Comparison of miR-181a transfected K562 cells and mock transfected K562 cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE3605 | GEO | 2005/11/18
SECONDARY ACCESSION(S): PRJNA93707
REPOSITORIES: GEO
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Project description:This series consists of samples taken from two groups of K562 cells(miR-181a transfected group and control group ) and harvested 48 hours later. We used Agilent human 1A oligo microarray identified the changes in gene expression profile of K562 cells after miR-181a transfection experiment. Further studies aimed to find target mRNA of miR-181a in mammalian cells and its biological function. Experiment Overall Design: The K562 cells in our research were divided into tranfected group and mock-transfected control according to Lim et al. Experiment Overall Design: To transfect, we diluted 60 pmol miR-181 a duplex into 12 µL Opti-Mem each well, then we diluted 3 µL Oligofectamine(Invitrogen) into 48 µL Opti-Mem for each sample, incubated for 5 min at room temperature and add 10 µL of diluted transfection mixture to wells and incubate for another 20 min at room temperature. We next added 100 µL of diluted cells suspension mixture containing 50000 cells on top of the complex. Four hours after transfection, FBS was added to the cells at a final concentration of 10% and the cells were incubated for 48 hours. And control group was transfected only with Oligofectamine but no RNA. Each individual transfection experiment was performed in 10 replicate wells. Experiment Overall Design: After transfection experiments, total RNAs from above two group K562 cells were purified using the RNeasy mini kit (Qiagen, Heidelberg, Germany) in accordance with the manufacturerâs protocols. RNA concentration and purity were estimated spectrophotometrically and the quality further assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Experiment Overall Design: Total RNA from control group and transfected group were respectively reverse transcription, in vitro transcribed and labeled (Cy3 and Cy5) according to the Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol, green is control group, red is transfected group. The array hybridization, washing, and scanning procedures were performed in our lab according to Agilent 60-mer oligo microarray processing protocol using 0.75 µg samples of labeled, fragmented cRNA on Agilent Human 1A oligo microarray with 22575 probes (Agilent Technologies). Labeled cRNA is incubated with the microarray in a hybridization chamber at 60ºC for 17 hours, 4 rpm/min. The microarray was scanned using Agilent 2565BA DNA microarray scanner (Agilent Technologies). Data collection was performed by Agilent DNA microarray scanner. Using the Agilent Feature Extraction Software(v.7.5), spots were identified, measured for fluorescence intensity, local background, and arraywide parameters used to normalize the signals. Outliers and saturated spots were rejected according to 1.42xIQR, and local backgroud subtracted from the signals. Signals were then normalized to remove dye bias and arraywide drift in flourescence. Experiment Overall Design: The following two tests are performed to determined the significance of a feature: (1) Log ratio P-value of the red over green processed signals was calculated using two-sided t-test with P<0.01 considered significant, and (2) a boolean flag of 1 indicated that the feature BGSubSignal is well above background and passes the IsPosAndSignif test. Experiment Overall Design: We used Agilent human 1A oligo microarray identified the changes in gene expression profile of K562 cells after miR-181a transfection experiment. Experiment Overall Design: Further studies aimed to find target mRNA of miR-181a in mammalian cells and the function of miR-181a in cancers.
2008-06-12 | E-GEOD-3605 | biostudies-arrayexpress
Project description:Background: Oral squamous cell carcinoma (OSCC) is a common malignant tumor associated with poor prognosis. MicroRNAs (miRNAs) play crucial regulatory roles in the cancer development. However, the role of miRNAs in OSCC development and progression is not well understood. Methods: We sought to establish a dynamic Chinese hamster OSCC animal model, construct miRNA differential expression profiles of its occurrence and development, predict its targets, and perform functional analysis and validation in vitro. Results: Using expression and functional analyses, the key candidate miRNA (miR-181a-5p) was selected for further functional research, and the expression of miR-181a-5p in OSCC tissues and cell lines was detected. Subsequently, transfection technology and a nude mouse tumorigenic model were used to explore potential molecular mechanisms. miR-181a-5p was significantly downregulated in human OSCC specimens and cell lines, and decreased miR-181a-5p expression was observed in multiple stages of the Chinese hamster OSCC animal model. Moreover, upregulated miR-181a-5p significantly inhibited OSCC cell proliferation, colony formation, invasion, and migration; blocked the cell cycle; and promoted apoptosis. BCL2 was identified as a target of miR-181a-5p. BCL2 may interact with apoptosis- (BAX), invasion- and migration- (TIMP1, MMP2, and MMP9), and cell cycle-related genes (KI67, E2F1, CYCLIND1, and CDK6) to further regulate biological behavior. Tumor xenograft analysis indicated that tumor growth was significantly inhibited in the high miR-181a-5p expression group. Conclusions: Our findings indicate that miR-181a-5p can be used as a potential biomarker and provide a novel animal model for mechanistic research on oral cancer.
