Function and mechanism of long non-coding RNA Gm21284 in the development of hippocampal cholinergic neurons.
ABSTRACT: Background:Increasing evidence has revealed that long non-coding RNAs (lncRNAs) play a pivotal role in the development of nervous system. Our previous studies have demonstrated that enhanced cholinergic neurogenesis occurs in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) after cholinergic denervation, which is closely associated with the core transcription factor Lhx8. This study aimed to identify novel lncRNAs in a denervated hippocampal niche, which may affect cholinergic neurogenesis, and to explore the molecular mechanisms underlying cholinergic neurogenesis. Methods:The gene expression profiles of the denervated hippocampus were examined by microarray analysis, and targeted lncRNAs were filtered using bioinformatics analysis. The lncRNA Gm21284 was predicted to be associated with Lhx8. RT-PCR and FISH were used to observe the expression and localization of Gm21284 in vitro and in vivo. The interaction between Gm21284 and Lhx8 and miR-30e-3P was verified using the luciferase reporter gene assay. Cell proliferation and differentiation was observed to reveal the effects of Gm21284 in cholinergic neurogenesis. Results:Microarray analysis demonstrated 482 up-regulated and 135 down-regulated mRNAs, 125 up-regulated and 55 down-regulated lncRNAs, and 10 up-regulated and 3 down-regulated miRNAs in the denervated hippocampal niche. Overall, 32 lncRNAs were differentially expressed in the denervated hippocampal niche, which could interact with miR-30e-3p, miR-431, and miR-147. Among these 32 lncRNAs, Gm21284 and Adarb1 were identified after interleaving with lncRNAs in a co-expression network and WGCNA. Gm21284 was mainly located in the hippocampal DG. Furthermore, Gm21284-positive cells were considerably increased in the denervated hippocampus than in the normal side. EdU proliferation assay revealed that the proliferation of neural stem cells was repressed after the overexpression of Gm21284. Compared with the control group, the proportion of ChAT-positive cells increased at 7 days of differentiation of NSCs overexpressing Gm21284. Conclusion:Thus, Gm21284 functions as a competing endogenous RNA, which inhibits the proliferation of hippocampal NSCs and promotes their differentiation toward cholinergic neurons by inhibiting miR-30e-3P competitively.
Project description:Background:Ovarian cancer (OC) is a common female reproductive malignancy with a high mortality rate. Although LAMA4 was observed to be downregulated in OC cells, its mechanism in regulating OC metastasis is still unknown. This study aimed to investigate the effect of LAMA4 and its mechanism on OC. Methods:To achieve this aim, a microarray analysis was performed to screen out the key genes involved in OC pathogenesis. Western-blot and qRT-PCR assays were also carried out to detect protein and mRNA expressions, respectively. A luciferase reporter assay was further used to confirm the direct interaction of miR-30e-3p with MEG3, and the direct interaction of miR-30e-3p with LAMA4 mRNA. Cytological experiments (CCK8, colony formation assay, wound-healing assay etc.) were then performed to explore the roles of miR-30e-3p, MEG3, and LAMA4 in OC cells. Results:After carrying out microarray analysis, LAMA4 was confirmed as a key gene associated with OC pathogenesis. Research results proved that miR-30e-3p was markedly upregulated, while MEG3 and LAMA4 were noticeably downregulated in OC tissues and cells. The overexpression of LAMA4 significantly impaired the proliferation, migration, and invasion of OC cells. However, the upregulation of MEG3 increased the expression of LAMA4 by sponging miR-30e-3p, which alleviated the malignancy of OC cells. Conclusions:Observations showed that forced LAMA4 overexpression could inhibit OC progression, which was regulated by MEG3 via sponging miR-30e-3p. The findings of this research could provide new insights into the mechanism by which MEG3 and LAMA4 exert their anti-oncogenic roles in OC progression.Trial registration Not applicable.
