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MiR-19a regulates PTEN expression to mediate glycogen synthesis in hepatocytes.

Dou L, Meng X, Sui X, Wang S, Shen T, Huang X, Guo J, Fang W, Man Y, Xi J, Li J - Sci Rep (2015)

Bottom Line: Here, we define the impact of miR-19a on glycogen synthesis and IL-6-induced reduced glycogenesis in hepatocytes and its underlying mechanisms.We found that over-expression of miR-19a in NCTC 1469 cells and HEP 1-6 cells led to increased activation of the AKT/GSK pathway and synthesis of glycogen, whereas down-regulation of miR-19a impaired AKT/GSK phosphorylation and glycogenesis.Over-expression of miR-19a ameliorated IL-6-induced reduced glycogen synthesis in hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, China.

ABSTRACT
MiR-19a, a member of mir-17-92 microRNA clusters, has been demonstrated to promote cell proliferation and angiogenesis via regulating the PI3K/AKT pathway, the major insulin signaling pathway. However, whether miR-19a plays an important role in glycogen synthesis in hepatocytes remains unknown. Here, we define the impact of miR-19a on glycogen synthesis and IL-6-induced reduced glycogenesis in hepatocytes and its underlying mechanisms. Our studies indicate that miR-19a was down-regulated in the livers of db/db mice and mice injected with IL-6, as well as mouse NCTC 1469 hepatocytes and HEP 1-6 hepatocytes treated by IL-6. We found that over-expression of miR-19a in NCTC 1469 cells and HEP 1-6 cells led to increased activation of the AKT/GSK pathway and synthesis of glycogen, whereas down-regulation of miR-19a impaired AKT/GSK phosphorylation and glycogenesis. Over-expression of miR-19a ameliorated IL-6-induced reduced glycogen synthesis in hepatocytes. Moreover, we identified PTEN as the target of miR-19a by a luciferase assay. Down-regulation of PTEN rescued the effects of miR-19a suppression on the activation of the AKT/GSK pathway and improved glycogenesis in NTC 1469 cells. These findings show for the first time that miR-19a might activate the AKT/GSK pathway and glycogenesis via down-regulation of PTEN expression.

No MeSH data available.


Related in: MedlinePlus

PTEN is identified as a target gene of miR-19a.(a) Several binding sites of miR-19a on 3’-UTR of PTEN, as analyzed by miRNA target prediction databases, including Miranda, TargetScan and PicTar. The fragment of PTEN 3’-UTR from 500 nt to 1600 nt was cloned and inserted into pmiRGLO vector (PTEN500). A luciferase reporter assay was used to assess whether miR-19a can directly bind to the 3’-UTR of PTEN. (b and c) MiR-19a mimics significantly inhibited the luciferase activity in the NCTC1469 cells and HEP 1–6 cells transfected with PTEN500 vector compared with pmiRGLO (left). Transfection with miR-19a mimics dramatically reduced the luciferase activity in the NCTC1469 cells and HEP 1–6 cells transfected with the luciferase reporter vector containing the 3’-UTR of PTEN compared with those transfected with miRNA mimic control (middle). MiR-19a inhibitor did not affect luciferase activity (right). (d and e) Decreased level of PTEN in the NCTC1469 cells and HEP 1–6 cells transfected with miR-19a mimics. (f and g) Increased level of PTEN in the NCTC1469 cells and HEP 1–6 cells transfected with miR-19a inhibitor. Data represent the mean ± S.D. N = 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001by ANOVAtest (vs. control). Full-length blots are presented in the supplementary information (supplementary Figure S4).
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f4: PTEN is identified as a target gene of miR-19a.(a) Several binding sites of miR-19a on 3’-UTR of PTEN, as analyzed by miRNA target prediction databases, including Miranda, TargetScan and PicTar. The fragment of PTEN 3’-UTR from 500 nt to 1600 nt was cloned and inserted into pmiRGLO vector (PTEN500). A luciferase reporter assay was used to assess whether miR-19a can directly bind to the 3’-UTR of PTEN. (b and c) MiR-19a mimics significantly inhibited the luciferase activity in the NCTC1469 cells and HEP 1–6 cells transfected with PTEN500 vector compared with pmiRGLO (left). Transfection with miR-19a mimics dramatically reduced the luciferase activity in the NCTC1469 cells and HEP 1–6 cells transfected with the luciferase reporter vector containing the 3’-UTR of PTEN compared with those transfected with miRNA mimic control (middle). MiR-19a inhibitor did not affect luciferase activity (right). (d and e) Decreased level of PTEN in the NCTC1469 cells and HEP 1–6 cells transfected with miR-19a mimics. (f and g) Increased level of PTEN in the NCTC1469 cells and HEP 1–6 cells transfected with miR-19a inhibitor. Data represent the mean ± S.D. N = 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001by ANOVAtest (vs. control). Full-length blots are presented in the supplementary information (supplementary Figure S4).