2023-01-10 | GSE222429 | GEO
Project description:HEK293T cell expression data from four conditions: mock transfection, Ago2 transfection, Ago2 & miR-1 transfection, and Ago2 & miR-124 transfection. RNA was isolated directly from 10cm dishes using Trizol reagent and amplified. Set of arrays that are part of repeated experiments Compound Based Treatment: FLAG-Ago2 transfection Keywords: Biological Replicate
2008-04-08 | GSE11080 | GEO
Project description:Overexpression of miR-181a in AML blasts: DNA Microarray was performed to compare overall gene expression profiles of KG-1 Anti-miR-CT and U937 Anti-miR-CT cells compared respectively to KG-1 Anti-miR-181a and U937 Anti-miR-181a.
2014-12-30 | E-MTAB-2837 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) are expressed from a class of small non-protein coding RNA molecules. Growing evidence shows that miRNAs are potent mediators of post-transcriptional gene silencing and emerged to be critical in the regulation of innate and adaptive immune responses (Ansel KM, Immunol Rev 2013, p5; Baumjohann D, Nature Rev Immunol 2013, p666). MicroRNA-181a (miR-181a) constitutes the most prominently expressed miRNA species in DP thymocytes (Neilson JR, Genes Dev 2007, p578; Kirigin FF, JI 2012, p3257) and has been associated with modulating TCR signal strength via targeting serine/threonine as well as tyrosine phosphatases (Li Q-J, Cell 2007, p147). Consequently, elevated expression of miR-181a results in reduced phosphatase activity and increased TCR signal strength. The effect of aberrant expression of miR-181a on TCR signaling has been already analyzed employing short-term assays and in vitro organ cultures. Our group started to investigate the consequences of miR-181a/b-1 deletion on T cell development in vivo in the steady state. To this end, we developed a new mouse model - miR-181a/b-1 knockout mice (Zietara N et al, PNAS 2013b). During investigations of the immune system of miR-181a/b-1 deficient mice we discovered that these miRNAs are critical for the development of invariant natural killer (NK) T cells. Such cells are known to be selected by a high affinity agonist ligands, thus they are particularly sensitive to the modulation of TCR signaling thresholds, which is achieved by miR-181a/b-1 (Zietara N et al, PNAS 2013b). Furthermore we hypothesized that similar regulation might apply for the other T cell populations selected under high TCR signal strength, like Treg cells. Thus our current research focuses on the role of miR-181a/b-1 during Treg cell development in the thymus as well as on their function in the periphery.
