MicroRNA Expression in Alpha and Beta Cells of Human Pancreatic Islets (original) (raw)
Figure 4
Over-expression of β-miRNAs in α-TC6 cells. A.
miR-200c targets the 3′UTR of cMaf. Luciferase reporter assay performed in α-TC6 cells transfected with luciferase firefly reporter vector containing the 3′UTR of mouse cMaf and exogenous miR-200c or irrelevant negative control mimics. Luciferase activity was standardized by the Renilla luciferase, mean ± SD (n = 3), * p = 0.002 (t-test, 2 tails). B. miR-200c inhibits Maf, Gcg expression in α-TC6 cells. 72 hrs overe-xpression of mimic miR-200c (50 nM) inhibits the expression of endogenous cMaf (RNA and protein) and RNA Gcg, (n = 8), * p = 0.0078 and 0.0495 (Gcg), Wilcoxon signed rank test-2 tails. Densitometry analysis of cMaf western blots (n = 5) * p = 0.032 Wilcoxon signed rank test-2 tails. C. miR-200c inhibits Zfpm2 at RNA and protein levels, (n = 8), * p = 0.0078, Wilcoxon signed rank test-2 tails. Densitometry analysis of Zfpm2 western blots (n = 5), * p = 0.032. D. miR-125b inhibits c-Maf and Gcg expression in α-TC6 cells. Over-expression of miR-125b mimics (50 nM) for 72 hs inhibits endogenous cMaf and Gcg mRNAs, (n = 5), * p = 0.05 and 0.002 respectively, paired-2 tailed t test. Densitometry analysis of cMaf western blots (n = 5), *p = 0.001, paired -2 tailed t test. E. miR-182 inhibits cMaf and Gcg expression in α-TC6 cells. Over expression of miR-182 (50 nM) for 72 hs inhibits endogenous cMaf and Gcg mRNA, (n = 6), * p = 0.036 for cMaf and Gcg, Wilcoxon signed rank test-2 tails. Densitometry analysis of cMaf Western blots (n = 3), * p = 0.029. RNA assessment experiments are expressed as RQ = 2−ΔCt, ΔCt = (CtRNA gene−CtRNA Ctrl). Beta actin was used as endogenous control. Results are shown as mean ± SD. For densitometry calculations representative Western blots shown.