Identification and functional analysis of microRNAs in rats following focal cerebral ischemia injury.
ABSTRACT: MicroRNA sequencing (miRNA?seq) was performed in the present study to investigate miRNA expression profiles in infarcted brain areas following focal cerebral ischemia induced by middle cerebral artery occlusion in rats. In total, 20 miRNAs were identified to be upregulated and 17 to be downregulated in the infarct area. The expression levels of six differentially expressed miRNAs (DEmiRs), miR?211?5p, miR?183?5p, miR?10b?3p, miR?182, miR?217?5p and miR?96?5p, were examined by reverse transcription??quantitative polymerase chain reaction. Subsequently, a miRNA?mRNA network was constructed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to investigate the functions of the mRNAs targeted by these DEmiRs. The present study aimed to investigate the association between miRNAs and cerebral ischemia to provide potential insight into the molecular mechanisms underlying ischemic stroke.
Project description:MicroRNAs (miRNAs) are small non-coding RNAs that recognize sites of complementarity of target messenger RNAs, resulting in transcriptional regulation and translational repression of target genes. In Huntington's disease (HD), a neurodegenerative disease caused by a trinucleotide repeat expansion, miRNA dyregulation has been reported, which may impact gene expression and modify the progression and severity of HD.We performed next-generation miRNA sequence analysis in prefrontal cortex (Brodmann Area 9) from 26 HD, 2 HD gene positive, and 36 control brains. Neuropathological information was available for all HD brains, including age at disease onset, CAG-repeat size, Vonsattel grade, and Hadzi-Vonsattel striatal and cortical scores, a continuous measure of the extent of neurodegeneration. Linear models were performed to examine the relationship of miRNA expression to these clinical features, and messenger RNA targets of associated miRNAs were tested for gene ontology term enrichment.We identified 75 miRNAs differentially expressed in HD brain (FDR q-value <0.05). Among the HD brains, nine miRNAs were significantly associated with Vonsattel grade of neuropathological involvement and three of these, miR-10b-5p, miR-10b-3p, and miR-302a-3p, significantly related to the Hadzi-Vonsattel striatal score (a continuous measure of striatal involvement) after adjustment for CAG length. Five miRNAs (miR-10b-5p, miR-196a-5p, miR-196b-5p, miR-10b-3p, and miR-106a-5p) were identified as having a significant relationship to CAG length-adjusted age of onset including miR-10b-5p, the mostly strongly over-expressed miRNA in HD cases. Although prefrontal cortex was the source of tissue profiled in these studies, the relationship of miR-10b-5p expression to striatal involvement in the disease was independent of cortical involvement. Correlation of miRNAs to the clinical features clustered by direction of effect and the gene targets of the observed miRNAs showed association to processes relating to nervous system development and transcriptional regulation.These results demonstrate that miRNA expression in cortical BA9 provides insight into striatal involvement and support a role for these miRNAs, particularly miR-10b-5p, in HD pathogenicity. The miRNAs identified in our studies of postmortem brain tissue may be detectable in peripheral fluids and thus warrant consideration as accessible biomarkers for disease stage, rate of progression, and other important clinical characteristics of HD.
Project description:BACKGROUND:The aim of this study was to gain further investigation of non-small cell lung cancer (NSCLC) tumorigenesis and identify biomarkers for clinical management of patients through comprehensive bioinformatics analysis. METHODS:miRNA and mRNA microarray datasets were downloaded from GEO (Gene Expression Omnibus) database under the accession number GSE102286 and GSE101929, respectively. Genes and miRNAs with differential expression were identified in NSCLC samples compared with controls, respectively. The interaction between differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRs) was predicted, followed by functional enrichment analysis, and construction of miRNA-gene regulatory network, protein-protein interaction (PPI) network, and competing endogenous RNA (ceRNA) network. Through comprehensive bioinformatics analysis, we anticipate to find novel therapeutic targets and biomarkers for NSCLC. RESULTS:A total of 123 DEmiRs (5 up- and 118 down-regulated miRNAs) and 924 DEGs (309 up- and 615 down-regulated genes) were identified. These genes and miRNAs were significantly involved in different pathways including adherens junction, relaxin signaling pathway, and axon guidance. Furthermore, hsa-miR-9-5p, has-miR-196a-5p and hsa-miR-31-5p, as well as hsa-miR-1, hsa-miR-218-5p and hsa-miR-135a-5p were shown to have higher degree in the miRNA-gene regulatory network and ceRNA network, respectively. Furthermore, BIRC5 and FGF2, as well as RTKN2 and SLIT3 were hubs in the PPI network and ceRNA network, respectively. CONCLUSION:Several pathways (adherens junction, relaxin signaling pathway, and axon guidance) miRNAs (hsa-miR-9-5p, has-miR-196a-5p, hsa-miR-31-5p, hsa-miR-1, hsa-miR-218-5p and hsa-miR-135a-5p) and genes (BIRC5, FGF2, RTKN2 and SLIT3) may play important roles in the pathogenesis of NSCLC.
