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.     

MicroRNA expression signatures of bladder cancer revealed by deep sequencing

Background

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression. They are aberrantly expressed in many types of cancers. In this study, we determined the genome-wide miRNA profiles in bladder urothelial carcinoma by deep sequencing.

Methodology/Principal Findings

We detected 656 differentially expressed known human miRNAs and miRNA antisense sequences (miRNA*s) in nine bladder urothelial carcinoma patients by deep sequencing. Many miRNAs and miRNA*s were significantly upregulated or downregulated in bladder urothelial carcinoma compared to matched histologically normal urothelium. hsa-miR-96 was the most significantly upregulated miRNA and hsa-miR-490-5p was the most significantly downregulated one. Upregulated miRNAs were more common than downregulated ones. The hsa-miR-183, hsa-miR-200b∼429, hsa-miR-200c∼141 and hsa-miR-17∼92 clusters were significantly upregulated. The hsa-miR-143∼145 cluster was significantly downregulated. hsa-miR-182, hsa-miR-183, hsa-miR-200a, hsa-miR-143 and hsa-miR-195 were evaluated by Real-Time qPCR in a total of fifty-one bladder urothelial carcinoma patients. They were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium (p<0.001 for each miRNA).

Conclusions/Significance

To date, this is the first study to determine genome-wide miRNA expression patterns in human bladder urothelial carcinoma by deep sequencing. We found that a collection of miRNAs were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium, suggesting that they might play roles as oncogenes or tumor suppressors in the development and/or progression of this cancer. Our data provide novel insights into cancer biology.     

High-Throughput Sequencing of Plasma MicroRNA in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Background

MicroRNAs (miRNAs) are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME.

Results

Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation of three miRNAs (hsa-miR-127-3p, hsa-miR-142-5p and hsa-miR-143-3p) in the CFS/ME patients.

Conclusion

Our study is the first to identify circulating miRNAs from CFS/ME patients and also to confirm three differentially expressed circulating miRNAs in CFS/ME patients, providing a basis for further study to find useful CFS/ME biomarkers.     

Mirna expression profiles identify drivers in colorectal and pancreatic cancers

Background and Aim

Altered expression of microRNAs (miRNAs) hallmarks many cancer types. The study of the associations of miRNA expression profile and cancer phenotype could help identify the links between deregulation of miRNA expression and oncogenic pathways.

Methods

Expression profiling of 866 human miRNAs in 19 colorectal and 17 pancreatic cancers and in matched adjacent normal tissues was investigated. Classical paired t-test and random forest analyses were applied to identify miRNAs associated with tissue-specific tumors. Network analysis based on a computational approach to mine associations between cancer types and miRNAs was performed.

Results

The merge between the two statistical methods used to intersect the miRNAs differentially expressed in colon and pancreatic cancers allowed the identification of cancer-specific miRNA alterations. By miRNA-network analysis, tissue-specific patterns of miRNA deregulation were traced: the driving miRNAs were miR-195, miR-1280, miR-140-3p and miR-1246 in colorectal tumors, and miR-103, miR-23a and miR-15b in pancreatic cancers.

Conclusion

MiRNA expression profiles may identify cancer-specific signatures and potentially useful biomarkers for the diagnosis of tissue specific cancers. miRNA-network analysis help identify altered miRNA regulatory networks that could play a role in tumor pathogenesis.     

Reorganization of metastamiRs in the evolution of metastatic aggressive neuroblastoma cells

Background

MetastamiRs have momentous clinical relevance and have been correlated with disease progression in many tumors. In this study, we identified neuroblastoma metastamiRs exploiting unique mouse models of favorable and high-risk metastatic human neuroblastoma. Further, we related their deregulation to the modulation of target proteins and established their association with clinical outcomes.

Results

Whole genome miRNA microarray analysis identified 74 metastamiRs across the manifold of metastatic tumors. RT-qPCR on select miRNAs validated profile expression. Results from bio-informatics across the ingenuity pathway, miRCancer, and literature data-mining endorsed the expression of these miRNAs in multiple tumor systems and showed their role in metastasis, identifying them as metastamiRs. Immunoblotting and TMA-IHC analyses revealed alterations in the expression/phosphorylation of metastamiRs’ targets, including ADAMTS-1, AKT1/2/3, ASK1, AURKβ, Birc1, Birc2, Bric5, β-CATENIN, CASP8, CD54, CDK4, CREB, CTGF, CXCR4, CYCLIN-D1, EGFR, ELK1, ESR1, CFOS, FOSB, FRA, GRB10, GSK3β, IL1α, JUND, kRAS, KRTAP1, MCP1, MEGF10, MMP2, MMP3, MMP9, MMP10, MTA2, MYB, cMYC, NF2, NOS3, P21, pP38, PTPN3, CLEAVED PARP, PKC, SDF-1β, SEMA3D, SELE, STAT3, TLR3, TNFα, TNFR1, and VEGF in aggressive cells ex vivo and in a manifold of metastatic tumors in vivo. miRNA mimic (hsa-miR-125b, hsa-miR-27b, hsa-miR-93, hsa-miR-20a) and inhibitor (hsa-miR-1224-3p, hsa-miR-1260) approach for select miRNAs revealed the direct influence of the altered metastamiRs in the regulation of identified protein targets. Clinical outcome association analysis with the validated metastamiRs’ targets corresponded strongly with poor overall and relapse-free survival.

