes of miR-148a, and the intersection of the three results as gene set was analyzed by GO analysis and pathway analysis.Results:1) miR-148a was highly expressed in differentiated HMSC-Ad, Pre-Ad and3T3-L1cells;2) miR-148a was expressed higher in overweight or obesity subjects than in normal, which was positively correlated with BMI;3) Epididymal adipose tissue from mice fed the HFD expressed a significant increase in miR-148a expression compared to animals fed the SD (Standard Diet);4) miR-148a is a high conserved miRNA, the functions of the target genes were involved in the Wnt signaling, TGF-β signaling, insulin signaling, adipoctokine signaling, cell cycle and DNA methylation.Conclusion:miR-148a was highly expressed in multiple obesity models, indicating miR-148a was closely related to obesity. The target genes set of miR-148a enrich in multiple biological process were related with the obesity, which lays the foundations for the further study in the development of obesity.Part Ⅱ:The mechanism and the effect of overexpression of miR-148a on adipogenesis in HMSC-AdObjective:To observe the effect of overexpression of miR-148a on adipogenesis in HMSC-Ad and understand the mechanism underlying the regulation of adipogenesis by miR-148a.Methods:HMSC-Ad were transduced with a lentivirus expressing miR-148a or empty virus for48h before transfer to differentiation medium and then difffferentiated into mature adipocytes. Adipogenesis as indicated by oil red O staining, triacylglycerol content and GPDH activity. Realtime PCR and western blotting analyses indicated that the adipocyte-specific factors PPARγ2, C/EBP-α and FABP4. Additionally, canonical Wnt signaling molecular (GSK-3β, p-GSK-3β, β-catenin) protein level was verified by western blotting. Multi-pathway reporters-scan and target assay were using the Dual Luciferase Assay.Results:1) Overexpression of miR-148a promoted adipogenesis as indicated by oil red O staining, triacylglycerol content and GPDH activity. Additionally, Realtime PCR and western blotting analyses indicated that the adipocyte-specific factors PPARy2, C/EBP-a and FABP4increased significantly after transduction with the miR-148a-expressing lentivirus;2) miR-148a increased PPREx3-TK activity approximately, whereas, TOPFlash activity normalized to either Renilla-TK or FOPFlash was inhibited;3) miR-148a inhibited luciferase activity of Wntl and decreased expression of Wntl protein;4) Canonical Wnt signaling was repressed following miR-148a expression, as indicated by the marked reduction of Wntl protein, increased phosphorylation GSK-3β (p-GSK-3β) and reduced nuclear β-catenin;5) Overexpression of miR-148a partially restored differentiation in Wntl-suppressed HMSC-Ad as assessed by lipid accumulation as well as PPARy2, C/EBP-αand Fabp4mRNA expression. Furthermore, down-regulation of endogenous Wntl promoted adipogenesis in HMSC-Adand increased the expression of the adipocyte-related genes PPARγ2, C/EBP-α and Fabp4, which was consistent with the results of miR-148a overexpression in HMSC-Ad.Conclusion:1) Exogenously expression of miR-148a in HMSC-Ad significantly promotes adipogenesis;2) Wntl as a direct translational target of miR-148a, miR-148a regulated Wntl expression at post-transcriptional level and rescued the negative effect of Wntl expression in HMSC-Ad. These findings suggest that miR-148a is up-stream regulator rather than an effector of adipogenesis.Part III:The mechanism of transcription of miR-148a in HMSC-AdObjective:To find the core transcriptional region of miR-148a and clarify the transcriptional mechanism of miR-148a in HMSC-Ad.Methods:The upstream region of miR-148a was examined using the NCBI Mapviewer and UCSC genome browser (http://genome.ucsc.edu/), and the predicted response elements which were related adipocyte differeniation were determined with TFSEARCH; promoter reporter plasmids with cherry protein were transfect into HEK293T cells and then the fluorescence was observed by fluorescence microscopy; promoter activity was measured in transfecting cells with promoter-Firefly luciferase reporter construct by the Dual Luciferase Reporter Assay System; EMSA and ChIP-qPCR nalysis demonstrated that which transcriptional factors could bind to the miR-148a promoter in HMSC-Ad, we examined whether endogenous miR-148a expression was regulated by CREB in HMSC-Ad by Realtime PCR.Results:1) miR-148a was intergenic miRNA. TSS, CpG Island and TFBS existed in the genome upstream and downstream within3kb, which contained CREB, E2F and CEBP response element;2) Analysis of the activity of these response elements using a luciferase reporter plasmid revealed that sequence-2947to-2687(pro-148a2-CREB) is the core promoter region with putative CREB binding sites in the core promoter region;3) EMSA revealed that CREB and E2F bound to promoter regions-2947to-2687and-800to-304, respectively. Additionally, we observed that IP of CREB was enriched320-fold when compared to input DNA and that CREB, bound to the miR-148a promoter sequence;4) Following transduction of HMS C-Ad with a lentivirus expressing CREB, the level of miR-148a increased2.6-fold in comparison to control cells. In contrast, transduction of HMSC-Ad with a lentivirus silencing CREB, the level of miR-148a decreased about70percent when compare to control cells.Conclusion:CREB (a transcriptional factor, which is very closely related with adipocyte differentiation.) can bind to the promoter of miR-148a and influence the endogenious miR-148a expression, suggesting CREB response element was required for miR-148a expression in HMSC-Ad.