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In silico analysis of mRNA:miR-3099 interaction
Get PDFIntroduction: MicroRNAs (miRNA) are small non-coding RNAs and have crucial role in gene expression and protein synthesis regulation, especially in nervous system and brain development. A novel miR-3099 was found highly express throughout embryogenesis especially in the developing central nervous system. Moreover, miR-3099 was also expressed upon neuronal differentiation in in vitro system suggesting that miR-3099 is a potential regulator during neuronal development. Therefore, objective of this study is to predict target genes of miR-3099 via in-silico analysis. These analyses will predict potential downstream targets for miR-3099 and their relationship to signalling pathways with special focus on neuronal function and brain development.
Methods: Four different prediction software, miRDB, miRanda, TargetScan and DIANA micro-T, were employed to identify the candidate target genes of miR-3099. The predicted downstream targeted genes were selected based on the database criteria, prior to BioVenn clustering to identify the common targeted genes. The targeted genes that were predicted by at least three different databases were subjected to DAVID bioinformatics analysis to understand the biological process and function of these targeted genes.
Results: Based on the analysis, a total of 1676 predicted genes were targeted by miR-3099. Of these, 73 genes were predicted by three software and 22 genes were predicted by all the four software. Majority of the targeted genes were annotated as involved in positive regulation of transcription activity and were identified as related to neuronal and brain development. Hence, the predicted downstream targets of miR-3099 warrant further investigation to validate the in silico analysis
miR-3099 promotes neurogenesis and inhibits astrogliogenesis during murine neural development
Get PDFMicroRNA-3099 is highly expressed during neuronal differentiation and development of the central nervous system. Here we characterised the role of miR-3099 during neural differentiation and embryonic brain development using a stable and regulatable mouse embryonic stem cell culture system for miR-3099 expression and in utero electroporation of miR-3099 expression construct into E15.5 embryonic mouse brains. In the in vitro system, miR-3099 overexpression upregulated gene related to neuronal markers such as Tuj1, NeuN, Gat1, vGluT1 and vGluT2. In contrast, gene related to astrocyte markers (Gfap, S100β and Slc1a3) were suppressed upon overexpression of miR-3099. Furthermore, miR-3099 overexpression between E15.5 and E18.5 mouse embryonic brains led to disorganised neuronal migration potentially due to significantly decreased Gfap+ cells. Collectively, our results indicated that miR-3099 plays a role in modulating and regulating expression of key markers involved in neuronal differentiation. In silico analysis was also performed to identify miR-3099 homologues in the human genome, and candidates were validated by stem-loop RT-qPCR. Analysis of the miR-3099 seed sequence AGGCUA against human transcriptomes revealed that a potential miRNA, mds21 (Chr21:39186698-39186677) (GenBank accession ID: MK521584), was 100% identical to the miR-3099 seed sequence. Mds21 expression was observed and validated in various human cell lines (293FT, human Wharton's jelly and dental pulp mesenchymal stem cells, and MCF-7, MDA-MB-231, C-Sert, SW780, RT112, 5637, EJ28 and SH-SY5Y cells), with the highest levels detected in human mesenchymal stem cell lines. The analysis validated mds21 as a novel miRNA and a novel homologue of miR-3099 in the human genome
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The expression profile of miR-3099 during neural development of Ts1Cje mouse model of down syndrome
Get PDFMicroRNA-3099 (miR-3099) plays a crucial role in regulating neuronal differentiation and development of the central nervous system (CNS). The miR-3099 is a pro-neuronal miRNA that promotes neural stem/progenitor cell (NSPC) differentiation into neuronal lineage by suppressing astrogliogenesis. Down syndrome (DS) brain exhibited increased astrogliogenesis and reduced neuronal cell density. The involvement of miR-3099 in the neurodevelopment of DS has not been investigated and potentially responsible for the neurogenic-to-gliogenic shift phenomenon observed in DS brain. To investigate the role of miR-3099 during DS brain development, neural/progenitor cell proliferation and differentiation, we profiled miR-3099 expression level in the Ts1Cje, a mouse model for DS. We analysed the Ts1Cje whole brain at embryonic day (E) 10.5, E14.5 and P1.5, proliferating neurospheres and differentiating neurospheres at 3, 9 and 15 days in vitro (DIV). Expression of miR-3099 in both the developing mouse brain and the differentiating neurosphere was not significantly different between Ts1Cje and wild type controls. In contrast, the expression level of miR-3099 was significantly higher (p<0.05) in proliferating NSPC derived from the Ts1Cje compared to wild-type. Further molecular profiling of NPSC and glial cell markers indicated that the expression of Sox2 (p<0.01) and Gfap (p<0.05) were significantly downregulated in Ts1Cje neurospheres as compared to that of wild type, respectively. While there were no significant differences in Tuj1 and Nestin expression levels between the Ts1Cje and wild type neurospheres, their expression levels were ~3-fold upregulated and ~2.6 downregulated Ts1Cje group, respectively. The findings suggest that dysregulation of miR-3099 affects NSPC lineage commitment as indicated by altered postmitotic neuronal cell markers. Further molecular characterisation and gene expression profiling of other neuronal and glial markers will help refine the analysis of gene-gene interactions underlying the neuropathologies of DS
MiR-3099 is overexpressed in differentiating 46c mouse embryonic stem cells upon neural induction
No full textBackground: MicroRNAs (miRNAs) have a crucial role in gene expression regulation and protein synthesis, especially in the central nervous system. In developing mouse embryos a novel miRNA, miR-3099, is highly expressed, particularly in the central nervous system. This study aims to determine the expression of miR-3099 during cellular differentiation of 46C mouse embryonic stem cells after neural induction with N2/B27 medium.
