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Integrated analyses to reconstruct microRNA-mediated regulatory networks in mouse liver using high-throughput profiling.

ABSTRACT: MicroRNAs (miRNAs) simultaneously target many transcripts through partial complementarity binding, and have emerged as a key type of post-transcriptional regulator for gene expression. How miRNA accomplishes its pleiotropic effects largely depends on its expression and its target repertoire. Previous studies discovered thousands of miRNAs and numerous miRNA target genes mainly through computation and prediction methods which produced high rates of false positive prediction. The development of Argonaute cross-linked immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) provides a system to effectively determine miRNA target genes. Likewise, the accuracy of dissecting the transcriptional regulation of miRNA genes has been greatly improved by chromatin immunoprecipitation of the transcription factors coupled with sequencing (ChIP-Seq). Elucidation of the miRNA target repertoire will provide an in-depth understanding of the functional roles of microRNA pathways. To reliably reconstruct a miRNA-mediated regulatory network, we established a computational framework using publicly available, sequence-based transcription factor-miRNA databases, including ChIPBase and TransmiR for the TF-miRNA interactions, along with miRNA-target databases, including miRTarBase, TarBase and starBase, for the miRNA-target interactions. We applied the computational framework to elucidate the miRNA-mediated regulatory network in the Mir122a?/? mouse model, which has an altered transcriptome and progressive liver disease.We applied our computational framework to the expression profiles of miRNA/mRNA of Mir122a?/? mutant mice and wild-type mice. The miRNA-mediated network involves 40 curated TFs contributing to the aberrant expression of 65 miRNAs and 723 curated miRNA target genes, of which 56% was found in the differentially-expressed genes of Mir122a--mice. Hence, the regulatory network disclosed previously-known and also many previously-unidentified miRNA-mediated regulations in mutant mice. Moreover, we demonstrate that loss of imprinting at the chromosome 12qF1 region is associated with miRNA overexpression in human hepatocellular carcinoma and stem cells, suggesting initiation of precancerous changes in young mice deficient in miR-122. A group of 9 miRNAs was found to share miR-122 target genes, indicating synergy between miRNAs and target genes by way of multiplicity and cooperativity.The study provides significant insight into miRNA-mediated regulatory networks. Based on experimentally verified data, this network is highly reliable and effective in revealing previously-undetermined disease-associated molecular mechanisms. This computational framework can be applied to explore the significant TF-miRNA-miRNA target interactions in any complex biological systems with high degrees of confidence.

SUBMITTER: Hsu SD

PROVIDER: S-EPMC4331712 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Integrated analyses to reconstruct microRNA-mediated regulatory networks in mouse liver using high-throughput profiling.

Hsu Sheng-Da SD Huang Hsi-Yuan HY Chou Chih-Hung CH Sun Yi-Ming YM Hsu Ming-Ta MT Tsou Ann-Ping AP

BMC genomics 20150121

<h4>Background</h4>MicroRNAs (miRNAs) simultaneously target many transcripts through partial complementarity binding, and have emerged as a key type of post-transcriptional regulator for gene expression. How miRNA accomplishes its pleiotropic effects largely depends on its expression and its target repertoire. Previous studies discovered thousands of miRNAs and numerous miRNA target genes mainly through computation and prediction methods which produced high rates of false positive prediction. Th ...[more]

PMID: 25707768

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Project description:BackgroundAristolochic Acid (AA), a natural component of Aristolochia plants that is found in a variety of herbal remedies and health supplements, is classified as a Group 1 carcinogen by the International Agency for Research on Cancer. Given that microRNAs (miRNAs) are involved in cancer initiation and progression and their role remains unknown in AA-induced carcinogenesis, we examined genome-wide AA-induced dysregulation of miRNAs as well as the regulation of miRNAs on their target gene expression in rat kidney.ResultsWe treated rats with 10 mg/kg AA and vehicle control for 12 weeks and eight kidney samples (4 for the treatment and 4 for the control) were used for examining miRNA and mRNA expression by deep sequencing, and protein expression by proteomics. AA treatment resulted in significant differential expression of miRNAs, mRNAs and proteins as measured by both principal component analysis (PCA) and hierarchical clustering analysis (HCA). Specially, 63 miRNAs (adjusted p value < 0.05 and fold change > 1.5), 6,794 mRNAs (adjusted p value < 0.05 and fold change > 2.0), and 800 proteins (fold change > 2.0) were significantly altered by AA treatment. The expression of 6 selected miRNAs was validated by quantitative real-time PCR analysis. Ingenuity Pathways Analysis (IPA) showed that cancer is the top network and disease associated with those dysregulated miRNAs. To further investigate the influence of miRNAs on kidney mRNA and protein expression, we combined proteomic and transcriptomic data in conjunction with miRNA target selection as confirmed and reported in miRTarBase. In addition to translational repression and transcriptional destabilization, we also found that miRNAs and their target genes were expressed in the same direction at levels of transcription (169) or translation (227). Furthermore, we identified that up-regulation of 13 oncogenic miRNAs was associated with translational activation of 45 out of 54 cancer-related targets.ConclusionsOur findings suggest that dysregulated miRNA expression plays an important role in AA-induced carcinogenesis in rat kidney, and that the integrated approach of multiple profiling provides a new insight into a post-transcriptional regulation of miRNAs on their target repression and activation in a genome-wide scale.