Mentions: Three binding sites of miR-19a on the 3'-UTR of PTEN were analyzed by miRNA target prediction databases, including Miranda, TargetScan and PicTar (Fig. 4a). The fragment of PTEN 3'-UTR from 500 nt to 1600 nt was cloned and inserted into pmiRGLO vector (PTEN500). A luciferase reporter assay was used to assess whether miR-19a can directly bind to the 3'-UTR of PTEN. As shown in Fig. 4b,c, over-expression of miR-19a dramatically reduced the luciferase activity in the NCTC1469 cells and HEP 1–6 cells transfected with the luciferase reporter vector containing the 3'-UTR of PTEN. Moreover, the level of PTEN was decreased in the NCTC1469 cells and HEP 1–6 cells transfected with miR-19a mimics (Fig. 4d,e), whereas down-regulation of miR-19a elevated the level of PTEN (Fig. 4f,g), suggesting that miR-19a negatively regulates the expression of PTEN by directly binding to its 3’-UTR.


MiR-19a regulates PTEN expression to mediate glycogen synthesis in hepatocytes.

Dou L, Meng X, Sui X, Wang S, Shen T, Huang X, Guo J, Fang W, Man Y, Xi J, Li J - Sci Rep (2015)

PTEN is identified as a target gene of miR-19a.(a) Several binding sites of miR-19a on 3’-UTR of PTEN, as analyzed by miRNA target prediction databases, including Miranda, TargetScan and PicTar. The fragment of PTEN 3’-UTR from 500 nt to 1600 nt was cloned and inserted into pmiRGLO vector (PTEN500). A luciferase reporter assay was used to assess whether miR-19a can directly bind to the 3’-UTR of PTEN. (b and c) MiR-19a mimics significantly inhibited the luciferase activity in the NCTC1469 cells and HEP 1–6 cells transfected with PTEN500 vector compared with pmiRGLO (left). Transfection with miR-19a mimics dramatically reduced the luciferase activity in the NCTC1469 cells and HEP 1–6 cells transfected with the luciferase reporter vector containing the 3’-UTR of PTEN compared with those transfected with miRNA mimic control (middle). MiR-19a inhibitor did not affect luciferase activity (right). (d and e) Decreased level of PTEN in the NCTC1469 cells and HEP 1–6 cells transfected with miR-19a mimics. (f and g) Increased level of PTEN in the NCTC1469 cells and HEP 1–6 cells transfected with miR-19a inhibitor. Data represent the mean ± S.D. N = 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001by ANOVAtest (vs. control). Full-length blots are presented in the supplementary information (supplementary Figure S4).
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f4: PTEN is identified as a target gene of miR-19a.(a) Several binding sites of miR-19a on 3’-UTR of PTEN, as analyzed by miRNA target prediction databases, including Miranda, TargetScan and PicTar. The fragment of PTEN 3’-UTR from 500 nt to 1600 nt was cloned and inserted into pmiRGLO vector (PTEN500). A luciferase reporter assay was used to assess whether miR-19a can directly bind to the 3’-UTR of PTEN. (b and c) MiR-19a mimics significantly inhibited the luciferase activity in the NCTC1469 cells and HEP 1–6 cells transfected with PTEN500 vector compared with pmiRGLO (left). Transfection with miR-19a mimics dramatically reduced the luciferase activity in the NCTC1469 cells and HEP 1–6 cells transfected with the luciferase reporter vector containing the 3’-UTR of PTEN compared with those transfected with miRNA mimic control (middle). MiR-19a inhibitor did not affect luciferase activity (right). (d and e) Decreased level of PTEN in the NCTC1469 cells and HEP 1–6 cells transfected with miR-19a mimics. (f and g) Increased level of PTEN in the NCTC1469 cells and HEP 1–6 cells transfected with miR-19a inhibitor. Data represent the mean ± S.D. N = 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001by ANOVAtest (vs. control). Full-length blots are presented in the supplementary information (supplementary Figure S4).
