|
|
|
| MiR-26a Promoting 3t3-l1 Adipocyte Differentiation via Targeting Regulatory PTEN |
|
|
|
|
Abstract Abstract As an endogenous small non-coding RNA (ncRNA), microRNAs (miRNAs) regulate gene expression mainly at post-transcriptional level. In recent years, numerous studies have demonstrated that miRNA could have an impact on adipocyte differentiation by modulating the expression of adipogenic transcription factors and signaling molecules. This study showed that the expression of miR-26a gradually rose on day 4 and reached the maximum level on day 8 during 3T3-L1 cell differentiation. In order to investigate the regulatory effects and mechanism of miR-26a on adipogenesis in 3T3-L1 preadipocyte of mouse (Mus musculus), the miR-26a agomir and antagomir were transfected into 3T3-L1 cells to perform miR-26a overexpression and knockdown, respectively. The result showed that miR-26a was significantly overexpressed following agomir transfection on day 2 of differentiation, and the elevated miR-26a expression was maintained until the eighth day after differentiation (P＜0.01). In contrast, miR-26a was effectively inhibited on day 2, 4 and 8 after induction when the antagomir was transfected into 3T3-L1 cells (P＜0.01). And overexpression of miR-26a significantly accelerated the relative mRNA expression of genes associated with adipogenesis such as peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS) and lipoprotein lipase (LPL) (P＜0.01, P＜0.05) on day 2, 4 and 8, and also increased the protein level of PPARγ and FAS (P＜0.05) on day 8 after differentiation. MiR-26a overexpression also led to a notable increase in lipid accumulation. In contrast, inhibition of miR-26a expression decreased the relative mRNA expression (on day 2, 4 and 8) and the protein level (on day 8) of PPARγ and FAS (P＜0.01, P＜0.05) after differentiation. Although there was no significant change in LPL mRNA on day 8 compared to ago-NC group, a significant decrease was detected on day 2 and 4 after differentiation (P＜0.05). Photomicrograph and quantitative analysis of Oil Red O on day 8 of differentiation also revealed that knocking down miR-26a decreased the formation of neutral lipid droplets. Next, to further determine the function of miR-26a, bioinformatics analysis was carried out to predict direct targets of miR-26a, the result showed that the 3' UTR of phosphatase and tensin homolog (PTEN) contained the target sites for miR-26a. By luciferase reporter assay, the firefly luciferase activity was reduced when co-transfection of pmirGLO-PTEN-3' UTR and miR-26a agomirs (P＜0.01), but remained unchanged in other combinations. To better understand the mechanism of miR-26a during adipogenic differentiation in 3T3-L1, the effect of miR-26a on the expression of PTEN had been further validated and the result revealed that overexpression of miR-26a lead to an obvious decrease in both the mRNA (P＜0.01) and protein levels (P＜0.05) of PTEN on day 8 of differentiation. In contrast, when the endogenous miR-26a was knocked down with the synthetic miR-26a antagomirs, the PTEN mRNA (P＜0.01) and protein (P＜0.05) levels were increased compared to the ant-NC group. Together, it was concluded that miR-26a positively regulated 3T3-L1 cell differentiation by directly inhibiting PTEN. Therefore, miR-26a and its target genes may play a role in the pathological progression of obesity, which may provide a novel research direction for biological therapy of obesity-related diseases.
|
|
Received: 09 January 2017
Published: 20 July 2017
|
| Fund:; the National High Technology Research and Development Program of China (863 Program) |
|
|
|
| [1]鞠大鹏，詹丽杏.脂肪细胞分化及其调控的研究进展[J].中国细胞生物学学报, 2010, 32(5):690-695
[2]Ambros V.The functions of animal microRNAs[J].Nature, 2004, 431(7006):350-355
[3]Arias N, Aguirre L, Fernández-Quintela A, et al.MicroRNAs involved in the browning process of adipocytes [J].Journal of Physiology and Biochemistry, 2016, 72(3):1-13
[4]Bartel D P. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function[J].Cell, 2004, 116(2):281-297
[5]Brennecke J, Hipfner D R, Stark A, et al.bantam Encodes a Developmentally Regulated microRNA that Controls Cell Proliferation and Regulates the Proapoptotic Gene hid in Drosophila[J].Cell, 2003, 116(2):281-297
[6]Calle E E, Kaaks R.Overweight, obesity and cancer: epidemiological evidence and proposed mechanisms[J].Nature Reviews Cancer, 2004, 4(8):579-591
