Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

Background

Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.

Methodology

Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays.

Results

When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression.

Conclusion

Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.     

Expression and localization of microRNAs in perinatal rat pancreas: role of miR-21 in regulation of cholesterol metabolism

Objective

To investigate the expression of pancreatic microRNAs (miRNAs) during the period of perinatal beta-cell expansion and maturation in rats, determine the localization of these miRNAs and perform a pathway analysis with predicted target mRNAs expressed in perinatal pancreas.

Research Design and Methods

RNA was extracted from whole pancreas at embryonic day 20 (E20), on the day of birth (P0) and two days after birth (P2) and hybridized to miRNA microarrays. Differentially expressed miRNAs were verified by northern blotting and their pancreatic localization determined by in situ hybridization. Pathway analysis was done using regulated sets of mRNAs predicted as targets of the miRNAs. Possible target genes were tested using reporter-gene analysis in INS-1E cells.

Results

Nine miRNAs were differentially expressed perinatally, seven were confirmed to be regulated at the level of the mature miRNA. The localization studies showed endocrine localization of six of these miRNAs (miR-21, -23a, -29a, -125b-5p, -376b-3p and -451), and all were expressed in exocrine cells at one time point at least. Pathways involving metabolic processes, terpenoid and sterol metabolism were selectively affected by concomitant regulation by miRNAs and mRNAs, and Srebf1 was validated as a target of miR-21.

Conclusions

The findings suggest that miRNAs are involved in the functional maturation of pancreatic exocrine and endocrine tissue following birth. Pathway analysis of target genes identify changes in sterol metabolism around birth as being selectively affected by differential miRNA expression during this period.     

Microarray-Based Approach Identifies Differentially Expressed MicroRNAs in Porcine Sexually Immature and Mature Testes

Background

MicroRNAs (miRNAs) are short non-coding RNA molecules which are proved to be involved in mammalian spermatogenesis. Their expression and function in the porcine germ cells are not fully understood.

Methodology

We employed a miRNA microarray containing 1260 unique miRNA probes to evaluate the miRNA expression patterns between sexually immature (60-day) and mature (180-day) pig testes. One hundred and twenty nine miRNAs representing 164 reporter miRNAs were expressed differently (p<0.1). Fifty one miRNAs were significantly up-regulated and 78 miRNAs were down-regulated in mature testes. Nine of these differentially expressed miRNAs were validated using quantitative RT-PCR assay. Totally 15919 putative miRNA-target sites were detected by using RNA22 method to align 445 NCBI pig cDNA sequences with these 129 differentially expressed miRNAs, and seven putative target genes involved in spermatogenesis including DAZL, RNF4 gene were simply confirmed by quantitative RT-PCR.

Conclusions

Overall, the results of this study indicated specific miRNAs expression in porcine testes and suggested that miRNAs had a role in regulating spermatogenesis.     

The induction of microRNA targeting IRS-1 is involved in the development of insulin resistance under conditions of mitochondrial dysfunction in hepatocytes

Background

Mitochondrial dysfunction induces insulin resistance in myocytes via a reduction of insulin receptor substrate-1 (IRS-1) expression. However, the effect of mitochondrial dysfunction on insulin sensitivity is not understood well in hepatocytes. Although research has implicated the translational repression of target genes by endogenous non-coding microRNAs (miRNA) in the pathogenesis of various diseases, the identity and role of the miRNAs that are involved in the development of insulin resistance also remain largely unknown.

Methodology

To determine whether mitochondrial dysfunction induced by genetic or metabolic inhibition causes insulin resistance in hepatocytes, we analyzed the expression and insulin-stimulated phosphorylation of insulin signaling intermediates in SK-Hep1 hepatocytes. We used qRT-PCR to measure cellular levels of selected miRNAs that are thought to target IRS-1 3′ untranslated regions (3′UTR). Using overexpression of miR-126, we determined whether IRS-1-targeting miRNA causes insulin resistance in hepatocytes.

