Role of miR-30d in insulin gene expression in pancreatic beta cells

描述(由申请人提供)： 我的长期目标是研究microRNAs(MiRNAs)在调节β细胞功能中的作用，并阐明miRNAs在II型糖尿病发展中的作用。MiRNAs是一种小的非编码核苷酸，与mRNAs结合，在哺乳动物中主要作为翻译抑制因子发挥作用。MiRNAs被认为在许多疾病中发挥作用，包括糖尿病。一些报告表明miRNAs在胰岛素分泌和胰岛β细胞发育中具有重要作用。我们最近在胰腺β细胞系MIN6中进行了一项筛选，以确定随着葡萄糖浓度的变化而改变丰度的miRNAs。这一筛选结果从MIN6细胞中可检测到的108个miRNAs中鉴定出50多个受葡萄糖调节的miRNAs。许多已鉴定的miRNAs，包括miR-124a、miR-107和miR-30d在高糖存在下表达上调。有趣的是，我们发现miR-30d的过表达增加了胰岛素基因的转录。此外，miR-30d的过表达诱导胰岛素与β细胞特异性转录因子MafA的表达增加有关。提示miR-30d可能是MafA/Ins基因表达的负调控基因。为了确定miR-30d在胰岛素基因转录中的功能，我将：1)寻找参与胰岛素基因转录调控的靶基因。2)鉴定miR-30d在胰岛β细胞系和原代小鼠胰岛中的生物学功能。验证的靶点将通过siRNA敲除技术分析它们在胰岛素基因表达中的作用。此外，还将表征miR-30d对目标下行信令的影响。最后，将利用表达miR-30d的重组腺病毒证实miR-30d在原代小鼠胰岛中的功能。这一发现将有助于我们理解miRNA在胰岛素基因转录中的功能。 公共卫生相关性：开发新的方法来恢复胰岛β细胞的胰岛素产生和分泌是糖尿病研究的主要目标。由于我们的初步结果表明，MIR-30d可以诱导葡萄糖刺激的胰岛素基因表达，因此特别有趣。我在这个提案中的目标是识别miR30d靶基因，并表征它们在胰岛素产生中的功能。考虑到miR-30d也诱导MafA的表达，这种miRNA成为一个重要的靶点，可能有助于增强胰岛功能，从而可能有助于设计预防和治疗糖尿病的替代疗法。

DESCRIPTION (provided by applicant): My long-term goal is to study the role of microRNAs (miRNAs) in regulating beta-cell function and to elucidate the role of miRNAs in the development of type-II diabetes. MiRNAs are small noncoding ribonucleotides that bind mRNAs and function mainly as translational repressors in mammals. miRNAs have been implicated to play a role in many diseases, including diabetes. Several reports indicate an important function for miRNAs in insulin secretion and pancreatic beta cell development. We have recently carried out a screen in the pancreatic beta cell line MIN6 to identify miRNAs with altered abundance in response to changes in glucose concentrations. This screen resulted in identification of more than 50 glucose-regulated miRNAs from a total of 108 miRNAs detectable in MIN6 cells. Many of the identified miRNAs, including miR-124a, miR-107 and miR-30d were upregulated in the presence of high glucose. Interestingly, we found that overexpression of miR-30d increased insulin gene transcription. Moreover, the induction of insulin by overexpression of miR-30d is associated with increased expression of the beta-cell specific transcription factor MafA. This suggests that the putative target genes of miR-30d may be negative regulators of MafA/insulin gene expression. To determine the function of miR-30d in insulin gene transcription, I will: 1) Identify miR-30d target genes involved in regulation of insulin gene transcription. 2) Characterize the biological function of miR-30d in pancreatic beta cell lines and primary mouse islets. The verified targets will be analyzed for their role in insulin gene expression by siRNA knock-down techniques. Moreover, the effect of miR-30d on the target downstream signaling will be characterized. Finally, the function of miR-30d in primary mouse islets will be confirmed using a recombinant adenovirus expressing miR-30d. The finding from this proposal will contribute to our understanding of miRNA function in insulin gene transcription. PUBLIC HEALTH RELEVANCE: Developing new approaches to restore insulin production and secretion from pancreatic beta cells is a major goal in diabetes research. MiR-30d is particularly interesting since our preliminary results indicate that it induces glucose-stimulated insulin gene expression. My objective in this proposal is to identify miR30d target genes and to characterize their function in insulin production. Taking into account that miR-30d also induces MafA expression, this miRNA emerges as an important target that may be beneficial in enhancing islet function and may thus aid in the design of alternative therapies for the prevention and treatment of diabetes.