Project description:Osteoporosis has become a major disease that threatened post-menopausal women and elder people. Circulating micorRNAs (miRNA) could provide useful information for diagnosis and therapeutics. The study employed RT-real time PCR to detect the circulating miRNAs between osteoporotic patients and healthy controls. Human and mouse osteoblast cell lines were used to test the differential induction effects by miRNAs. Alkaline phosphatase activity and Alizarin red staining were examined after miRNA mimics stimulation. The authors found 14 of 150 tested miRNAs were significantly aberrant expressed between patients and healthy controls. Results showed miR-328-3p, let-7g-5p, miR-133b, miR-22-3p, miR-2861, miR-518 miR-100 were down-regulated osteoporotic patient, while miR-10b-5p, miR-21, miR-125b and miR-127 were up-regulated. MiR-10b-3p, miR-328-3p, miR-100 and let-7 showed tight association with Wnt pathway. MiR-10b-5p increased ALP activity and mineral deposition in human and mouse osteoblast cells, indicating miR-10b-3p promoted osteoblast cell differentiation. MiR-328-3p and let-7g-5p decreased ALP activity and suppressed mineral deposition in both cell lines. Conclusively, miR-10b-5p promoted osteoblast cells differentiation; miR-328-3p, miR-100 and let-7 inhibited osteoblast cells differentiation.
Project description:Post-transcriptional regulation by miRNAs plays an important role in the pathogenesis of rheumatoid arthritis (RA), however, the roles of specific miRNAs in RA pathogenesis remain largely unclear. This study performed dual-omics (miRNA and mRNA) integration analysis and in-depth cellular and molecular functional exploration to identify novel RA-associated miRNAs and to understand their underlying pathogenic mechanism. Based on the miRNA and mRNA expression profiles in peripheral blood mononuclear cells (PBMCs) from a discovery sample set (25 RA cases and 18 healthy controls), 18 differentially expressed miRNAs (DEMIRs) (|Fold-change|>2 and P < 0.05) were identified and corresponding interaction networks of DEMIRs and mRNA were constructed. After the expression validation of the DEMIRs in a validation sample set (35 RA cases and 35 healthy controls), miR-99b-5p was highlighted. The over-expression of newly discovered miR-99b-5p is able to suppress T cell apoptosis, promote cell proliferation and activation, increase expression of proinflammatory cytokines (IL-2, IL-6, TNF-?, and IFN-?), and inhibit expression of its target genes mTOR and RASSF4. This study comprehensively identified PBMC-expressed miRNAs along with corresponding regulatory networks significant for RA and discovered miR-99b-5p as a novel post-transcriptional mediator involved in RA pathogenesis. The findings improved our understanding of RA pathogenesis and provided novel insights into the molecular mechanisms underlying RA pathogenesis.
Project description:Accumulating evidence has demonstrated that voltage-gated potassium channels (Kv channels) were associated with regulating cell proliferation and apoptosis in tumor cells. Our previous study proved that the Kv channel blocker 4-aminopyridine (4-AP) could inhibit cell proliferation and induce apoptosis in glioma. However, the precise mechanisms were not clear yet. MicroRNAs (miRNAs) are small noncoding RNAs that act as key mediators in the progression of tumor, so the aim of this study was to investigate the role of miRNAs in the apoptosis-promoting effect of 4-AP in glioma cells. Using a microRNA array, we found that 4-AP altered the miRNA expression in glioma cells, and the down-regulation of miR-10b-5p induced by 4-AP was verified by real-time PCR. Transfection of miR-10b-5p mimic significantly inhibited 4-AP-induced caspases activation and apoptosis. Moreover, we verified that apoptosis-related molecule Apaf-1 was the direct target of miR-10b-5p. Furthermore, miR-10b-5p mimic significantly inhibited 4-AP-induced up-regulation of Apaf-1 and its downstream apoptosis-related proteins, such as cleaved caspase-3. In conclusion, Kv channel blocker 4-AP may exert its anti-tumor effect by down-regulating the expression of miR-10b-5p and then raised expression of Apaf-1 and its downstream apoptosis-related proteins. Current data provide evidence that miRNAs play important roles in Kv channels-mediated cell proliferation and apoptosis.