Principal Findings

Mitochondrial dysfunction resulting from genetic (mitochondrial DNA depletion) or metabolic inhibition (Rotenone or Antimycin A) induced insulin resistance in hepatocytes via a reduction in the expression of IRS-1 protein. In addition, we observed a significant up-regulation of several miRNAs presumed to target IRS-1 3′UTR in hepatocytes with mitochondrial dysfunction. Using reporter gene assay we confirmed that miR-126 directly targeted to IRS-1 3′UTR. Furthermore, the overexpression of miR-126 in hepatocytes caused a substantial reduction in IRS-1 protein expression, and a consequent impairment in insulin signaling.

Conclusions/Significance

We demonstrated that miR-126 was actively involved in the development of insulin resistance induced by mitochondrial dysfunction. These data provide novel insights into the molecular basis of insulin resistance, and implicate miRNA in the development of metabolic disease.     

Identification of Wolbachia-responsive microRNAs in the two-spotted spider mite,Tetranychus urticae

Background

The two-spotted spider mite, Tetranychus urticae, is infected with Wolbachia, which have the ability to manipulate host reproduction and fitness. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes such as development, reproduction and host-pathogen interactions. Although miRNA was observed to involve in Wolbachia-host interactions in the other insect systems, its roles have not been fully deciphered in the two-spotted spider mite.

Results

Small RNA libraries of infected and uninfected T. urticae for both sexes (in total four libraries) were constructed. By integrating the mRNA data originated from the same samples, the target genes of the differentially expressed miRNAs were predicted. Then, GO and pathway analyses were performed for the target genes. Comparison of libraries showed that Wolbachia infection significantly regulated 91 miRNAs in females and 20 miRNAs in males, with an overall suppression of miRNAs in Wolbachia-infected libraries. A comparison of the miRNA and mRNA data predicted that the differentially expressed miRNAs negatively regulated 90 mRNAs in females and 9 mRNAs in males. An analysis of target genes showed that Wolbachia-responsive miRNAs regulated genes with function in sphingolipid metabolism, lysosome function, apoptosis and lipid transporting in both sexes, as well as reproduction in females.

Conclusion

Comparisons of the miRNA and mRNA data can help to identify miRNAs and miRNA target genes involving in Wolbachia-host interactions. The molecular targets identified in this study should be useful in further functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1122) contains supplementary material, which is available to authorized users.     

miR-29b,miR-205 and miR-221 Enhance Chemosensitivity to Gemcitabine in HuH28 Human Cholangiocarcinoma Cells

Background and Aims

Cholangiocarcinoma (CCA) is highly resistant to chemotherapy, including gemcitabine (Gem) treatment. MicroRNAs (miRNAs) are endogenous, non-coding, short RNAs that can regulate multiple genes expression. Some miRNAs play important roles in the chemosensitivity of tumors. Here, we examined the relationship between miRNA expression and the sensitivity of CCA cells to Gem.

Methods

Microarray analysis was used to determine the miRNA expression profiles of two CCA cell lines, HuH28 and HuCCT1. To determine the effect of candidate miRNAs on Gem sensitivity, expression of each candidate miRNA was modified via either transfection of a miRNA mimic or transfection of an anti-oligonucleotide. Ontology-based programs were used to identify potential target genes of candidate miRNAs that were confirmed to affect the Gem sensitivity of CCA cells.

Results

HuCCT1 cells were more sensitive to Gem than were HuH28 cells, and 18 miRNAs were differentially expressed whose ratios over ± 2log2 between HuH28 and HuCCT1. Among these 18 miRNAs, ectopic overexpression of each of three downregulated miRNAs in HuH28 (miR-29b, miR-205, miR-221) restored Gem sensitivity to HuH28. Suppression of one upregulated miRNA in HuH28, miR-125a-5p, inhibited HuH28 cell proliferation independently to Gem treatment. Selective siRNA-mediated downregulation of either of two software-predicted targets, PIK3R1 (target of miR-29b and miR-221) or MMP-2 (target of miR-29b), also conferred Gem sensitivity to HuH28.

