Regulation Of MicroRNAs On Cell Signal Transduction

With great changes in human lifestyle in recent years, the incidence of metabolic and endocrine diseases is rising yearly. Diabetes is one of the most common chronic diseases characterized by disordered glucose metabolism. Clinically, more than 95% diabetic patients suffer from typeⅡdiabetes (T2DM). Recent studies have found that the main reason for typeⅡdiabetes is insulin resistance, which runs through the whole process and plays an important role in typeⅡdiabetes. Although many findings suggest that insulin resistance is the result of congenerous effects including hormonal factors, inflammation and endoplasmic reticulum stress, the specific mechanism of insulin resistance remains unclear. MicroRNAs (miRs) are a large class of 20-25 nucleotides that evolutionarily conserve non-protein-coding small RNAs that repress target gene expression by translational inhibition or mRNA degradation. There is a closely relationship between miRNAs and the development of typeⅡdiabetes. The latest study of Zampetaki et al found that people with diabetes were significantly different in plasma volume and normal expression of miRNAs. One of the miRNAs on the list is blood vessel-promoting miR-126, which is significantly reduced in diabetic patients. More interestingly, these miRNAs are predictive of disease, because their levels are altered in normal individuals who later go on to develop diabetes. These studies suggest that there is a closely relationship between miR-126 and the development of typeⅡdiabetes. However, the relationship between miR-126 and the pathogenesis of diabetes remains unclear. The current study aims to investigate the regulation of miR-126 on INS-1 cell insulin signaling pathway.ObjectiveThe current study aims to investigate the regulation of miR-126 on INS-1 cell insulin signaling pathway in an attempt to provide new theoretical and potential therapeutic targets in typeⅡdiabetes.MethodsMiR-126 target genes were predicted by bioinformatics methods. INS-1 cells were transfected with miR-126 mimics, miR-126 mutation, miRNA control mimics or Si-IRS1 for 48 h and then stimulated with Insulin (100nM) for 12 h. IRS-1 and PIK3R2 protein expression was detected by Western blot. The GAPDH expression was used for protein-level normalization. The transfection efficiency was tested by real-time PCR and fluorescence microscopy. MTT was used to investigate the regulation of miR-126 on INS-1 cell proliferation.ResultsTo understand the role of miR-126 in INS-1 cells, PicTar or BibiServ was used to search for the potential direct mRNA targets of miR-126. It was found that IRS-1 and PIK3R2 3'-UTR contained potential miR-126 binding sites. The free energy of these two potential binding complexes was ?19.3 and ?19.4 kcal/mol. To regulate miRNA expression in the cells, miR-126 mimics were transfected into INS-1 cells, and miRNA expression level was measured by quantitative PCR. The results showed that artificial synthetic miRNA mimics effectively regulated miRNA expression in vitro. To see whether miR-126 could regulate IRS-1 and PIK3R2 expression in INS-1 cells, INS-1 cells were transfected with miR-126 mimics, miR-126 mutation, miRNA control mimics or Si-IRS1 for 48 h and then stimulated with Insulin (100nM) for 12 h. Western blot showed that IRS-1, PIK3R2 and Akt protein expressions were downregulated when either miR-126 mimics or Si-IRS1 was introduced into Insulin-stimulated INS-1 cells. Phospho-Akt (P-Akt) expression was upregulated in a time-dependent manner when INS-1 cells were stimulated with Insulin(100nM) for different time points (0, 1, 5, 10, 30 and 60 min), but Akt protein expression did not change significantly. To explore the functional relevance of miR-126 in INS-1 cells, the influence on proliferation of INS-1 cells was observed by MTT. It was found that the proliferation of miR-126-overexpressed INS-1 cells was impaired compared to miR-CTL in the presence of insulin for 12 h. The 490nm absorbance of miR-126-overexpressed INS-1 cells decreased by 30.05% compared to miR-CTL group.ConclusionsIRS-1 or PIK3R2 is a target of miR-126. MiR-126 decreased the expression of IRS-1, PIK3R2 and downstream Akt protein by acting on the specific target sequence of IRS-1 and PIK3R2 mRNA 3 '-UTR in INS-1 cells. Insulin promoted Akt phosphorylation in INS-1 cells. MiR-126 inhibited IRS-1/PI3K/Akt signaling pathway by targeting on IRS-1 and PIK3R2 mRNA, thereby inhibiting the proliferation of INS-1 cells. These findings present possible therapeutic targets in typeⅡdiabetes. Inflammation is observed in all stages of atherosclerosis, the initial stage of which is characterized by recruitment of leukocytes to activated endothelial cells (ECs). MicroRNAs (miRNAs) are a class of 20-25 nucleotides that evolutionarily conserve non-protein-coding small RNAs that repress target gene expression by translational inhibition or mRNA degradation. The link between miRNA and endothelial function is largely unknown. Northern blot showed that miR-155 and miR-221 were highly expressed in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs). Bioinformatics analysis proposed Ets-1, a key endothelial transcription factor for inflammation and tube formation, as a candidate target for miR-155 and miR-221/222 cluster. The effect was also demonstrated by Western blot. By using Western blot, we also confirmed that angiotensin II type 1 receptor (AT1R) is a target of miR-155 in HUVECs. Quantitative PCR showed that Ets-1 and its downstream genes, including VCAM1, MCP1 and FLT1, were upregulated in angiotensin II-stimulated HUVECs, and this effect was partially reversed by the overexpression of miR-155 and miR-221/222. In addition, by targeting AT1R, miR-155 could also decrease the HUVECs migration in response to Ang II. In summary, HUVECs highly expressed miR-155 may co-target AT1R and Ets-1 while miR-221/222 targets Ets-1, which indirectly regulates the expression of several inflammatory molecules of ECs, and therefore decreases HUVECs migration. These findings suggest possible therapeutic targets in atherosclerosis.