Objective

MicroRNAs (miRNAs) play an integral role in maintaining beta cell function and identity. Deciphering their targets and precise role, however, remains challenging. In this study, we aimed to identify miRNAs and their downstream targets involved in the regeneration of islet beta cells following partial pancreatectomy in mice.

Methods

RNA from laser capture microdissected (LCM) islets of partially pancreatectomized and sham-operated mice were profiled with microarrays to identify putative miRNAs implicated in beta cell regeneration. Altered expression of the selected miRNAs, including miR-132, was verified by RT-PCR. Potential targets of miR-132 were selected through bioinformatic data mining. Predicted miR-132 targets were validated for their changed RNA, protein expression levels, and signaling upon miR-132 knockdown and/or overexpression in mouse MIN6 and human EndoC-βH1 insulinoma cells. The ability of miR-132 to foster beta cell proliferation in vivo was further assessed in pancreatectomized miR-132^−/− and control mice.

Results

Partial pancreatectomy significantly increased the number of BrdU⁺/insulin⁺ islet cells. Microarray profiling revealed that 14 miRNAs, including miR-132 and -141, were significantly upregulated in the LCM islets of the partially pancreatectomized mice compared to the LCM islets of the control mice. In the same comparison, miR-760 was the only downregulated miRNA. The changed expression of these miRNAs in the islets of the partially pancreatectomized mice was confirmed by RT-PCR only in the case of miR-132 and -141. Based on previous knowledge of its function, we focused our attention on miR-132. Downregulation of miR-132 reduced the proliferation of MIN6 cells while enhancing the levels of pro-apoptotic cleaved caspase-9. The opposite was observed in miR-132 overexpressing MIN6 cells. Microarray profiling, RT-PCR, and immunoblotting of the latter cells demonstrated their downregulated expression of Pten with concomitant increased levels of pro-proliferative factors phospho-Akt and phospho-Creb and inactivation of pro-apoptotic Foxo3a via its phosphorylation. Downregulation of Pten was further confirmed in the LCM islets of pancreatectomized mice compared to the sham-operated mice. Moreover, overexpression of miR-132 correlated with increased proliferation of EndoC-βH1 cells. The regeneration of beta cells following partial pancreatectomy was lower in the miR-132/212^−/− mice than the control littermates.

Conclusions

This study provides compelling evidence about the critical role of miR-132 for the regeneration of mouse islet beta cells through the downregulation of its target Pten. Hence, the miR-132/Pten/Akt/Foxo3 signaling pathway may represent a suitable target to enhance beta cell mass.

中文翻译：

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MiR-132通过Pten / Akt / Foxo3信号传导控制胰腺β细胞的增殖和存活。

客观的

MicroRNA（miRNA）在维持β细胞功能和特性方面起着不可或缺的作用。但是，确定其目标和确切角色仍然充满挑战。在这项研究中，我们旨在鉴定小鼠部分胰腺切除术后胰岛β细胞再生中涉及的miRNA及其下游靶标。

方法

使用微阵列对部分胰腺切除和假手术小鼠的激光捕获显微解剖（LCM）胰岛的RNA进行分析，以鉴定与β细胞再生有关的假定miRNA。通过RT-PCR验证了包括miR-132在内的所选miRNA的表达变化。通过生物信息数据挖掘选择了miR-132的潜在靶标。验证了预测的miR-132目标的RNA，蛋白质表达水平以及在小鼠MIN6和人EndoC-βH1胰岛素瘤细胞中miR-132敲低和/或过表达后的信号传导。的能力的miR-132，以促进体内的β细胞增殖的进一步评估pancreatectomized的miR-132 ^{- / -} 和对照小鼠。

结果

胰腺部分切除术显着增加了BrdU ⁺ /胰岛素⁺胰岛细胞的数量。微阵列分析显示，与对照组小鼠的LCM胰岛相比，部分胰腺切除的小鼠的LCM胰岛中有14个miRNA（包括miR-132和-141）显着上调。在同一比较中，miR-760是唯一被下调的miRNA。仅在miR-132和-141的情况下，通过RT-PCR证实了在部分胰腺切除的小鼠的胰岛中这些miRNA的表达发生了变化。基于其功能的先前知识，我们将注意力集中在miR-132上。下调miR-132减少了MIN6细胞的增殖，同时提高了促凋亡裂解的caspase-9的水平。在过表达miR-132的MIN6细胞中观察到相反的情况。微阵列分析，RT-PCR和后一种细胞的免疫印迹证明，Pten的表达下调，同时促增殖因子磷酸化Akt和磷酸化Creb的水平升高，促凋亡的Foxo3a通过其磷酸化而失活。与假手术小鼠相比，在胰腺切除的小鼠的LCM胰岛中进一步证实了Pten的下调。此外，miR-132的过表达与EndoC-βH1细胞增殖增加有关。在miR-132 / 212 ^-/-小鼠中，部分胰腺切除术后的β细胞再生低于对照同窝仔。

结论

这项研究提供了令人信服的证据，证明miR-132通过下调其靶标Pten对小鼠胰岛β细胞再生的关键作用。因此，miR-132 / Pten / Akt / Foxo3信号通路可能代表增加β细胞质量的合适靶标。