Mentions: Three binding sites of miR-19a on the 3'-UTR of PTEN were analyzed by miRNA target prediction databases, including Miranda, TargetScan and PicTar (Fig. 4a). The fragment of PTEN 3'-UTR from 500 nt to 1600 nt was cloned and inserted into pmiRGLO vector (PTEN500). A luciferase reporter assay was used to assess whether miR-19a can directly bind to the 3'-UTR of PTEN. As shown in Fig. 4b,c, over-expression of miR-19a dramatically reduced the luciferase activity in the NCTC1469 cells and HEP 1–6 cells transfected with the luciferase reporter vector containing the 3'-UTR of PTEN. Moreover, the level of PTEN was decreased in the NCTC1469 cells and HEP 1–6 cells transfected with miR-19a mimics (Fig. 4d,e), whereas down-regulation of miR-19a elevated the level of PTEN (Fig. 4f,g), suggesting that miR-19a negatively regulates the expression of PTEN by directly binding to its 3’-UTR.

Bottom Line: Here, we define the impact of miR-19a on glycogen synthesis and IL-6-induced reduced glycogenesis in hepatocytes and its underlying mechanisms.We found that over-expression of miR-19a in NCTC 1469 cells and HEP 1-6 cells led to increased activation of the AKT/GSK pathway and synthesis of glycogen, whereas down-regulation of miR-19a impaired AKT/GSK phosphorylation and glycogenesis.Over-expression of miR-19a ameliorated IL-6-induced reduced glycogen synthesis in hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, China.

ABSTRACT
MiR-19a, a member of mir-17-92 microRNA clusters, has been demonstrated to promote cell proliferation and angiogenesis via regulating the PI3K/AKT pathway, the major insulin signaling pathway. However, whether miR-19a plays an important role in glycogen synthesis in hepatocytes remains unknown. Here, we define the impact of miR-19a on glycogen synthesis and IL-6-induced reduced glycogenesis in hepatocytes and its underlying mechanisms. Our studies indicate that miR-19a was down-regulated in the livers of db/db mice and mice injected with IL-6, as well as mouse NCTC 1469 hepatocytes and HEP 1-6 hepatocytes treated by IL-6. We found that over-expression of miR-19a in NCTC 1469 cells and HEP 1-6 cells led to increased activation of the AKT/GSK pathway and synthesis of glycogen, whereas down-regulation of miR-19a impaired AKT/GSK phosphorylation and glycogenesis. Over-expression of miR-19a ameliorated IL-6-induced reduced glycogen synthesis in hepatocytes. Moreover, we identified PTEN as the target of miR-19a by a luciferase assay. Down-regulation of PTEN rescued the effects of miR-19a suppression on the activation of the AKT/GSK pathway and improved glycogenesis in NTC 1469 cells. These findings show for the first time that miR-19a might activate the AKT/GSK pathway and glycogenesis via down-regulation of PTEN expression.

No MeSH data available.


Related in: MedlinePlus