Effects of miR-26a expression and miRNA-sponge-mediated miR-26 family knockdown on the transcriptome of B cell precursors
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PROVIDER: PRJNA773055 | ENA |
REPOSITORIES: ENA
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Project description:Here, we have screened a miRNA expression library in a pre-B cell expression system to identify miRNAs with a potential oncogenic activity in early lymphocytes. We show that miR-26a is sufficient to transform B cell precursors in vitro as defined by growth factor independence, and demonstrate that its continuous expression is necessary to keep cells in the transformed state. Mechanistically, we find that expression of miR-26a protects against apoptosis induced by growth factor withdrawal as well as DNA damage, provides a competitve advantage and interferes with the differentiation of pre-B to immature B cells. In consequence, transformed cells express the pre-BCR on the cell surface, which appears to provide signals for ongoing proliferation and survival. Using microarray analysis, we identified several previously known and novel putative target genes of miR-26a in B cell precursors. A key target appears to be PTEN, a well established tumor suppressor and regulator of PI3K signaling, as knockdown of Pten mRNA by shRNA at least partially phenocopies miR-26a overexpression. To gain inside into the physiological role of miR-26 in early B cell develobment, in contrast to the oncogenic situation if aberrantly overexpressed, we further more sequestered endogenous miR-26a and b by expression of a miRNA sponge. This functional knockdown indeed induced the reciprocal phenotype relative to the overexpression, i.e. cells had a proliferative disadvantage and differentiated more efficient into immature B cells. Surprisingly, however, transcriptional changes induced by miR-26 sequestration were very mild. We therefoer hypothesize that most of the sponge-mediated deregulation is below detection level while still being functional relevant.
2022-01-01 | GSE186236 | GEO
Project description:To reveal the potential regulation target genes of miR-26a and miR-23a/b clusters in articular chondrocytes, we performed a multi-omics analysis of LC-MSMS and RNA-seq using cultured chondrocytes samples, which were primarily isolated from 3-week-old wild-type, miR-26a -/- (with or without miR-26a mimic transfection afterwards) or miR-23a/b cluster flox/flox;Col2a1-cre mice. For LC-MSMS, protein from TRIZOL reagent was extracted, nanoLC-MSMS was performed. An expression list was made to further explore the regulation targets of miR-26a and miR-23a/b clusters.
2023-01-25 | PXD031868 | Pride
Project description:A common experimental setup to investigate the function of a microRNA is a â??gain-of-functionâ?? approach, in which the microRNA of interest is overexpressed to analyze its impact on a given process. However, a major drawback of this technique is that microRNAs, when expressed significantly above endogenous levels, may target genes that are not affected under physiological conditions. To circumvent this problem, we have generated a retroviral microRNA-knockdown library encompassing the majority of the microRNA families expressed throughout lymphocyte development. MicroRNA knockdown is mediated by sponges, mRNAs that possess multiple binding sites for a certain microRNA family in their 3â??-UTR region. MiRNA sponges function as competitive inhibitors that sequester all specific microRNA/RISC complexes, thereby de-repressing the endogenous target genes. Using this library in pre-B cells as a model system, we have characterized all microRNAs expressed in early B cells development according to their impact on processes such as apoptosis and differentiation. In doing so, we show that functional knockdown of the known tumor-suppressive miR-15 family has only slight effects under cellular steady-state conditions. Upon withdrawal of the growth factor IL-7, however, loss of miR-15 function almost completely inhibits pre-B to immature B cell differentiation, partially protects from apoptosis and enables prolonged proliferation. In correspondence with that, knockdown of the miR-15 family confers a competitive advantage in suboptimal IL-7 concentrations, indicating that levels of miR-15 determine the sensitivity of cells towards growth factor receptor signaling. Moreover, IL-7 withdrawal seems to increase the activity of endogenous miR-15 family members in pre-B cells. Using microarray analysis, we have identified cyclin E1 as a direct and cyclin D3 as an indirect putative target gene of the miR-15 family in B cell precursors. Exogenous expression of cyclin E1 or cyclin D3 recapitulates the miR-15 loss-of-function phenotype in B cell precursors, validating their biological importance. Together, this suggests that the miR-15 family functions as a central element in a positive feedback loop that reinforces cell-cycle arrest at low growth factor concentrations, which is considered a prerequisite for light chain gene recombination and differentiation. Gene expression in cells expressing a scrambled sponge as a control vector or a sponge designed to sequester all members of the miR-15 family (miR-15a, miR-15b, miR-16, miR-497, miR-195, miR-322) was measured either in the presence or 36 hours after withdrawal of the growth factor IL-7. Two independent experiments were performed, using independently generated cell clones for each repetition.