[7]Chalhoub N, Baker S J.PTEN and the PI3-kinase pathway in cancer[J].Pathology: Mechanisms of Disease, 2009, 4(1):127-150
[8]Dey N, Bera A, Das F, et al.High glucose enhances microRNA-26a to activate mTORC1 for mesangial cell hypertrophy and matrix protein expression[J].Cellular Signalling, 2015, 27(7):1276-1285
[9]Esquela-Kerscher A, Slack F J.Oncomirs - microRNAs with a role in cancer[J].Nature Reviews Cancer, 2006, 6(4):259-269
[10]Gagnon A, Chen C S, Sorisky A. Activation of protein kinase B and induction of adipogenesis by insulin in 3T3-L1 preadipocytes: contribution of phosphoinositide-3, 4, 5-trisphosphate versus phosphoinositide-3, 4-bisphosphate[J].Diabetes, 1999, 48(4):691-698
[11]Gimeno R E, Klaman L D.Adipose tissue as an active endocrine organ: recent advances[J].Current Opinion in Pharmacology, 2005, 5(2):122-128
[12]He M L, Kung H F, Lu J, et al.MiR-26a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of EZH2[J].Cancer Research, 2011, 71(1):225-233
[13]Huse J T, Brennan C, Hambardzumyan D, et al.The PTEN-regulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis[J].Genes & Development, 2009, 23(11):1327-1337
[14]Kajimoto K, Naraba H, Iwai N.MicroRNA and 3T3-L1 pre-adipocyte differentiation[J].RNA, 2006, 12(9):1626-1632
[15]Kopelman P G.Obesity as a medical problem[J].Nature, 2000, 404(6778):635-643
[16]Li B, Sun H.MiR-26a promotes neurite outgrowth by repressing PTEN expression[J].Molecular Medicine Reports, 2013, 8(2):676-680
[17]Lynn F C.Meta-regulation: microRNA regulation of glucose and lipid metabolism[J].Trends in Endocrinology & Metabolism Tem, 2009, 20(9):452-459
[18]Maehama T, Dixon J E.The Tumor Suppressor, PTEN/MMAC1, Dephosphorylates the Lipid Second Messenger, Phosphatidylinositol 3, 4, 5-Trisphosphate[J].Journal of Biological Chemistry, 1998, 273(22):13375-13378
[19]Nakashima N, Sharma P M, Imamura T, et al.The tumor suppressor PTEN negatively regulates insulin signaling in 3T3-L1 adipocytes[J].Journal of Biological Chemistry, 2000, 275(17):12889-12895
[20]Ono H, Katagiri H, Funaki M, et al.Regulation of phosphoinositide metabolism, Akt phosphorylation, and glucose transport by PTEN (phosphatase and tensin homolog deleted on chromosome 10) in 3T3-L1 adipocytes[J].Molecular Endocrinology, 2001, 15(8):1411-1422
[21]Ortegamolina A, Efeyan A, Lopezguadamillas E, et al.Pten positively regulates brown adipose function, energy expenditure, and longevity[J].Cell Metabolism, 2012, 15(3):382-394
[22]Peng Y, Yu S, Li H, et al.MicroRNAs: emerging roles in adipogenesis and obesity[J].Cellular Signalling, 2014, 26(9):1888-1896
[23]Rajala M W, Scherer P E.Minireview: The adipocyte--at the crossroads of energy homeostasis, inflammation, and atherosclerosis[J].Endocrinology, 2003, 144(9):3765-3773
[24]Rosen E D, Spiegelman B M.Adipocytes as regulators of energy balance and glucose homeostasis[J].Nature, 2006, 444(7121):847-853
[25]Salmena L, Carracedo A, Pandolfi P P.Tenets of PTEN tumor suppression[J].Cell, 2008, 133(3):403-414
[26]Sk?rn M, Naml?s H M, Noordhuis P, et al.Adipocyte differentiation of human bone marrow-derived stromal cells is modulated by microRNA-155, microRNA-221, and microRNA-222[J].Stem Cells & Development, 2012, 21(6):873-883
[27]Stambolic V, Suzuki A, Pompa J L D L, et al.Negative Regulation of PKB/Akt-Dependent Cell Survival by the Tumor Suppressor PTEN[J].Cell, 1998, 95(1):29-39
[28]Sun K, Kusminski C M, Scherer P E.Adipose tissue remodeling and obesity[J].Journal of Clinical Investigation, 2011, 121(6):2094-2101
[29]Tomiyama K, Nakata H, Sasa H, et al.Wortmannin, a specific phosphatidylinositol 3-kinase inhibitor, inhibits adipocytic differentiation of 3T3-L1 cells[J].Biochemical & Biophysical Research Communications, 1995, 212(1):263-269
[30]Wang H, Luo J, Zhang T, et al.MicroRNA-26a/b and their host genes synergistically regulate triacylglycerol synthesis by targeting the INSIG1 gene[J].Rna Biology, 2016, 13(5):500-510
[31]Wang Y, Lee C G.MicroRNA and cancer--focus on apoptosis[J].Journal of Cellular & Molecular Medicine, 2009, 13(1):12-23
[32]Yao J, Wang Y, Wang W, et al.Solexa sequencing analysis of chicken pre-adipocyte microRNAs[J].Agricultural and Biological Chemistry, 2011, 75(1):54-61
[33]Zhu Y, Lu Y, Zhang Q, et al.MicroRNA-26a/b and their host genes cooperate to inhibit the G1/S transition by activating the pRb protein[J].Nucleic Acids Research, 2011, 40(10):4615-4625
|
|
|
|