Project description:Background:Microvascular obstruction (MVO) can result in coronary microcirculation embolism and myocardial microinfarction. Myocardial injury induced by MVO is characterized by continuous ischemia and hypoxia of cardiomyocytes. Autophagy and apoptosis are closely associated with various cardiovascular diseases. Based on our previous study, we observed a decrease in miR-30e-3p expression and an increase in Egr-1 expression in a rat coronary microembolization model. However, the specific function of miR-30e-3p in regulating autophagy and apoptosis in an ischemia/hypoxia (IH) environment remains to be deciphered. We exposed cardiomyocytes to an IH environment and then determined whether miR-30e-3p was involved in promoting cardiomyocyte autophagy and inhibiting apoptosis by regulating Egr-1. Methods:Cardiomyocytes were isolated from rats for our in vitro study. miR-30e-3p was either overexpressed or inhibited by transfection with lentiviral vectors into cardiomyocytes. 3-Methyladenine (3-MA) was used to inhibit autophagy. RT-qPCR and western blotting were used to determine the expression levels of miR-30e-3p, Egr-1, and proteins related to the autophagy and apoptosis process. Autophagic vacuoles and autophagic flux were evaluated using transmission electron microscopy (TEM) and confocal microscopy, respectively. Cardiomyocyte viability was evaluated using the MTS assay. Cell injury was assessed by lactate dehydrogenase (LDH) leakage, and apoptosis was determined by flow cytometry. Results:Both miR-30e-3p expression and autophagy were significantly inhibited, and apoptosis was increased in cardiomyocytes after 9 hours of IH exposure. Overexpression of miR-30e-3p increased autophagy and inhibited apoptosis, as well as suppressed Egr-1 expression and decreased cell injury. In addition, inhibition of miR-30e-3p reduced autophagy and increased apoptosis and cell injury. Conclusions:miR-30e-3p may be involved in promoting cardiomyocyte autophagy and inhibiting apoptosis by indirectly regulating Egr-1 expression in an IH environment.
Project description:BACKGROUND: MicroRNA (miRNA) expression is known to be deregulated in ovarian carcinomas. However, limited data is available about the miRNA expression pattern for the benign or borderline ovarian tumors as well as differential miRNA expression pattern associated with histological types, grades or clinical stages in ovarian carcinomas. We defined patterns of microRNA expression in tissues from normal, benign, borderline, and malignant ovarian tumors and explored the relationship between frequently deregulated miRNAs and clinicopathologic findings, response to therapy, survival, and association with Her-2/neu status in ovarian carcinomas. METHODS: We measured the expression of nine miRNAs (miR-181d, miR-30a-3p, miR-30c, miR-30d, miR-30e-3p, miR-368, miR-370, miR-493-5p, miR-532-5p) in 171 formalin-fixed, paraffin-embedded ovarian tissue blocks as well as six normal human ovarian surface epithelial (HOSE) cell lines using Taqman-based real-time PCR assays. Her-2/neu overexpression was assessed in ovarian carcinomas (n = 109 cases) by immunohistochemistry analysis. RESULTS: Expression of four miRNAs (miR-30c, miR-30d, miR-30e-3p, miR-370) was significantly different between carcinomas and benign ovarian tissues as well as between carcinoma and borderline tissues. An additional three miRNAs (miR-181d, miR-30a-3p, miR-532-5p) were significantly different between borderline and carcinoma tissues. Expression of miR-532-5p was significantly lower in borderline than in benign tissues. Among ovarian carcinomas, expression of four miRNAs (miR-30a-3p, miR-30c, miR-30d, miR-30e-3p) was lowest in mucinous and highest in clear cell samples. Expression of miR-30a-3p was higher in well-differentiated compared to poorly differentiated tumors (P = 0.02), and expression of miR-370 was higher in stage I/II compared to stage III/IV samples (P = 0.03). In multivariate analyses, higher expression of miR-181d, miR-30c, miR-30d, and miR-30e-3p was associated with significantly better disease-free or overall survival. Finally, lower expression of miR-30c, miR-30d, miR-30e-3p and miR-532-5p was significantly associated with overexpression of Her-2/neu. CONCLUSIONS: Aberrant expression of miRNAs is common in ovarian tumor suggesting involvement of miRNA in ovarian tumorigenesis. They are associated with histology, clinical stage, survival and oncogene expression in ovarian carcinoma.
Project description:The aim of this pilot investigation was to determine if microRNA expression differed in the presence or absence of obesity, comparing gingival biopsies obtained from patients with or without periodontal disease. Total RNA was extracted from gingival biopsy samples collected from 20 patients: 10 non-obese patients (BMI < 30 kg/m(2)) and 10 obese patients (BMI > 30 kg/m(2)), each group with 5 periodontally healthy sites and 5 chronic periodontitis sites. MicroRNA expression patterns were assessed with a quantitative microRNA PCR array to survey 88 candidate microRNA species. Four microRNA databases were used to identify potential relevant mRNA target genes of differentially expressed microRNAs. Two microRNA species (miR-18a, miR-30e) were up-regulated among obese individuals with a healthy periodontium. Two microRNA species (miR-30e, miR-106b) were up-regulated in non-obese individuals with periodontal disease. In the presence of periodontal disease and obesity, 9 of 11 listed microRNAs were significantly up-regulated (miR-15a, miR-18a, miR-22, miR-30d, miR-30e, miR-103, miR-106b, miR-130a, miR-142-3p, miR-185, and miR-210). Predicted targets include 69 different mRNAs from genes that comprise cytokines, chemokines, specific collagens, and regulators of glucose and lipid metabolism. The expression of specific microRNA species in obesity, which could also target and post-transcriptionally modulate cytokine mRNA, provides new insight into possible mechanisms of how risk factors might modify periodontal inflammation and may represent novel therapeutic targets.