2019-02-08 | GSE115391 | GEO
Project description:We have identified a single miRNA, miR-181a, that can modulate TGF-β signaling to induce and maintain EMT, and effect further downstream events of tumour cell survival, altered response to chemotherapy, migration, invasion and dissemination in vivo. Our present study provides an understanding of how enhanced expression of miR-181a can confer malignant and invasive traits through the modulation of a canonical signaling pathway and a consequent maintenance of a mesenchymal state. Furthermore, inhibition of miR-181a led to a reversion of EMT and subsequent events through decreased TGF-β signaling. Our data confirmed Smad7 as a functional target through which TGF-β-mediated EMT occurs; re-expression of Smad7 lacking its 3'UTR was able to rescue miR-181a-mediated phenotypes, deeming Smad7 as a critical mediator of miR-181a-induced EMT. Other recent studies support the crucial role(s) that miRNAs play in mediating EMT and consequent aggressive disease traits. For example, the miR-106b-25 cluster has also been shown to target Smad7 and mediate TGF-β-induced EMT downstream to Six1 in breast cancer34. miR-9 directly targets E-cadherin and inhibition of miR-9 had led to an inhibition of metastasis35. Conversely, the miR-200 and -205 family was shown to target transcriptional repressors of E-cadherin, ZEB1 and SIP1, and re-expression of these miRNAs led to a mesenchymal-to-epithelial transition and prevented TGF-β -induced EMT36. A2780 ovarian cancer cell lines stably expressing either pBABE (control vector), p181a#1( clone 1 expressing miR-181a) or p181a#2( clone 2 expressing miR-181a)
2013-12-01 | E-GEOD-52077 | biostudies-arrayexpress
Project description:HEK293T cell expression data from four conditions: mock transfection, Ago2 transfection, Ago2 & miR-1 transfection, and Ago2 & miR-124 transfection. RNA was isolated directly from 10cm dishes using Trizol reagent and amplified. Set of arrays that are part of repeated experiments Compound Based Treatment: FLAG-Ago2 transfection Keywords: Biological Replicate Biological Replicate Using regression correlation
2008-04-07 | E-GEOD-11080 | biostudies-arrayexpress
Project description:microRNAs are small (22-24 nucleotide), non-coding RNA transcripts that play a role in RNA silencing and post transcriptional regulation of gene expression. They have been implicated to have profound roles in many cancers. The role of microRNAs in chondrosarcoma progression is not well understood. Our lab has identified miR-181a to be up-regulated in chondrosarcoma. miR-181a modulates CXCR4 signaling by targeting RGS-16, leading to increased levels of VEGF and MMP-1, major factors in tumor invasion. In an effort to identify other targets of miR-181a apart from RGS-16, we carried out a gene array to compare gene expression between untreated/control CS-1(chondrosarcoma cell line) cells and CS-1 cells transduced with a lenti virus expression construct with anti-miR-181a sequence.
2018-06-19 | GSE70065 | GEO
Project description:The generation of highly-reactive T lymphocytes against tumor-associated antigens remains an obstacle for effective adoptive immunotherapy of cancer. Recently, we found that microRNA-181a (miR-181a) acts as an intrinsic modulator of T cell antigen sensitivity in a transgenic T cell line in vitro. This molecule is part of a growing family of evolutionarily selected small interfering RNA that control gene expression at the posttranscriptional level by targeting messenger RNA (mRNA) for degradation or translational repression. Here, we explored the use of miR-181a to improve anti-tumor T cell responsiveness against weakly immunogenic tumor-associated antigens. We found that genetic engineering of T lymphocytes with miR-181a dramatically augmented the function of poorly responsive human tumor-infiltrating lymphocytes and TCR-engineered peripheral blood lymphocytes, resulting in potent anti-tumor reactivity. Furthermore, in a mouse model, miR-181a increased the function of self/tumor-specific CD8+ T cells enabling effective tumor destruction in the absence of vaccination or exogenous cytokines that were otherwise essential requirements and thus represents a significant advance over previous approaches. Although miRNAs have been previously shown to play critical functional roles in the development and function of vertebrate immune systems and pathogenesis of cancer, this report comprises the first use of a miRNA gene as tool in the treatment of disease. Keywords: cellular modification design
2008-10-01 | GSE9240 | GEO
Project description:miR-34a is strongly induced upon TPA-induced megakaryocyte differentiation of K562 cells. To investigate the gene networks regulated by this miRNA during the process of differentiation we performed gene microarray analysis in K562 cells overexpressing miR-34a or a control sequence. Experiment Overall Design: K562 cells were transfected by nucleofection (Amaxa) with a miR-34a mimic (Dharmacon) or a control sequence with no homology to human genes. 24 hours post-transfection total RNA was extracted using trizol and hybridized on Affimetrix microarrays for comparing gene expression.
2009-07-19 | E-GEOD-16674 | biostudies-arrayexpress