2016-07-03 | E-GEOD-72767 | biostudies-arrayexpress
Project description:RNA sequencing technology has been carried out in order to evaluate mRNA expression changes after manipulation of miR-26a in both MCF-7 and MDA-MB-231 breast cancer cell lines. To evaluate the entire set of genes modulated by miR-26a in breast cancer, we performed RNA-seq after ectopic manipulation of this miRNA. We over-expressed miR-26a in MCF-7 epithelial cancer cell lines and also reduced its activity by stably transfecting MDA-MB-231 mesenchymal-like cancer cell lines with a specific sponge vector. GO terms and pathway enriched analysis of the transcripts that significantly change upon miR-26 ectopic manipulation implicates miR-26ab in cell cycle, apoptosis, cell spreading and cell adhesion in breast cancer
2016-06-30 | E-GEOD-43726 | biostudies-arrayexpress
Project description:In this work, we showed that the re-expression of miR-26a in DU-145 prostate cancer cells restored the tumor suppressor activity of miR-26a. To discover the genes and pathways elicited by miR-26a re-expression, we used the miRNA pull out assay to capture and the Next Generation Sequencing to identify the miR-26a targets. Data showed that: i) miR-26a captured both non-coding and coding RNAs; ii) 46% of transcripts were putative miR-26a targets according to target prediction algorithms; iii) 21 pathways were significantly enriched and the “Pathway in Cancer” was among those comprising the largest number of genes, including BIRC5 that we experimentally validated. Accordingly, the detection of cell proliferation-related events showed that miR-26a exerted its tumor suppressor activity at several levels, by decreasing the survival, impairing the migration of tumor cells and by inducing both apoptosis and cell cycle block. In conclusion, we showed that the collection of miR-26a interacting transcripts (miR-26a/targetome) represented a fruitful platform to decipher the miR-26a-dependent gene expression networks. In perspective the availability of miRNA-specific and tumor-specific targetomes will allow the discovery of new druggable tumor genes and pathways.
2017-04-04 | GSE85306 | GEO
Project description:Human multipotent adipose-derived stem (hMADS) cells were transfected at confluence with miR-26a or miR-C (non-targeting control microRNA) and cells were harvested two days later (=d0) to identify mRNAs that are downregulated by miR-26a versus miR-C, as such mRNAs could be direct miR-26a targets.
2013-11-25 | E-MARS-28 | biostudies-arrayexpress
Project description:We performed transcriptome analyses of miR-26a-overexpressing RAW264.7 cells (RAW264.7.miR-26a.OE) and NC-plasmid-transfected RAW264.7 cells (RAW264.7.NC), using RNA-seq . We identified 121 genes that were downregulated in RAW264.7.miR-26a.OE cells compared with RAW264.7.NC cells. Among the downregulated genes, we identified four putative target genes, using TargetScan (www.targetscan.org), including EphA2, Map3k9, Nhsl1, and Otud1, which all contain a putative miR-26a seed sequence.
2022-06-01 | GSE149842 | GEO
Project description:Through a series of mouse genetic studies, we concluded that miR-26 overexpression in intestinal epithelial cells inhibits tumor formation in Apcmin/+ mice. We isolated intestinal epithelial cells from M2rtTA and M2rtTA; eGFP.miR-26a to assess gene expression changes after the induction of miR-26a expression.
2014-10-30 | GSE62831 | GEO
Project description:miRNA sponge, a special class of miRNA target, has been emerging as a pivotal player in miRNA mediated regulatory network. Currently, the identified miRNA sponge genes mostly act on sequestering conserved miRNAs (e.g. miR-7, miR-145), however, the existence, potential function and evolutionary process of miRNA sponge genes for species-specific miRNA, especially for human specific miRNA, are largely unknown. In this study, we conducted a systematic analysis including sponge gene identification and subsequent function and evolutionary analyses for an authentic human-specific miRNA, miR-941.
2019-01-01 | GSE93716 | GEO
Project description:Objective: Trastuzumab has been used for the treatment of HER2-positive breast cancer (BC). However, a subset of BC patients exhibited resistance to trastuzumab therapy. Thus, clarifying the molecular mechanism of trastuzumab treatment will be beneficial to improve the treatment of HER2-positive BC patients. In this study, we identified trastuzumab-responsive microRNAs that are involved in the therapeutic effects of trastuzumab. Methods and results: RNA samples were obtained from HER2-positive (SKBR3 and BT474) and HER2-negetive (MCF7 and MDA-MB-231) cells with and without trastuzumab treatment for 6 days. Next, we conducted a microRNA profiling analysis using these samples to screen those microRNAs that were up- or downregulated only in HER2-positive cells. This analysis identified miR-26a and miR-30b as trastuzumab-inducible microRNAs. Transfecting miR-26a and miR-30b induced cell growth suppression in the BC cells by 40% and 32%, respectively. A cell cycle analysis showed that these microRNAs induced G1 arrest in HER2-positive BC cells as trastuzumab did. An Annexin-V assay revealed that miR-26a but not miR-30b induced apoptosis in HER2-positive BC cells. Using the prediction algorithms for microRNA targets, we identified cyclin E2 (CCNE2) as a target gene of miR-30b. A luciferase-based reporter assay demonstrated that miR-30b post-transcriptionally reduced 27% (p=0.005) of the gene expression by interacting with two binding sites in the 3’-UTR of CCNE2. Conclusion: In BC cells, trastuzumab modulated the expression of a subset of microRNAs, including miR-26a and miR-30b. The upregulation of miR-30b by trastuzumab may play a biological role in trastuzumab-induced cell growth inhibition by targeting CCNE2.
2012-06-03 | GSE38415 | GEO