Methods: 46C mouse embryonic stem cells were subjected to neural induction with N2/B27 medium. At 0, 3, 7, 11, 17, and 22 days after neural induction, the cells were screened for various pluripotent, progenitor, and differentiating/differentiated cells markers by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). Stem-loop pulse RT-PCR was performed to determine the expression of miR-3099 at all selected time points after neural induction.
Results: Our findings showed that after induction, mouse embryonic stem cells differentiated into heterogeneous pools of cells containing neurons, astrocytes, and oligodendrocytes. Mouse embryonic stem cells and neural progenitor/precursor cells were also present in culture up to day 22 as indicated by RT-PCR analysis. Elucidation of miR-3099 expression during in vitro neural induction revealed that this miRNA was expressed throughout the differentiation process of 46C mouse embryonic stem cells. miR-3099 was expressed at higher levels on day 11, 17, and 22 as compared to day 0, 3 and 7 after neural induction.
Conclusion: The level of miR-3099 expression was higher in differentiated mouse embryonic stem cells after neural induction. This finding suggested that miR-3099 might play a role in regulating neural stem cell differentiation. However, further characterisation of miR-3099 in a better characterised or optimised differentiated neural stem cell culture would provide increased understanding of the cellular function and molecular targets of miR-3099, especially in neuron development
MiR-3099 is overexpressed in differentiating 46c mouse embryonic stem cells upon neural induction
No full textBackground: MicroRNAs (miRNAs) have a crucial role in gene expression regulation and protein synthesis, especially in the central nervous system. In developing mouse embryos a novel miRNA, miR-3099, is highly expressed, particularly in the central nervous system. This study aims to determine the expression of miR-3099 during cellular differentiation of 46C mouse embryonic stem cells after neural induction with N2/B27 medium.
Methods: 46C mouse embryonic stem cells were subjected to neural induction with N2/B27 medium. At 0, 3, 7, 11, 17, and 22 days after neural induction, the cells were screened for various pluripotent, progenitor, and differentiating/differentiated cells markers by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). Stem-loop pulse RT-PCR was performed to determine the expressionof miR-3099 at all selected time points afterneuralinduction.
Results: Our findings showed that after induction, mouse embryonic stem cells differentiated into heterogeneous pools of cells containing neurons, astrocytes, and oligodendrocytes. Mouse embryonic stem cells and neural progenitor/precursor cells were also present in culture up to day 22 as indicated by RT-PCR analysis. Elucidation of miR-3099 expression during in vitro neural induction revealed that this miRNA was expressed throughout the differentiation process of 46C mouse embryonic stem cells. miR-3099 was expressed at higher levels on day 11, 17, and 22 as compared to day 0, 3 and 7 after neural induction.
Conclusion: The level of miR-3099 expression was higher in differentiated mouse embryonic stem cells after neural induction. This finding suggested that miR-3099 might play a role in regulating neural stem cell differentiation. However, further characterisation of miR-3099 in a better characterised or optimised differentiated neural stem cell culture would provide increased understanding of the cellular function and molecular targets of miR-3099, especially in neuron development
Characterization of miR-3099-mediated posttranscriptional of target genes regulation during neurogenesis in mice
Get PDFMicroRNAs (miRNAs) are a family of small non-coding RNAs with potent
regulatory roles in metabolism, neurodevelopment, neuroplasticity, apoptosis,
and other neurobiological processes. MiRNAs function through partial
complementary base-pairing with specific target mRNAs, resulting in the
repression of translational processes or the promotion of mRNA deadenylation
leading to degradation. In 2011, miR-3099 was found to be expressed as early as
in the blastocyst stage, in which the expression was maintained until the
developing E11.5 mouse brain. The expression of miR-3099 was further
restricted to the cortical plate of the developing mouse brain between E13.5
and E17.5, coinciding with the time that the majority of the cells are committed
to neuronal cell lineage. Moreover, the miR-3099 was also found to be highly
expressed in differentiating P19 cell (2-fold upregulation) when comparing to
the proliferating P19 cell. Therefore, this study aims to understand the role of
miR-3099 during neuro-differentiation and corticogenesis in the mouse model.
