Nature:miRNA给糖尿病患者带来希望

生物谷报道：本周Nature报道了miRNA的应用上一个重要发现：成功采用miRNA调节了胰岛素的分泌，这为糖尿病的治疗带来新的希望，也将为糖尿病的新药研究带来新的光明和思路。

MicroRNAs (miRNAs) constitute a growing class of non-coding RNAs that are thought to regulate gene expression by translational repression. Several miRNAs in animals exhibit tissue-specific or developmental-stage-specific expression, indicating that they could play important roles in many biological processes. To study the role of miRNAs in pancreatic endocrine cells we cloned and identified a novel, evolutionarily conserved and islet-specific miRNA (miR-375). Here we show that overexpression of miR-375 suppressed glucose-induced insulin secretion, and conversely, inhibition of endogenous miR-375 function enhanced insulin secretion. The mechanism by which secretion is modified by miR-375 is independent of changes in glucose metabolism or intracellular Ca²⁺-signalling but correlated with a direct effect on insulin exocytosis. Myotrophin (Mtpn) was predicted to be and validated as a target of miR-375. Inhibition of Mtpn by small interfering (si)RNA mimicked the effects of miR-375 on glucose-stimulated insulin secretion and exocytosis. Thus, miR-375 is a regulator of insulin secretion and may thereby constitute a novel pharmacological target for the treatment of diabetes.

Figure 1 miR-375 is expressed in pancreatic -cells and regulates insulin secretion. a, Northern blots of total RNA (10 µg) isolated from purified pancreatic islets, MIN6 cells and total pancreas. High expression levels were detected in mouse pancreatic islets. b, Tissue expression of miR-375 and miR-376. Total RNA (30 µg) was isolated from mouse tissues for northern blots and probed for the indicated miRNAs or transfer RNA (tRNA) as a loading control. c, Northern blots of total RNA (10 µg) isolated from purified MIN6 and TC1 cells. d, MIN6 cells were transiently transfected with synthetic siRNAs with homologous sequence to miR-375 (si-375) or a mutated miR-375 (si-375MUT), or siRNAs targeting glucokinase (si-Gck) or apoM (si-apoM). After 48 h, the cells were incubated under low (2.8 mM) and stimulatory concentrations of glucose (25 mM) and insulin was measured by RIA (Linco). e, Immunoblot analysis of Gck in MIN6 cells that were transfected with either si-apoM (control) or si-Gck. A 70% reduction in glucokinase protein expression was observed. TATA binding protein (Tbp) was used as a loading control. f, MIN6 cells were transfected with 2'-O-methyl oligoribonucleotides complementary to miR-375 (2'-O-me-375), or a control 2'-O-methyl oligoribonucleotide (2'-O-me-eGFP). Similarly, after 48 h, the cells were incubated at either 2.8 or 25 mM glucose and insulin was measured. Data represent three independent experiments s.e.m. with n = 3. *P = 0.05, **P = 0.01.

Figure 2 Expression of miR-375 using recombinant adenovirus (Ad-375) leads to impaired glucose-, KCl- and tolbutamide-induced insulin secretion in MIN6 cells. a, Northern blot analysis and dose-dependent expression of miR-375 following infection of MIN6 cells for 48 h with Ad-eGFP (control, lane 1) or Ad-375. The multiplicity of infection (MOI) is indicated. The precursor and mature miR-375 can be visualized at 64 and 22 nt, respectively. b–d, Insulin secretion of MIN6 cells following infection with Ad-eGFP and Ad-375 in response to 25 mM glucose (b), 30 mM KCl (c) and 500 µM tolbutamide (d). Insulin secretion from MIN6 cells was measured 48 h after infection with Ad-eGFP or Ad-375 and following incubation with the indicated concentrations of secretagogues. Data represent three independent experiments s.e.m. with n = 3. *P = 0.05, **P = 0.01.

Figure 3 No effect of miR-375 on intracellular Ca2+ signalling in -cells. Intracellular [Ca2+]i measurements of Ad-eGFP-infected (a) and Ad-375-infected pancreatic islets (b). The fluorescence signal has been calibrated and the approximate intracellular [Ca2+]i is indicated to the left. Traces are representative of five experiments in each group. c, Capacitance increases (Cm; lower) elicited by a train of ten depolarizations from -70 mV to 0 mV (V; top) in -cells infected with Ad-eGFP (left) or Ad-375 (right). d, Mean increase in membrane capacitance elicited by the individual depolarization of the train (Cm,n–Cm,n–1) displayed against pulse number (n). e, Total increase in membrane capacitance evoked by the train of depolarizations (Cm). Data are mean values s.e.m. of nine or ten experiments. **P < 0.01. f, Capacitance increase evoked by infusion of a Ca2+/EGTA buffer (free [Ca2+]i = 2 µM) in one control cell and one cell over-expressing miR-375. g, Summary of the experiments in f and similar experiments conducted on MIN6 cells (as indicated). Data are mean values s.e.m. of 15–17 experiments (primary -cells) and 7–12 measurements (MIN6 cells). **P < 0.01, ***P < 0.001.

Figure 4 Identification of target genes of miR-375. a, Western blot analysis of cells infected with Ad-eGFP or Ad-375 (MIN6 cultured for 5 days post-infection; N2A, 2 days) and probed for the expression of Mtpn (anti-Mtpn), Vti1a (anti-Vti1a) or TATA binding protein (anti-Tbp) as a loading control, using specific antisera. b, Immunoblot analysis of Mtpn in MIN6 cells that were transfected with either 2'-O-me-eGFP (control) or 2'-O-me-375. Expression levels of TATA binding protein (Tbp) were used as a loading control; c, d, RT–PCR analysis of Mtpn, Vti1a and GAPDH (loading control) in MIN6 and N2A cells. e, Sequence of the target site in the 3' UTR of Mtpn. The mutant sequence (Mtpn-MUT) is identical to the Mtpn-WT construct except for five point mutations disrupting base-pairing at the 5' end of miR-375 (indicated with a bar). f, Mutating the miR-375 target site in the 3' UTR of Mtpn abolishes inhibition of luciferase activity by endogenous miR-375 in MIN6 cells. MIN6 cells were transiently transfected with either reporter construct in addition to 2'-O-methyl-oligoribonucleotides complementary to miR-375 (2'-O-me-375) or a control 2'-oligoribonucleotide (2'-O-me-eGFP). Data represent three independent experiments s.e.m. with n = 6. g–i, Silencing of Mtpn by siRNA impairs insulin secretion. g, MIN6 cells transiently transfected with siRNAs designed against Mtpn (si-Mtpn) or Vti1a (si-Vti1a) for 48 h and lysed. After separation of proteins by SDS–polyacrylamide gel electrophoresis (PAGE), samples were immunoblotted for either Mtpn or Vti1a expression. The expression of TATA binding protein (Tbp) was analysed for a loading control. h, MIN6 cells were transiently transfected with si-apoM (control), si-Mtpn or si-Vti1a. After 48 h, the cells were incubated under low (2.8 mM) and stimulatory concentrations of glucose (25 mM). Data represent three independent experiments s.e.m. with n = 3. *P = 0.05, **P = 0.01. i, Capacitance measurements in MIN6 cells transfected with si-apoM (control), si-Mtpn or si-Vti1a. Data are mean values s.e.m. **P < 0.01 versus control (si-apoM).

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