Project description:BackgroundAlu elements are a group of repetitive elements that can influence gene expression through CpG residues and transcription factor binding. Altered gene expression and methylation profiles have been reported in various tissues and cell lines from individuals with autism spectrum disorder (ASD). However, the role of Alu elements in ASD remains unclear. We thus investigated whether Alu elements are associated with altered gene expression profiles in ASD.MethodsWe obtained five blood-based gene expression profiles from the Gene Expression Omnibus database and human Alu-inserted gene lists from the TranspoGene database. Differentially expressed genes (DEGs) in ASD were identified from each study and overlapped with the human Alu-inserted genes. The biological functions and networks of Alu-inserted DEGs were then predicted by Ingenuity Pathway Analysis (IPA). A combined bisulfite restriction analysis of lymphoblastoid cell lines (LCLs) derived from 36 ASD and 20 sex- and age-matched unaffected individuals was performed to assess the global DNA methylation levels within Alu elements, and the Alu expression levels were determined by quantitative RT-PCR.ResultsIn ASD blood or blood-derived cells, 320 Alu-inserted genes were reproducibly differentially expressed. Biological function and pathway analysis showed that these genes were significantly associated with neurodevelopmental disorders and neurological functions involved in ASD etiology. Interestingly, estrogen receptor and androgen signaling pathways implicated in the sex bias of ASD, as well as IL-6 signaling and neuroinflammation signaling pathways, were also highlighted. Alu methylation was not significantly different between the ASD and sex- and age-matched control groups. However, significantly altered Alu methylation patterns were observed in ASD cases sub-grouped based on Autism Diagnostic Interview-Revised scores compared with matched controls. Quantitative RT-PCR analysis of Alu expression also showed significant differences between ASD subgroups. Interestingly, Alu expression was correlated with methylation status in one phenotypic ASD subgroup.ConclusionAlu methylation and expression were altered in LCLs from ASD subgroups. Our findings highlight the association of Alu elements with gene dysregulation in ASD blood samples and warrant further investigation. Moreover, the classification of ASD individuals into subgroups based on phenotypes may be beneficial and could provide insights into the still unknown etiology and the underlying mechanisms of ASD.

Project description:BACKGROUND:The exact cause and mechanisms underlying the pathobiology of autism spectrum disorder (ASD) remain unclear. Dysregulation of long interspersed element-1 (LINE-1) has been reported in the brains of ASD-like mutant mice and ASD brain tissues. However, the role and methylation of LINE-1 in individuals with ASD remain unclear. In this study, we aimed to investigate whether LINE-1 insertion is associated with differentially expressed genes (DEGs) and to assess LINE-1 methylation in ASD. METHODS:To identify DEGs associated with LINE-1 in ASD, we reanalyzed previously published transcriptome profiles and overlapped them with the list of LINE-1-containing genes from the TranspoGene database. An Ingenuity Pathway Analysis (IPA) of DEGs associated with LINE-1 insertion was conducted. DNA methylation of LINE-1 was assessed via combined bisulfite restriction analysis (COBRA) of lymphoblastoid cell lines from ASD individuals and unaffected individuals, and the methylation levels were correlated with the expression levels of LINE-1 and two LINE-1-inserted DEGs, C1orf27 and ARMC8. RESULTS:We found that LINE-1 insertion was significantly associated with DEGs in ASD. The IPA showed that LINE-1-inserted DEGs were associated with ASD-related mechanisms, including sex hormone receptor signaling and axon guidance signaling. Moreover, we observed that the LINE-1 methylation level was significantly reduced in lymphoblastoid cell lines from ASD individuals with severe language impairment and was inversely correlated with the transcript level. The methylation level of LINE-1 was also correlated with the expression of the LINE-1-inserted DEG C1orf27 but not ARMC8. CONCLUSIONS:In ASD individuals with severe language impairment, LINE-1 methylation was reduced and correlated with the expression levels of LINE-1 and the LINE-1-inserted DEG C1orf27. Our findings highlight the association of LINE-1 with DEGs in ASD blood samples and warrant further investigation. The molecular mechanisms of LINE-1 and the effects of its methylation in ASD pathobiology deserve further study.

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Integrated analyses to reconstruct microRNA-mediated regulatory networks in mouse liver using high-throughput profiling.

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Integrated analyses to reconstruct microRNA-mediated regulatory networks in mouse liver using high-throughput profiling.

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