In Silico Prediction and Validation of Gfap as an miR-3099 Target in Mouse Brain
In Silico Prediction and Validation of Gfap as an miR-3099 Target in Mouse Brain
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DOI：
10.1007/s12264-017-0143-0
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发表时间：
2017-08-01
影响因子：
5.6
通讯作者：
Ling, King-Hwa
中科院分区：
文献类型：
--
作者：
Abidin, Shahidee Zainal;Leong, Jia-Wen;Ling, King-Hwa
MicroRNAs是一种小的非编码rna，在大脑发育过程中对基因表达和蛋白质合成的调节起着至关重要的作用。MiR-3099在整个胚胎发生过程中高度表达，特别是在发育中的中枢神经系统中。此外，在体外分化的神经元中，miR-3099的表达水平也更高，这表明它是神经元细胞发育过程中的潜在调节剂。本研究旨在通过四种独立的预测算法（miRDB、miRanda、TargetScan和DIANA-micro-T-CDS）通过计算机分析预测miR-3099的靶基因，重点关注与大脑发育和功能相关的靶基因。在此基础上，共预测了3174个miR-3099靶基因。通过至少三种算法（324个基因）预测的基因进行DAVID生物信息学分析，以了解其整体功能主题和代表性。分析显示，近70%的靶基因在神经系统中表达，其中很大一部分与转录调控和蛋白质泛素化机制有关。将已发表和内部生成的miR-3099原位杂交（ISH）表达模式与Allen Brain Atlas中靶基因的ISH切片进行比较，发现7个靶基因（Dnmt3a、Gabpa、Gfap、Itga4、Lxn、Smad7和Tbx18）在发育和成年小鼠脑样本中具有与miR-3099互补的表达模式。其中，我们使用荧光素酶报告基因系统验证了Gfap是miR-3099的直接下游靶标。总之，我们报告了利用生物信息学资源、基于功能本体的丰富注释和时空表达数据集的组合，成功预测和验证了Gfap作为miR-3099靶基因。
MicroRNAs are small non-coding RNAs that play crucial roles in the regulation of gene expression and protein synthesis during brain development. MiR-3099 is highly expressed throughout embryogenesis, especially in the developing central nervous system. Moreover, miR-3099 is also expressed at a higher level in differentiating neurons in vitro, suggesting that it is a potential regulator during neuronal cell development. This study aimed to predict the target genes of miR-3099 via in-silico analysis using four independent prediction algorithms (miRDB, miRanda, TargetScan, and DIANA-micro-T-CDS) with emphasis on target genes related to brain development and function. Based on the analysis, a total of 3,174 miR-3099 target genes were predicted. Those predicted by at least three algorithms (324 genes) were subjected to DAVID bioinformatics analysis to understand their overall functional themes and representation. The analysis revealed that nearly 70% of the target genes were expressed in the nervous system and a significant proportion were associated with transcriptional regulation and protein ubiquitination mechanisms. Comparison of in situ hybridization (ISH) expression patterns of miR-3099 in both published and in-house-generated ISH sections with the ISH sections of target genes from the Allen Brain Atlas identified 7 target genes (Dnmt3a, Gabpa, Gfap, Itga4, Lxn, Smad7, and Tbx18) having expression patterns complementary to miR-3099 in the developing and adult mouse brain samples. Of these, we validated Gfap as a direct downstream target of miR-3099 using the luciferase reporter gene system. In conclusion, we report the successful prediction and validation of Gfap as an miR-3099 target gene using a combination of bioinformatics resources with enrichment of annotations based on functional ontologies and a spatio-temporal expression dataset.