The expression of miR-3099 was found elevated by 2-3 folds in 46C mouse
embryonic stem (mES) cell upon neural induction. Then, predicted target gene
of miR-3099 was further analysed by using four different prediction algorithms
(miRDB, miRanda, TargetScan and DIANA-micro-T-CDS) and DAVID
bioinformatics analysis with emphasis on target genes related to brain
development and function. Based on the prediction, nearly 70% of the
predicted target genes were expressed in the nervous system. Of these
predicted target genes, Gfap was chosen as a candidate for downstream
validation because it had been implicated in an important pathway in the brain
known as the JAK-STAT signalling pathway, which controls the onset of
astrocyte formation. By using the luciferase reporter gene system, Gfap was
negatively inhibited by miR-3099. Furthermore, overexpression of miR-3099
was performed in vitro and in vivo for better understanding of the role of miR3099 during neuro-differentiation and brain development. In vitro, a transgenic mES cell that carried miR-3099 was overexpressed and differentiated for 17
days. The gene expression profile was carried out by using stem-loop RT-qPCR
for different marker analysis such as proliferative, neural progenitor, neuron,
astrocyte and oligodendrocyte markers. The analysis revealed that the
overexpression of miR-3099 promoted neuronal differentiation and suppressed
the astrogliogenesis in the in vitro system. In the in vivo system, the
overexpression of miR-3099 caused disorganised neuronal migration
potentially due to downregulation of Gfap. Heretofore, the human homologue
of miR-3099 has not been found or reported. In silico analysis via seed sequence
similarity search in GEO database found that mds21 to be novel miRNA that
has 100% identical at seed region and 64% closed to miR-3099 mature sequence.
Interestingly, the expression of mds21 was found to be expressed in various
human cell line and tissue, including the brain suggesting that mds21 might be
a potential miR-3099 homologue in the human genome. Collectively, this study
has shown that miR-3099 plays an essential role in modulating and regulating
key markers involved in neuronal differentiation and neural cell function. The
degree of functional conservation between miR-3099 and mds21 is not clear,
and further validations are needed to characterise them further
In silico prediction and validation of Gfap as miR-3099 target in the mouse brain
Get PDFMicroRNAs are small non-coding RNAs that play crucial roles in the regulation of gene expression and protein synthesis during brain development. MiR-3099 is highly expressed throughout embryogenesis, especially in the developing central nervous system. Moreover, miR-3099 is also expressed at a higher level in differentiating neurons in vitro, suggesting that it is a potential regulator during neuronal cell development. This study aimed to predict the target genes of miR-3099 via in-silico analysis using four independent prediction algorithms (miRDB, miRanda, TargetScan, and DIANA-micro-T-CDS) with emphasis on target genes related to brain development and function. Based on the analysis, a total of 3,174 miR-3099 target genes were predicted. Those predicted by at least three algorithms (324 genes) were subjected to DAVID bioinformatics analysis to understand their overall functional themes and representation. The analysis revealed that nearly 70% of the target genes were expressed in the nervous system and a significant proportion were associated with transcriptional regulation and protein ubiquitination mechanisms. Comparison of in situ hybridization (ISH) expression patterns of miR-3099 in both published and in-house-generated ISH sections with the ISH sections of target genes from the Allen Brain Atlas identified 7 target genes (Dnmt3a, Gabpa, Gfap, Itga4, Lxn, Smad7, and Tbx18) having expression patterns complementary to miR-3099 in the developing and adult mouse brain samples. Of these, we validated Gfap as a direct downstream target of miR-3099 using the luciferase reporter gene system. In conclusion, we report the successful prediction and validation of Gfap as an miR-3099 target gene using a combination of bioinformatics resources with enrichment of annotations based on functional ontologies and a spatio-temporal expression dataset
Construction and validation of a mammalian expression vector for in utero electroporation study of miR-3099 in the mouse neocortex
Get PDFIntroduction: MiR-3099 was reported to play a role in neuronal cell differentiation/function in the brain during late embryonic and early neonatal development. To further explore its potential regulatory effects on embryonic brain development, this study aims to construct and validate an expression vector of miR-3099 for future gain-of-function and loss-of-function studies. Methods: pCAG-eGFP vector was modified to include IRES2 and miR-3099 with 150bp upstream and downstream genomic sequences. The newly constructed vector, pCAG-miR-3099-IRES2-eGFP, consists of CAG promoter. The in vitro expression level of miR-3099 was measured using stem-loop RT-qPCR after it was transfected into 293FT cell. Later, the vector was electroporated into the embryonic brain at E15.5. Three days later, the E18.5 embryonic brain was harvested and cryopreserved. Immunohistochemistry was performed by using antibody against eGFP to validate the in utero expression of the transgene in the neocortex of the brain. Results: Our finding showed that, the expression level of miR-3099 was significantly upregulated (p<0.001) in cells transfected with miR-3099 vector as compared to both negative and empty plasmid control groups. In addition, the expression of eGFP was noted in the brain section indicating that the vectors with or without miR-3099 transgene were successfully transfected into and expressed in the neocortex upon electroporation. Conclusion: The bicistronic expression vector of miR-3099 which was driven by the CAG promoter was successfully constructed, validated and sufficiently delivered to brain cells via the in utero electroporation approach. The regulatory roles of miR-3099 in embryonic brain development can be manipulated using similar approach
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