The Biological Effects Of Hsa-miR-1908 In Human Adipocytes

Obesity has become a serious public health problem in the world. With economic development and lifestyle changes, China’s obese population increased sharply and there was a younger age trend. Obesity leads to metabolic syndrome, is a serious threat to human health. Mechanism of adipose-derived mesenchymal stem cells, human multipotent adipose-derived stem cells and preadipocytes proliferation and differentiation into mature adipocytes, which is adipogenic differentiation (adipogenesis), has been an important direction of obesity research. Although with a series of key adipogenic gene PPARy, C/EBPa found that people had more understanding on the mechanism of proliferation and adipogenesis, for these targets to carry out clinical drug development has not obtained a good effect. So obesity prevention is still not optimistic, looking for new targets for obesity prevention mechanisms and in-depth study has been one of the positive efforts of the direction of researcher.miR-1908, is a miRNA that expressed lower in adipose-derived mesenchymal stem cells and preadipocytes, and whose expression was significantly upregulated in adipocytes with high-throughput miRNA microarray found by our team previous. As we all know, Dicer is a cellular enzyme required for the processing of pre-miRNA molecules into mature miRNA. With Dicer fat-specific knockout mice can lead to the development of fat abnormalities reported, suggesting an important role of miRNA in adipocyte differentiation. Thus, we first discussed the relationship between miR-1908 and obesity, and then we examined the candidate target genes of miR-1908 by bioinformatic analysis, which provides experimental and theoretical basis for further study in the development of obesity. Additionally, we investigated the biological effects of miR-1908 in hMADSCs proliferation and adipogenesis, and preliminary explored possible mechanisms of those functions. It is expected to provide new clues and potential intervention targets for the prevention and treatment of obesity and its complications.PartⅠ:The relationship between miR-1908 and obesityObjective:By observing miR-1908 expression characteristics in adipocytes, obese or overweight subjects, and regulation in adipocytes by fat-derived cytokines, and miR-1908 biological information analysis, study the relationship between miR-1908 with obesity, to provide theoretical and experimental basis for elucidating the role of miR-1908 in obesity.Methods:Select human adipose tissue-derived HMSC-Ad, human preadipocytes, incubated with insulin, dexamethasone,3-isobutyl-l-methylxanthine (MIX) and rosiglitazone, then were differentiated into mature adipocytes, and intervened with fat-derived cytokines (TNF-a, IL-6, leptin, resistin) and free fatty acids (FFAs). Abdomen fat biopsies were prospectively collected from patients undergoing surgery, which were divided into two groups based on BMI:lean subjects (18.5 to 23.9 kg/m2, n=16)and obese or overweight subjects (BMI≥ 24 kg/m2, n=9). We detected the expression level of miR-1908 in adipocytes and obese or overweight subjects abdomen fat biopsies by Realtime PCR technique; analyzed the species conservation of miR-1908 by miRBase; searched the target genes of miR-1908 in TargetScan, Pictar and miRanda database, and proceed the GO Analysis and Pathway Analysis to the intersection of the three results.Results:1) miR-1908 was highly expressed in differentiated HMSC-Ad and Pre-Ad; 2) The expression of miR-1908 in differentiated Pre-Ad was regulated by fat-derived cytokines and FFAs; 3) miR-1908 was expressed higher in abdomen fat biopsies of obese or overweight subjects than normal; 4) miR-1908 is a conserved miRNA, the functions of the target genes were involved in signal pathways involved in obesity, insulin resistence, preadipocyte proliferation and adipogenesis, such as MAPK signaling pathway, insulin signaling and cell cycle.Conclusion:miR-1908 was highly expressed in adipocytes and regulated by fat-derived cytokines and FFAs. The target genes set of miR-1908 enrich in multiple biological process were related with the obesity, preadipocyte proliferation and adipogenesis, indicating miR-1908 was closely related to obesity.Part II:The effect of overexpression of miR-1908 in hMADSCsObjective:To observe the effect of overexpression of miR-1908 on proliferation, apoptosis, cell cycle and adipogenesis in hMADSCs.Methods:hMADSCs were transduced with a lentivirus expressing miR-1908 or empty virus for 72h before transfer to differentiation medium and then difffferentiated into mature adipocytes with insulin, dexamethasone, MIX and rosiglitazone. Proliferation was detected by CCK-8. Apoptosis and cell cycle were analyzed by flow cytometry. Adipogenesis was indicated by oil red O staining and triacylglycerol content. Realtime PCR and western blotting analyses indicated the adipocyte-specific factors PPARγ and C/EBP-a.Results:1) Overexpression of miR-1908 promoted cell proliferation, and had no effect on apoptosis and facilitated the process of the G1 to S phase in cell cycle, so that more cells can enter into S phase; 2) Although realtime PCR analysis indicated that the adipocyte-specific factors PPARy and C/EBP-a in adipocytes on the 15th day during adipocyte differentiation decreased after transduction with the overexpressing of miR-1908. Overexpression of miR-1908 had no significant effect on lipid droplets, triacylglycerol content and the protein level of PPARy and C/EBP-a.Conclusion:1) Exogenous expression of miR-1908 in hMADSCs significantly promoted cell proliferation; 2) Overexpression of miR-1908 had no significant effect on hMADSCs adipogenesis.Part III:The potential mechanism of miR-1908 in hMADSCsObjective:To preliminaryly discuss the possible mechanism of miR-1908 in hMADSCs.Methods:hMADSCs were transduced with a lentivirus expressing miR-1908 or empty virus for 72h before examined differentially expressed genes in miR-1908 overexpressed cells using cDNA microarrays. Realtime PCR was employed to further verify and validate the results obtained by microarrays.Results:1) The analysis of gene expression profiles indicated that 69 genes were up-regulated and 54 genes were down-regulated in miR-1908 overexpressed hMADSCs; 2) The molecular functions of differential expressed genes were involved in 15 different groups, and the Pathway analysis indicated that overexpression of miR-1908 influenced insulin signaling pathway and mTOR signaling pathway; 3) The expression levels of CORO6, ST14, and KCNC3 were significantly down-regulated in miR-1908 overexpressed Pre-Ad, consistent with the microarray results.Conclusion:1) Overall, the expression patterns obtained by qRT-PCR reflected the results obtained by microarrays, demonstrating the low false positive rate associated with microarrays in this experiment; 2) The mechanism of miR-1908 on the biological function of hMADSCs was complicated, might related with insulin signaling pathway and mTOR signaling pathway.