MicroRNA-7 regulates the mTOR pathway and proliferation in adult pancreatic β-cells

Abstract

Elucidating the mechanism underlying the poor proliferative capacity of adult pancreatic β-cells is critical to regenerative therapeutic approaches for diabetes. Here, we show that the microRNA (miR)-7/7ab family member miR-7a is enriched in mouse adult pancreatic islets compared with miR-7b. Remarkably, miR-7a targets five components of the mTOR signaling pathway. Further, inhibition of miR-7a activates mTOR signaling and promotes adult β-cell replication in mouse primary islets, which can be reversed by the treatment with a well-known mTOR inhibitor, rapamycin. These data suggest that miR-7 acts as a brake on adult β-cell proliferation. Most importantly, this miR-7-mTOR proliferation axis is conserved in primary human β-cells, implicating miR-7 as a therapeutic target for diabetes.

FIG. 1.

miR-7a is the major miR-7/7ab microRNA family member in mouse pancreatic β-cells. Relative mRNA levels of mature miR-7 (7a and 7b; upper panels) and primary miR-7 (7a-1 and 7a-2; lower panels) in cultured Min6 cells (A) and wild-type islets isolated from 6- to 7-week-old C57/Bl6 mice (B), as determined by quantitative RT-PCR. n = 3, **P < 0.01, ***P < 0.001.

FIG. 2.

miR-7a acts on the 3′-UTR of genes involved in the mTOR pathway. A: Growth curve analysis of miR-7a-deficient Min6 cells. Min6 β-cells were transfected with control (NC) or 7a oligonucleotide. Total cell number was counted on days 0, 2, and 4. n = 3, *P < 0.05. B: The predicted miR-7 binding site consensus in the 3′-UTR of genes involved in the mTOR pathway using TargetScan software and evolutionary conservation. Mmu, Mus musculus; Rno, Rattus norvegicus; Hsa, Homo sapiens; Ptr, Pan Troglodytes; Ocu, Oncorhynchus. C: Reporter assays performed in HEK-293T cells cotransfected with a plasmid encoding pri-miR-7a-2 and the luciferase reporter vector (pMIR-check2) containing the 3′-UTR for p70S6K, eIF4E, Mknk1, Mknk2, or Mapkap1 (n = 3, **P < 0.01). D: Reporter assays performed in Min6 cells cotransfected with antimiR-7a inhibitor and the same 3′-UTR luciferase reporter constructs (n = 3, *P < 0.05, **P < 0.01). (A high-quality color representation of this figure is available in the online issue.)

FIG. 3.

Inhibition of miR-7a activates the mTOR signaling pathway in Min6 β-cells. A and C: Representative Western blot analysis of the indicated mTOR pathway proteins in Min6 cells transfected with control (NC) or antimiR-7a oligonucleotide, performed in duplicate. Cyclophilin B protein was detected as a loading control. The same loading control applies to (A) and (C) and is only shown once in (A). B: Quantitative RT-PCR analysis of relative gene expression levels of potential miR-7a targets in antimiR-7a-transfected Min6 cells; all the P values were not statistically significant relative to NC. n = 3. D: Quantification analysis of Western blot results shown in (A) and (C). n = 4, *P < 0.05, **P < 0.01. NS, no statistical significance.

FIG. 4.

miR-7a targets mTOR signaling in primary mouse islets. A: Representative Western blot analysis of lysates prepared from primary islets transfected with antimiR-7a or control oligonucleotide for the indicated miR-7 targets, phospho-S6 and phospho-eIF4E, as well as the proliferation marker phospho-Histone H3. B: Quantification analysis of Western blot results from three independent experiments is shown in (A). The value of each control group was set to 1.00.

FIG. 5.

miR-7a regulates cell proliferation in primary mouse islets. A–D: Representative images of primary mouse β-cells costained with anti-insulin (red) and antiphospho-Histone H3 (magenta) (A) or anti-Ki67 (magenta) (C). Fluorescently labeled control (NC) or antimiR-7a oligonucleotide are shown in green. Percent adherent-transfected insulin-positive phospho-Histone H3 (B) or Ki67-positive (D) cells were quantified in 500 insulin-expressing cells per group (n = 3, *P < 0.05, **P < 0.01 relative to NC). E: miR-7a deficiency did not induce β-cell apoptosis in mouse islets. miR-7a inhibitor (antimiR-7a) or control oligonucleotide (NC) transfected mouse islets cells were costained with TUNEL and insulin. Total numbers of TUNEL-positive cells were quantified in 500 insulin-positive cells per group; there is no significant change between the groups (n = 3). F and G: miR-7a deficiency did not alter glucose-stimulated insulin secretion (GSIS) in mouse islets cells. Relative insulin secretion at <2.8 mmol/L or 16 mmol/L glucose and insulin content were shown. n = 3, **P < 0.01 relative to 2.8 mmol/L. NS, no statistical significance.

FIG. 6.

miR-7 deficiency promotes replication of human islet β-cells. Primary islets isolated from normoglycemic donors (15- and 24-year-old men, 48- and 49-year-old women, respectively) were prepared and transfected as for mouse islets. A: Representative images of insulin-positive (red) and Ki67-positive (blue) cells transfected with control (NC) or antimiR-7a oligonucleotide (green). B: Percent Ki67-positive cells were quantified in 6,000 insulin-expressing cells per group (n = 4, **P = 0.01 relative to NC).

FIG. 7.

The mTOR inhibitor, rapamycin, blocks β-cell proliferation induced by miR-7 deficiency in primary islets. After transfection with miR-7a inhibitor or control (NC) oligonucleotide as described, islet cells were treated with 10 nmol/L rapamycin for 24 h and fixed for costaining with Ki67 and insulin. A: Percent Ki67-positive cells were quantified in 1,000 insulin-expressing mouse islets cells per group (n = 3, **P < 0.01, ## P < 0.01). B: Primary islets were isolated from normoglycemic donors (a 48-year-old woman and 15- and 24-year-old men, respectively). Ki67-positive cells were quantified in 6,000 insulin-expressing cells per group (n = 3, *P < 0.05). C: Schematic model for miR-7a regulation of the mTOR signaling pathway. Inhibitory relationships are depicted in red, activating relationships are depicted in black/gray. Direct targets of miR-7a are shaded. (A high-quality color representation of this figure is available in the online issue.)