Effect of miR-34a overexpression on the transcriptome of HEK293T cells
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PROVIDER: PRJNA1145337 | ENA |
REPOSITORIES: ENA
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Project description:MicroRNAs regulate gene expression post-transcriptionally through binding to target sites in mRNAs. Predictions of target site efficiency are limited by our understanding of the structure-function relationship of microRNA-target complexes. Here, we overexpressed miR-34a in HEK293T cells and performed RNA-sequencing in order to study how different structural features of predicted miRNA-target complexes correlate with changes in gene expression.
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Project description:MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of nearly all biological processes. Global miRNA biogenesis is altered in many cancers and RNA-binding proteins (RBPs) have been shown to play a role in this process, presenting a promising avenue for targeting miRNA dysregulation in disease. miR-34a exhibits tumor-suppressive functions by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor Bcl-2, among other regulatory pathways such as Wnt, TGF-, and Notch signaling. Many cancers show downregulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo; however, the post-transcriptional mechanisms by which miR-34a is lost in cancer are not entirely understood. Here, we have used a proteomics-mediated approach to identify Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a-binding protein. SART3 is a spliceosome recycling factor and nuclear RBP with no previously reported role in miRNA regulation. We demonstrate that SART3 binds pre-miR-34a with specificity over pre-let-7d and begin to elucidate a new functional role for this protein in non-small lung cancer cells. Overexpression of SART3 led to increased miR-34a levels, downregulation of the miR-34a target genes CDK4 and CDK6, and cell cycle arrest in the G1 phase. In vitro binding studies showed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Collectively, our results present evidence for an influential role of SART3 in miR-34a biogenesis and cell cycle progression.
2019-10-14 | PXD015278 | Pride
Project description:Here we show that biotin-labelled miR-34a can be loaded to AGO2, and AGO2 immunoprecipitation can pulldown biotinylated miR-34a (Bio-miR pulldown). RNA-sequencing (RNA-seq) of the Bio-miR pulldown RNAs efficiently identified miR-34a mRNA targets, which could be verified with luciferase assays. In contrast to the approach of Bio-miR pulldown, RNA-seq of miR-34a overexpression samples had limited value in identifying direct targets of miR-34a. It seems that pulldown of 30 -Biotin-tagged miRNA can identify bona fide microRNA targets at least for miR34a.
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Project description:Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. Through a gene expression profile (GEP) study we recently highlighted the upregulationof miR-34a-5p in PMF versus healthy donor (HD) CD34+ hematopoietic progenitor cells (HPCs). To shed some light into the role of miR-34a-5p in PMF pathogenesis, here we unravelled the effects of the overexpression of miR-34a-5p in HPCs forcing its expression in HPCs. We showed that enforced expression of miR-34a-5p blocks proliferation and favours the megakaryocyte and monocyte/macrophage commitment of HPCs. Interestingly, we identified lymphoid enhancer-binding factor 1 (LEF1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts as miR-34a-5p-targets downregulated after miR-34a-5p overexpression in HPCs as well as in PMF compared with HD HPCs. Remarkably, the knockdown of NR4A2 in HPCs mimicked the antiproliferative effects of miR-34a-5p overexpression, while the silencing of LEF1 phenocopied the effects of miR-34a-5p overexpression in HPCs lineage choice, by stimulating the megakaryocyte and monocyte/macrophage commitment.
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Project description:MiR-34a appears to be protective against cancer. Blocking miR-34a results in pro-tumorigenic phenotypic changes. The purpose of this experiment was to identify gene expression changes that could potentially explain the pro-tumorigenic effects of anti-miR 34a transfection.
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Project description:miR-34a and miR-34c were found up-regulated at wound-edges of human venous ulcer compared to nomal wound and the intact skin; however their biological role in keratinocytes during wound repair has not been studied. To study the genes regulated by miR-34a and miR-34c, we transfected miR-34a and miR-34c mimic into human primary epidermal keratinocytes to overexpress them. We performed a global transcriptome analysis of keratinocytes upon overexpression of miR-34a or miR-34c using Affymetrix arrays.
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Project description:Previous studies have evaluated the role of miRNAs in the initiation and progression of cancer. MiR-34a was found to be downregulated in several tumors, including medulloblastoma. We here analysed the function of miR-34a in vivo by targeted transgenesis to generate mice with constitutive deletion of the miR-34a gene, which resulted in the absence of mir-34a in all analysed tissues. Nevertheless, these mice were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters performed by the German Mouse Clinic revealed no apparent phenotype. Analysis of miR-34a expression in human medulloblastomas and medulloblastoma cell lines revealed significant downregulation as compared to human cerebellum. Re-expression of mir-34a in human medulloblastoma cells in vitro reduced cell viability, cell proliferation and induced apoptosis. Among the targets downregulated by miR-34a in human medulloblastoma cells were NMYC and SIRT1. Activation of the Shh pathway by targeted overexpression of SmoA1 causes medulloblastoma in mice, which is dependent on the presence and upregulation of NMYC. Analysis of miR-34a in ND2:SmoA1-derived medulloblastomas revealed significant suppression of miR-34a compared to normal cerebellum. Crossbreeding these mice with miR-34a knockout mice significantly accelerated medulloblastoma growth in mice deficient for miR-34a. Interestingly, NMYC and SIRT1 were highly expressed in medulloblastomas derived from these mice. We here demonstrate that miR-34a is dispensable for normal development, but that its loss accelerates medulloblastoma. Strategies aiming to re-express miR-34a in tumors could therefore represent an efficient therapy option.
2014-09-22 | GSE40511 | GEO
Project description:In order to examine the consequences of human miR-34a induction on the transcriptome, HCT116 cells (a colon cancer cell line) were infected with a retrovirus that produces miR-34a. Gene expression profiles were then monitored using Affymetrix microarrays. Affymetrix microarrays were used to examine the transcriptomes of HCT116 cells infected with an empty retroviral vector (pMSCV-PIG) or a retroviral vector that expresses human miR-34a. Keywords: comparison of cells with or without enforced miR-34a expression
2007-06-07 | GSE7754 | GEO
Project description:To clarify the effect of miRNAs, we carried out a gene expression microarray analysis using GIST-T1 cells transfected with a miR-34a mimic or a negative control. We found that 2,621 probe sets (1,933 unique genes) were downregulated (>1.5-fold) by ectopic miR-34a expression, including PDGFRA gene which was previously reported as a miR-34a target gene.
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Project description:miR-34a and miR-34b/c genes are frequently epigenetically silenced in primary CRCs. However, the in vivo relevance of miR-34a/b/c for suppression of intestinal tumor formation has not been analyzed by genetic approaches. ApcMin/+ mice with deletion of the miR-34a and miR-34b/c genes were generated and analyzed. The mRNA expression profiles of intestinal adenomas with and without functional miR-34a/b/c genes were compared.
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