Project description:Malignant Pleural Mesothelioma (MPM) is an aggressive cancer mainly caused by asbestos exposure and refractory to current therapies. Specific diagnostic markers for early MPM diagnosis are needed. Changes in miRNA expression have been implicated in several diseases and cancers, including MPM. We examined if a specific miRNA signature in plasmatic extracellular vesicles (EV) may help to discriminate between malignant pleural mesothelioma patients (MPM) and subjects with Past Asbestos Exposure (PAE).We investigated 23 MPM patients and 19 cancer-free subjects with past asbestos exposure (PAE). We screened 754 miRNAs in plasmatic EVs by OpenArray and found 55 differential miRNAs using logistic regression models adjusted for age, sex, BMI, and smoking. Among the top-20 differential miRNAs chosen for validation by Real time PCR, 16 were confirmed. Using receiver operating characteristic (ROC) curve analysis, the most discriminating miRNA combination was given by miR-103a-3p + miR-30e-3p, which generated an AUC of 0.942 (95% CI 0.87-1.00), with a sensitivity of 95.5% and a specificity of 80.0%. Using multivariate Cox regression analysis including gender, age, BMI and smoking we found a Hazard Ratio for miR-103a-3p above the median of 0.37 (95%CI 0.13-1.13) and of 0.51 (95%CI 0.17-1.52) for miR-30e-3p.This study suggests EV-associated miR-103a-3p and miR-30e-3p are able to discriminate MPM from PAE subjects. Larger and prospective studies are needed to confirm these two-miRNA signature alone or in combination with other biomarkers as diagnostic tools for MPM.
Project description:Borna disease virus 1 (BoDV-1) is neurotropic prototype of Bornaviruses causing neurological diseases and maintaining persistent infection in brain cells of mammalian species. Long non-coding RNA (lncRNA) is transcript of more than 200 nucleotides without protein-coding function regulating various biological processes as proliferation, apoptosis, cell migration and viral infection. However, regulatory of lncRNAs in BoDV-1 infection remains unknown. To identify differential expression profiles and predict functions of lncRNA in BoDV-1 infection, microarray data showed that 3528 lncRNAs and 2661 lncRNAs were differentially expressed in Strain V and Hu-H1 BoDV-infected groups compared with control groups, respectively. Gene Ontology (GO) and pathway analysis suggested that differential lncRNAs may be involved in regulation of metabolic, biological regulation, cellular process, endocytosis, viral infections and cell adhesion processes, cancer in both BoDV-infected strains. ENSMUST00000128469 was found down-regulated in both BoDV-infected groups compared with control groups consistent with microarray (p < 0.05). ceRNA analysis indicated possible interaction networks as ENSMUST00000128469/miR-22-5p, miR-206-3p, miR-302b-5p, miR-302c-3p, miR-1a-3p/Igf1. Igf1 was found up-regulated in both BoDV-infected groups compared with control groups (p < 0.05). Possible functions of predicted target mRNAs and miRNAs of ENSMUST00000128469 were involved in cell proliferation, transcriptional misregulation and proteoglycan pathways enriched in cancer. lncRNA may be involved in regulation of Hu-H1 inhibited cell proliferation and promoted apoptosis through NF-kB, JNK/MAPK signaling, BCL2 and CDK6/E2F1 pathways different from Strain V. Possible interaction networks as ENSMUST00000128469/miR-22-5p, miR-206-3p, miR-302b-5p, miR-302c-3p, miR-1a-3p/Igf1 may involve in regulation of cell proliferation, apoptosis, and cancer.
Project description:Forebrain cholinergic neurons play important roles as striatal local circuit neurons and basal telencephalic projection neurons. The genetic mechanisms that control development of these neurons suggest that most of them are derived from the basal telencephalon where Lhx8, a LIM-homeobox gene, is expressed. Here we report that mice with a null mutation of Lhx8 are deficient in the development of forebrain cholinergic neurons. Lhx8 mutants lack the nucleus basalis, a major source of the cholinergic input to the cerebral cortex. In addition, the number of cholinergic neurons is reduced in several other areas of the subcortical forebrain in Lhx8 mutants, including the caudate-putamen, medial septal nucleus, nucleus of the diagonal band, and magnocellular preoptic nucleus. Although cholinergic neurons are not formed, initial steps in their specification appear to be preserved, as indicated by a presence of cells expressing a truncated Lhx8 mRNA and mRNA of the homeobox gene Gbx1. These results provide genetic evidence supporting an important role for Lhx8 in development of cholinergic neurons in the forebrain.
