MicroRNA-20a-5p contributes to hepatic glycogen synthesis through targeting p63 to regulate p53 and PTEN expression

Abstract

Recently, it is implicated that aberrant expression of microRNAs (miRs) is associated with insulin resistance. However, the role of miR-17 family in hepatic insulin resistance and its underlying mechanisms remain unknown. In this study, we provided mechanistic insight into the effects of miR-20a-5p, a member of miR-17 family, on the regulation of AKT/GSK pathway and glycogenesis in hepatocytes. MiR-20a-5p was down-regulated in the liver of db/db mice, and NCTC1469 cells and Hep1-6 cells treated with high glucose, accompanied by reduced glycogen content and impaired insulin signalling. Notably, inhibition of miR-20a-5p significantly reduced glycogen synthesis and AKT/GSK activation, whereas overexpression of miR-20a-5p led to elevated glycogenesis and activated AKT/GSK signalling pathway. In addition, miR-20a-5p mimic could reverse high glucose-induced impaired glycogenesis and AKT/GSK activation in NCTC1469 and Hep1-6 cells. P63 was identified as a target of miR-20a-5p by bioinformatics analysis and luciferase reporter assay. Knockdown of p63 in the NCTC1469 cells and the Hep1-6 cells by transfecting with siRNA targeting p63 could increase glycogen content and reverse miR-20a-5p inhibition-induced reduced glycogenesis and activation of AKT and GSK, suggesting that p63 participated in miR-20a-5p-mediated glycogenesis in hepatocytes. Moreover, our results indicate that p63 might directly bind to p53, thereby regulating PTEN expression and in turn participating in glycogenesis. In conclusion, we found novel evidence suggesting that as a member of miR-17 family, miR-20a-5p contributes to hepatic glycogen synthesis through targeting p63 to regulate p53 and PTEN expression.

Keywords: PTEN; glycogen synthesis; miR-20a-5p; p53; p63.

Figure 1

Down‐regulation of miR‐20a‐5p is accompanied by reduced glycogen synthesis. The levels of miR‐20a‐5p and other five members of miR‐17 family including miR‐20b‐5p, miR‐106a‐5p, miR‐106b‐5p, miR‐17‐5p and miR‐93‐5p were measured in the liver of db/db mice (A). The level of miR‐20a‐5p was detected in patients with NAFLD (B). Murine NCTC 1469 and Hep1‐6 hepatocytes were stimulated with 33.3 mmol/l glucose for 48 hrs, 0.25 mmol/l palmitate for 24 hrs, 10 nmol/l IL‐6 or 10 nmol/l TNF‐α for 24 hrs respectively. The level of miR‐20a‐5p was determined in both cells (C). The changes of miR‐17 family members were detected in NCTC 1469 and Hep1‐6 cells stimulated with 33.3 mmol/l glucose for 48 hrs (D). Genes related to hepatic glycogen synthesis, glycogen level (E and F) and activation of AKT and GSK (G and H) were analysed in NCTC 1469 and Hep1‐6cells treated with 33.3 mmol/l glucose for 48 hrs. Data represent the mean ± S.D. N = 5 mice or N = 3 independent experiments. *P < 0.05; **P < 0.01 (versus control).

Figure 2

MiR‐20a‐5p contributes to glycogenesis in hepatocytes. The levels of miR‐20a‐5p (A), glycogen synthesis (B and C) and phosphorylation of AKT and GSK (D and E) were analysed in NCTC1469 cells and Hep1‐6 cells transfected with miR‐20a‐5p inhibitor. The transfected efficiency of miR‐20a‐5p mimic was measured in NCTC1469 cells and Hep1‐6 cells (F). Moreover, miR‐20a‐5p mimic was transfected in NCTC1469 cells and Hep1‐6 cells treated with 33.3 mmol/l glucose for 48 hrs. The levels of glycogen synthesis (G and H) and phosphorylation of AKT and GSK (I and J) were measured. Data represent the mean ± S.D. N = 3 independent experiments. *P < 0.05; **P < 0.01;***P < 0.001 (versus control).

Figure 3

P63 is identified as a target of miR‐20a‐5p. A binding site of miR‐20a‐5p on the 3′UTR of p63 was analysed by TargetScan (A). The 3′UTR of p63 containing the predicted binding site was cloned into pGLOmiR vector. The luciferase activity in the 293T cells transfected with pmirGLO‐p63‐3′UTR or pmirGLO‐p63‐3′UTR mutant and miR‐20a‐5p mimic was tested by Dual‐luciferase reporter assay (B). The p63 expression was measured in NCTC146 cells and Hep1‐6 cells transfected with miR‐20a‐5p mimic (C and D) or miR‐20a‐5p inhibitor (E and F). In addition, the fluorescence intensity of p63 was attenuated in the NCTC1469 cells treated with miR‐20a‐5p mimic for 48 hrs, as shown by immunofluorescence assay (G). Scale bar represents 20 μm. Data represent the mean ± S.D. N = 3 independent experiments. *P < 0.05; **P < 0.01(versus control).

Figure 4

Knockdown of p63 improves glycogen synthesis. Two specific siRNA targeting p63 was selected (A). Genes related to hepatic glycogen synthesis, glycogen content (B and C) and phosphorylation levels of AKT and GSK (D and E) were measured in the NCTC1469 cells and the Hep1‐6 cells transfected with siRNA targeting p63. Moreover, p63 knockdown could reverse miR‐20a‐5p inhibition‐induced reduced glycogenesis and activation of AKT and GSK (F and G). Data represent the mean ± S.D. N = 3 independent experiments. *P < 0.05; **P < 0.01(versus control).

Figure 5

P63 regulates expression of PTEN by directly binding to p53. A direct interaction between p63 and p53 was observed by co‐immunoprecipitation (A). The levels of p63, p53 and PTEN protein were measured in the liver of db/db mice (B), NCTC1469 cells (C) and Hep1‐6 cells (D) treated with high glucose as well as NCTC1469 cells (E) and Hep1‐6 cells (F) transfected with siRNA targeting p63. Overexpression of miR‐20a‐5p led to decreased protein levels of p53 and PTEN (G and H), whereas inhibition of miR‐20a‐5p increased the expression of p53 and PTEN (I and J). Transfection of miR‐20a‐5p mimic significantly decreased the fluorescence intensity of p53 and PTEN, as shown by immunofluorescence assay (K). Moreover, overexpression of miR‐20a‐5p in NCTC1469 cells and Hep1‐6 cells could reverse high glucose‐induced increased expression of p63, p53 and PTEN (L and M). Moreover, p53 knockdown could reverse miR‐20a‐5p inhibition‐induced reduced expression of genes related to hepatic glycogen synthesis such as UPG2, G6PC and GBE1 (N) and hepatic glycogen synthesis (O), and activation of AKT and GSK (P) in the NCTC1469 cells treated by miR‐20a‐5p inhibition. Scale bar represents 20 μm. Data represent the mean ± S.D. N = 5 mice or N = 3 independent experiments. *P < 0.05; **P < 0.01 (versus control).

Figure 6

Proposed mechanisms by which miR‐20a‐5p contributes to glycogenesis in hepatocytes. MiR‐20a‐5p targets p63 to regulate p53 and PTEN expression, in turn contributes to glycogenesis in hepatocytes.