Project description:Dietary microRNAs (miRNAs), notably those found in milk, are currently being investigated for their potential to elicit biological effects via canonical binding to human messenger RNA targets once ingested. Besides milk, beef and other bovine tissue-derived ingredients could also be a relevant source of potentially bioactive dietary miRNAs. In this study, we characterized the human homologous miRNA profiles in food-grade, bovine-sourced sirloin, heart and adrenal tissue (raw, cooked, and pasteurized, freeze-dried extracts) via deep-sequencing and quantitative reverse transcription PCR (RT-qPCR). A total of 198 human homologous miRNAs were detected at 10 or more normalized reads in all replicates (n = 3) of at least one preparation method. Tissue origin rather than preparation method was the major differentiating factor of miRNA profiles, and adrenal-based miRNA profiles were the most distinct. The ten most prevalent miRNAs in each tissue represented 71-93% of the total normalized counts for all annotated miRNAs. In cooked sirloin, the most abundant miRNAs were miR-10b-5p, (48.8% of total annotated miRNA reads) along with the muscle-specific miR-1 (24.1%) and miR-206 (4.8%). In dried heart extracts, miR-1 (17.0%), miR-100-5p (16.1%) and miR-99a-5p (11.0%) gave the highest normalized read counts. In dried adrenal extracts, miR-10b-5p (71.2%) was the most prominent followed by miR-143-3p (7.1%) and 146b-5p (3.7%). Sequencing results for five detected and two undetected miRNAs were successfully validated by RT-qPCR. We conclude that edible, bovine tissues contain unique profiles of human homologous dietary miRNAs that survive heat-based preparation methods.
Project description:Background:Osteosarcoma, which originates in the mesenchymal tissue, is the prevalent primary solid malignancy of the bone. It is of great importance to explore the mechanisms of metastasis and recurrence, which are two primary reasons accounting for the high death rate in osteosarcoma. Data and methods:Three miRNA expression profiles related to osteosarcoma were downloaded from GEO DataSets. Differentially expressed miRNAs (DEmiRs) were screened using MetaDE.ES of the MetaDE package. A support vector machine (SVM) classifier was constructed using optimal miRNAs, and its prediction efficiency for recurrence was detected in independent datasets. Finally, a co-expression network was constructed based on the DEmiRs and their target genes. Results:In total, 78 significantly DEmiRs were screened. The SVM classifier constructed by 15 miRNAs could accurately classify 58 samples in 65 samples (89.2%) in the GSE39040 database, which was validated in another two databases, GSE39052 (84.62%, 22/26) and GSE79181 (91.3%, 21/23). Cox regression showed that four miRNAs, including hsa-miR-10b, hsa-miR-1227, hsa-miR-146b-3p, and hsa-miR-873, significantly correlated with tumor recurrence time. There were 137, 147, 145, and 77 target genes of the above four miRNAs, respectively, which were assigned to 17 gene ontology functionally annotated terms and 14 Kyoto Encyclopedia of Genes and Genomes pathways. Among them, the "Osteoclast differentiation" pathway contained a total of seven target genes and was analyzed further. Conclusion:The 15-miRNAs-based SVM classifier provides a potential useful tool to predict the recurrence of osteosarcoma. Our results suggest the possible mechanisms of osteosarcoma metastasis and recurrence and provide fresh DEmiRs as potential biomarkers or therapeutic targets for osteosarcoma.