Project description:MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally control the expression of their target genes via RNA interference. There is increasing evidence that expression of miRNAs is dysregulated in neuronal disorders, including epilepsy, a chronic neurological disorder characterized by spontaneous recurrent seizures. Mesial temporal lobe epilepsy (MTLE) is a common type of focal epilepsy in which disease-induced abnormalities of hippocampal neurogenesis in the subgranular zone as well as gliosis and neuronal cell loss in the cornu ammonis area are reported. We hypothesized that in MTLE altered miRNA-mediated regulation of target genes could be involved in hippocampal cell remodeling. A miRNA screen was performed in hippocampal focal and non-focal brain tissue samples obtained from the temporal neocortex (both n=8) of MTLE patients. Out of 215 detected miRNAs, two were differentially expressed (hsa-miR-34c-5p: mean increase of 5.7 fold (p=0.014), hsa-miR-212-3p: mean decrease of 76.9% (p=0.0014)). After in-silico target gene analysis and filtering, reporter gene assays confirmed RNA interference for hsa-miR-34c-5p with 3'-UTR sequences of GABRA3, GRM7 and GABBR2 and for hsa-miR-212-3p with 3'-UTR sequences of SOX11, MECP2, ADCY1 and ABCG2. Reporter gene assays with mutated 3'-UTR sequences of the transcription factor SOX11 identified two different binding sites for hsa-miR-212-3p and its primary transcript partner hsa-miR-132-3p. Additionally, there was an inverse time-dependent expression of Sox11 and miR-212-3p as well as miR-132-3p in rat neonatal cortical neurons. Transfection of neurons with anti-miRs for miR-212-3p and miR-132-3p suggest that both miRNAs work synergistically to control Sox11 expression. Taken together, these results suggest that differential miRNA expression in neurons could contribute to an altered function of the transcription factor SOX11 and other genes in the setting of epilepsy, resulting not only in impaired neural differentiation, but also in imbalanced neuronal excitability and accelerated drug export.
Project description:Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral "sponge" to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.
Project description:BACKGROUND:Ischemic retinopathies (IRs) are leading causes of visual impairment. They are characterized by an initial phase of microvascular degeneration and a second phase of aberrant pre-retinal neovascularization (NV). microRNAs (miRNAs) regulate gene expression, and a number play a role in normal and pathological NV. But, post-transcriptional modulation of miRNAs in the eye during the development of IRs has not been systematically evaluated. AIMS & METHODS:Using Next Generation Sequencing (NGS) we profiled miRNA expression in the retina and choroid during vasodegenerative and NV phases of oxygen-induced retinopathy (OIR). RESULTS:Approximately 20% of total miRNAs exhibited altered expression (up- or down-regulation); 6% of miRNA were found highly expressed in retina and choroid of rats subjected to OIR. During OIR-induced vessel degeneration phase, miR-199a-3p, -199a-5p, -1b, -126a-3p displayed a robust decreased expression (> 85%) in the retina. While in the choroid, miR-152-3p, -142-3p, -148a-3p, -532-3p were upregulated (>200%) and miR-96-5p, -124-3p, -9a-3p, -190b-5p, -181a-1-3p, -9a-5p, -183-5p were downregulated (>70%) compared to controls. During peak pathological NV, miR-30a-5p, -30e-5p and 190b-5p were markedly reduced (>70%), and miR-30e-3p, miR-335, -30b-5p strongly augmented (by up to 300%) in the retina. Whereas in choroid, miR-let-7f-5p, miR-126a-5p and miR-101a-3p were downregulated by (>81%), and miR-125a-5p, let-7e-5p and let-7g-5p were upregulated by (>570%) during NV. Changes in miRNA observed using NGS were validated using qRT-PCR for the 24 most modulated miRNAs. In silico approach to predict miRNA target genes (using algorithms of miRSystem database) identified potential new target genes with pro-inflammatory, apoptotic and angiogenic properties. CONCLUSION:The present study is the first comprehensive description of retinal/choroidal miRNAs profiling in OIR (using NGS technology). Our results provide a valuable framework for the characterization and possible therapeutic potential of specific miRNAs involved in ocular IR-triggered inflammation, angiogenesis and degeneration.