Conclusions

miRNA expression profiling was used to identify key miRNAs that regulate Gem sensitivity in CCA cells, and software that predicts miRNA targets was used to identify promising target genes for anti-tumor therapies.     

Inferring the perturbed microRNA regulatory networks from gene expression data using a network propagation based method

Background

MicroRNAs (miRNAs) are a class of endogenous small regulatory RNAs. Identifications of the dys-regulated or perturbed miRNAs and their key target genes are important for understanding the regulatory networks associated with the studied cellular processes. Several computational methods have been developed to infer the perturbed miRNA regulatory networks by integrating genome-wide gene expression data and sequence-based miRNA-target predictions. However, most of them only use the expression information of the miRNA direct targets, rarely considering the secondary effects of miRNA perturbation on the global gene regulatory networks.

Results

We proposed a network propagation based method to infer the perturbed miRNAs and their key target genes by integrating gene expressions and global gene regulatory network information. The method used random walk with restart in gene regulatory networks to model the network effects of the miRNA perturbation. Then, it evaluated the significance of the correlation between the network effects of the miRNA perturbation and the gene differential expression levels with a forward searching strategy. Results show that our method outperformed several compared methods in rediscovering the experimentally perturbed miRNAs in cancer cell lines. Then, we applied it on a gene expression dataset of colorectal cancer clinical patient samples and inferred the perturbed miRNA regulatory networks of colorectal cancer, including several known oncogenic or tumor-suppressive miRNAs, such as miR-17, miR-26 and miR-145.

Conclusions

Our network propagation based method takes advantage of the network effect of the miRNA perturbation on its target genes. It is a useful approach to infer the perturbed miRNAs and their key target genes associated with the studied biological processes using gene expression data.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-255) contains supplementary material, which is available to authorized users.     

MicroRNA expression profiling of the fifth-instar posterior silk gland of Bombyx mori

Background

The growth and development of the posterior silk gland and the biosynthesis of the silk core protein at the fifth larval instar stage of Bombyx mori are of paramount importance for silk production.

Results

Here, aided by next-generation sequencing and microarry assay, we profile 1,229 microRNAs (miRNAs), including 728 novel miRNAs and 110 miRNA/miRNA* duplexes, of the posterior silk gland at the fifth larval instar. Target gene prediction yields 14,222 unique target genes from 1,195 miRNAs. Functional categorization classifies the targets into complex pathways that include both cellular and metabolic processes, especially protein synthesis and processing.

Conclusion

The enrichment of target genes in the ribosome-related pathway indicates that miRNAs may directly regulate translation. Our findings pave a way for further functional elucidation of these miRNAs and their targets in silk production.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-410) contains supplementary material, which is available to authorized users.     

Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

Background

The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer.

Results

We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein.

Conclusions

Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer.     

Identification of Conserved and Novel microRNAs from Liriodendron chinense Floral Tissues

Background

Liriodendron chinense (L. chinense) is an endangered basal angiosperm plant in China because of its low reproductive efficiency. Recently, miRNAs have obtained great attention because they can play important roles. Through high throughput sequencing technique, large amount of miRNAs were identified from different plant species. But there were few studies about the miRNAs in the basal angiosperms especially in the sexual reproduction process.

Results

Deep sequencing technology was applied to discover miRNAs in L. chinense flowers at different stages. After bioinformatic analysis, 496 putative conserved miRNAs representing 97 families and 2 novel miRNAs were found. Among them, one is previously regarded as gymnosperm specific. Their expressions were further validated by Real-time PCR for 13 selected miRNAs. Putative targeting genes were predicted and categorized with gene ontology (GO) analysis. About ten percents of the targets are involved in the reproduction process. Further expressional analysis showed that many of these miRNAs were highly related to the reproductive growth.

Conclusions

This is the first comprehensive identification of conserved and novel miRNAs in L. chinense. The data presented here might not only help to fill the gap of miRNA registered about basal angiosperm plants but also contribute to understanding the evolution of miRNAs. The differential expression of some of the miRNAs and the prediction of their target genes are also helpful in understanding the regulation of L. chinense sexual reproduction.