Conclusions

For the first time, these results identified a comprehensive list of neuroblastoma metastamiRs, related their deregulation to altered expression of protein targets, and established their association with poor clinical outcomes. The identified set of distinctive neuroblastoma metastamiRs could serve as potential candidates for diagnostic markers for the switch from favorable to high-risk metastatic disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1642-x) contains supplementary material, which is available to authorized users.     

Whole blood-derived microRNA signatures in mice exposed to lipopolysaccharides

Background

Lipopolysaccharide (LPS) is recognized as the most potent microbial mediator presaging the threat of invasion of Gram-negative bacteria that implicated in the pathogenesis of sepsis and septic shock. This study was designed to examine the microRNA (miRNA) expression in whole blood from mice injected with intraperitoneal LPS.

Methods

C57BL/6 mice received intraperitoneal injections of varying concentrations (range, 10–1000 μg) of LPS from different bacteria, including Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enterica, and Serratia marcescens and were killed 2, 6, 24, and 72 h after LPS injection. Whole blood samples were obtained and tissues, including lung, brain, liver, and spleen, were harvested for miRNA expression analysis using an miRNA array (Phalanx miRNA OneArray® 1.0). Upregulated expression of miRNA targets in the whole blood of C57BL/6 and Tlr4^−/− mice injected with LPS was quantified using real-time RT-PCR and compared with that in the whole blood of C57BL/6 mice injected with lipoteichoic acid (LTA) from Staphylococcus aureus.

Results

Following LPS injection, a significant increase of 15 miRNAs was observed in the whole blood. Among them, only 3 miRNAs showed up-regulated expression in the lung, but no miRNAs showed a high expression level in the other examined tissues. Upregulated expression of the miRNA targets (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107 and miR-451) following LPS injection on real-time RT-PCR was dose- and time-dependent. miRNA induction occurred after 2 h and persisted for at least 6 h. Exposure to LPS from different bacteria did not induce significantly different expression of these miRNA targets. Additionally, significantly lower expression levels of let-7d, miR-25, miR-92a, miR-103, and miR-107 were observed in whole blood of Tlr4^−/− mice. In contrast, LTA exposure induced moderate expression of miR-451 but not of the other 7 miRNA targets.

Conclusions

We identified a specific whole blood–derived miRNA signature in mice exposed to LPS, but not to LTA, from different gram-negative bacteria. These whole blood-derived miRNAs are promising as biomarkers for LPS exposure.     

Genome-Wide Analyses of Radioresistance-Associated miRNA Expression Profile in Nasopharyngeal Carcinoma Using Next Generation Deep Sequencing

Background

Rapidly growing evidence suggests that microRNAs (miRNAs) are involved in a wide range of cancer malignant behaviours including radioresistance. Therefore, the present study was designed to investigate miRNA expression patterns associated with radioresistance in NPC.

Methods

The differential expression profiles of miRNAs and mRNAs associated with NPC radioresistance were constructed. The predicted target mRNAs of miRNAs and their enriched signaling pathways were analyzed via biological informatical algorithms. Finally, partial miRNAs and pathways-correlated target mRNAs were validated in two NPC radioreisitant cell models.

Results

50 known and 9 novel miRNAs with significant difference were identified, and their target mRNAs were narrowed down to 53 nasopharyngeal-/NPC-specific mRNAs. Subsequent KEGG analyses demonstrated that the 53 mRNAs were enriched in 37 signaling pathways. Further qRT-PCR assays confirmed 3 down-regulated miRNAs (miR-324-3p, miR-93-3p and miR-4501), 3 up-regulated miRNAs (miR-371a-5p, miR-34c-5p and miR-1323) and 2 novel miRNAs. Additionally, corresponding alterations of pathways-correlated target mRNAs were observed including 5 up-regulated mRNAs (ICAM1, WNT2B, MYC, HLA-F and TGF-β1) and 3 down-regulated mRNAs (CDH1, PTENP1 and HSP90AA1).

Conclusions

Our study provides an overview of miRNA expression profile and the interactions between miRNA and their target mRNAs, which will deepen our understanding of the important roles of miRNAs in NPC radioresistance.     

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.     

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.     

Pre-miRNA loop nucleotides control the distinct activities of mir-181a-1 and mir-181c in early T cell development

Background

Mature miRNAs can often be classified into large families, consisting of members with identical seeds (nucleotides 2 through 7 of the mature miRNAs) and highly homologous ∼21-nucleotide (nt) mature miRNA sequences. However, it is unclear whether members of a miRNA gene family, which encode identical or nearly identical mature miRNAs, are functionally interchangeable in vivo.

Methods and Findings

We show that mir-181a-1, but not mir-181c, can promote CD4 and CD8 double-positive (DP) T cell development when ectopically expressed in thymic progenitor cells. The distinct activities of mir-181a-1 and mir-181c are largely determined by their unique pre-miRNA loop nucleotides—not by the one-nucleotide difference in their mature miRNA sequences. Moreover, the activity of mir-181a-1 on DP cell development can be quantitatively influenced by nucleotide changes in its pre-miRNA loop region. We find that both the strength and the functional specificity of miRNA genes can be controlled by the pre-miRNA loop nucleotides. Intriguingly, we note that mutations in the pre-miRNA loop regions affect pre-miRNA and mature miRNA processing, but find no consistent correlation between the effects of pre-miRNA loop mutations on the levels of mature miRNAs and the activities of the mir-181a-1/c genes.

Conclusions

These results demonstrate that pre-miRNA loop nucleotides play a critical role in controlling the activity of miRNA genes and that members of the same miRNA gene families could have evolved to achieve different activities via alterations in their pre-miRNA loop sequences, while maintaining identical or nearly identical mature miRNA sequences.     

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.