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Neurosphere cultures transfected with nonsense & miRNA-335 knockout reagents

ABSTRACT: To understand the mechanism by which miR-335 acts anti proliferative factor, we profiled mRNA expression using microarray platform ( codelink array). Embryonic day 12.5 neural cortical epithelium was isolated and grown as neurospheres in defined medium. Cultures were transfected with 2'-O-methyl (2'OMe)-base modified miRNA-335 antisense (2'OMe-335) and control (2' O Me-EGFP). To verify the suppression of miR-335 after the 2'-OMe treatment, we used real time RT-PCR which confirmed the down regulation of miR-335 up to ~4.5 cycles (~24 fold). For the microarray experiment, mRNA isolated from these cells was hybridized to codelink microarrays. Three independent replicate were done for treatment and control, six independent single channel intensity values were uploaded after normalization for analysis. Our array results indicated that there is general up-regulation in gene expression due to miR-335 Knock Out ( KO). We identified 116 genes statistically significant genes (adjusted p value < 0.05), out of these 95.7% were up-regulated genes. The highest fold difference we observed was between 2 and 3 fold, indicating the role of this miRNA in moderately controlling expression hundreds of genes. Of the several genes the highly expressed are important in maturation of neural stem cells and downstream signaling pathway genes

ORGANISM(S): Mus musculus

PROVIDER: GSE14624 | GEO | 2019/07/31

REPOSITORIES: GEO

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Project description:The role of microRNAs (miRNAs) in multiple myeloma (MM) has yet to be fully elucidated. To identify miRNAs that are potentially deregulated in MM, we investigated those mapping within transcription units, based on evidence that intronic miRNAs are frequently coexpressed with their host genes. To this end, we monitored host transcript expression values in a panel of 20 human MM cell lines (HMCLs) profiled on Affymetrix U133A oligonucleotide microarrays and focused on transcripts whose expression varied significantly across the dataset. We identified transcripts specific to six miRNA host genes (CCPG1, GULP1, EVL, TACSTD1, MEST, and TNIK) whose average changes in expression varied at least 2-fold from the mean of the examined dataset. We evaluated the expression levels of the corresponding intronic miRNAs by quantitative real-time RT-PCR. There was a significant correlation between the expression levels of MEST, EVL, and GULP1 and those of the corresponding miRNAs miR-335, miR-342-3p, and miR-561, respectively. Genome-wide profiling of the 20 HMCLs with the Affymetrix GeneChip human mapping 250K array set indicated that the increased expression of the three host genes and their corresponding intronic miRNAs was not correlated with local copy number variations. Notably, miRNAs and their host genes were overexpressed in a fraction of primary tumors with respect to normal plasma cells; however, this finding was not correlated with known molecular myeloma groups. The predicted putative miRNA targets, as well as the transcriptional profiles associated with the primary tumors, suggest that MEST/miR-335 and EVL/miR-342-3p may play a role in plasma cell homing and/or interactions with the bone marrow microenvironment. Our data support the idea that intronic miRNAs and their host genes are regulated dependently, and may contribute to the understanding of their biological roles in cancer. To our knowledge, this is the first evidence of deregulated miRNA expression in MM, providing insights that may lead to the identification of new biomarkers and altered molecular pathways of the disease. Keywords: integrative genomic analysis of miR-335, miR-342, and miR-561 in multiple myeloma

Project description:Human mesenchymal stem cells (hMSC) have an extensive potential for clinical applications in cell therapy. However, very little is known of the specific molecular regulatory mechanisms that control the therapeutical properties of these cells. We aimed to identify microRNAs (miRNAs) that could be involved in controlling the transition between the self-renewing (undifferentiated) and the reparative (differentiated) phenotypes of hMSCs. MicroRNA microarrays were used to identify miRNAs that are upregulated in undifferentiated hMSCs. For that, we compared the miRNA expression profiles of undifferentiated bone marrow-derived hMSCs with the same primary cell lines after 9 days of in vitro adipogenic or osteogenic induction. We also compared the miRNA expression profiles of undifferentiated hMSCs with skin fibroblasts (a mesenchymal cell lineage with a more restricted differentiation potential). These experiments allowed us to identify miR-335 as the only miRNA downregulated upon MSC differentiation as well as in MSCs in comparison with skin fibroblasts. Gene expression microarrays were used to identify genes that are downregulated in hMSCs overexpressing miR-335. We compared the miRNA expression profiles of hMSCs transduced with a lentiviral vector encoding miR-335 with MSCs transduced with a control lentiviral vector. Our results suggest miR-335 downregulation could be one of the triggers for the initiation of MSCs activities involved in tissue repair and remodelling, including cell migration and differentiation. We compared the miRNA expression profiles of undifferentiated bone marrow-derived hMSCs with the same primary cell lines after 9 days of adipogenic or osteogenic induction, as well as with skin fibroblasts. A total of four independent samples were used for each condition. For the adipogenic/osteogenic vs. undifferentiated MSC comparison, the RNA samples were pooled (two independent samples/pool) before labeling. We also compared the miRNA expression profiles of hMSCs transduced with the lentiviral vector pLV-EmGFP-MIRN335 with MSCs transduced with the control vector pLV-EmGFP-Mock. For the gene expression microarrays, a total of three independent samples were used for each condition.

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Neurosphere cultures transfected with nonsense & miRNA-335 knockout reagents

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