Project description:Rationale: Triple-negative breast cancer (TNBC), which has the highest recurrence rate and shortest survival time of all breast cancers, is in urgent need of a risk assessment method to determine an accurate treatment course. Recently, miRNA expression patterns have been identified as potential biomarkers for diagnosis, prognosis, and personalized therapy. Here, we investigate a combination of candidate miRNAs as a clinically applicable signature that can precisely predict relapse in TNBC patients after surgery. Methods: Four total cohorts of training (TCGA_TNBC and GEOD-40525) and validation (GSE40049 and GSE19783) datasets were analyzed with logistic regression and Gaussian mixture analyses. We established a miRNA signature risk model and identified an 8-miRNA signature for the prediction of TNBC relapse. Results: The miRNA signature risk model identified ten candidate miRNAs in the training set. By combining 8 of the 10 miRNAs (miR-139-5p, miR-10b-5p, miR-486-5p, miR-455-3p, miR-107, miR-146b-5p, miR-324-5p and miR-20a-5p), an accurate predictive model of relapse in TNBC patients was established and was highly correlated with prognosis (AUC of 0.80). Subsequently, this 8-miRNA signature prognosticated relapse in the two validation sets with AUCs of 0.89 and 0.90. Conclusion: The 8-miRNA signature predictive model may help clinicians provide a prognosis for TNBC patients with a high risk of recurrence after surgery and provide further personalized treatment to decrease the chance of relapse.
Project description:Transcriptional dysregulation has long been recognized as central to the pathogenesis of Huntington's disease (HD). MicroRNAs (miRNAs) represent a major system of post-transcriptional regulation, by either preventing translational initiation or by targeting transcripts for storage or for degradation. Using next-generation miRNA sequencing in prefrontal cortex (Brodmann Area 9) of twelve HD and nine controls, we identified five miRNAs (miR-10b-5p, miR-196a-5p, miR-196b-5p, miR-615-3p and miR-1247-5p) up-regulated in HD at genome-wide significance (FDR q-value<0.05). Three of these, miR-196a-5p, miR-196b-5p and miR-615-3p, were expressed at near zero levels in control brains. Expression was verified for all five miRNAs using reverse transcription quantitative PCR and all but miR-1247-5p were replicated in an independent sample (8HD/8C). Ectopic miR-10b-5p expression in PC12 HTT-Q73 cells increased survival by MTT assay and cell viability staining suggesting increased expression may be a protective response. All of the miRNAs but miR-1247-5p are located in intergenic regions of Hox clusters. Total mRNA sequencing in the same samples identified fifteen of 55 genes within the Hox cluster gene regions as differentially expressed in HD, and the Hox genes immediately adjacent to the four Hox cluster miRNAs as up-regulated. Pathway analysis of mRNA targets of these miRNAs implicated functions for neuronal differentiation, neurite outgrowth, cell death and survival. In regression models among the HD brains, huntingtin CAG repeat size, onset age and age at death were independently found to be inversely related to miR-10b-5p levels. CAG repeat size and onset age were independently inversely related to miR-196a-5p, onset age was inversely related to miR-196b-5p and age at death was inversely related to miR-615-3p expression. These results suggest these Hox-related miRNAs may be involved in neuroprotective response in HD. Recently, miRNAs have shown promise as biomarkers for human diseases and given their relationship to disease expression, these miRNAs are biomarker candidates in HD.
Project description:Purpose:This research explores the aberrant expression of the long non-coding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) in pterygium. A competitive endogenous RNA (ceRNA) network was constructed to elucidate the molecular mechanisms in pterygium. Methods:We obtained the differentially expressed mRNAs based on three datasets (GSE2513, GSE51995, and GSE83627), and summarized the differentially expressed miRNAs (DEmiRs) and differentially expressed lncRNAs (DELs) data by published literature. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, protein-protein interaction (PPI), and gene set enrichment analysis (GSEA) analysis were performed. DEmiRs were verified in GSE21346, and the regulatory network of hub mRNAs, DELs, and DEmiRs were constructed. Results:Overall, 40 upregulated and 40 downregulated differentially expressed genes (DEGs) were obtained. The KEGG enrichment showed the DEGs mainly involved in extracellular matrix (ECM)-receptor interaction, focal adhesion, and PI3K-Akt signaling pathway. The GSEA results showed that cornification, keratinization, and cornified envelope were significantly enriched. The validation outcome confirmed six upregulated DEmiRs (miR-766-3p, miR-184, miR-143-3p, miR-138-5p, miR-518b, and miR-1236-3p) and two downregulated DEmiRs (miR-200b-3p and miR-200a-3p). Then, a ceRNA regulatory network was constructed with 22 upregulated and 15 downregulated DEmiRs, 4 downregulated DELs, and 26 upregulated and 33 downregulated DEGs. The network showed that lncRNA SNHG1/miR-766-3p/FOS and some miRNA-mRNA axes were dysregulated in pterygium. Conclusions:Our study provides a novel perspective on the regulatory mechanism of pterygium, and lncRNA SNHG1/miR-766-3p/FOS may contribute to pterygium development.