MicroRNAs miR-186 and miR-150 Down-regulate Expression of the Pro-apoptotic Purinergic P2X7 Receptor by Activation of Instability Sites at the 3′-Untranslated Region of the Gene That Decrease Steady-state Levels of the Transcript

Department of Reproductive Biology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2008; 283(42):28274-86. DOI: 10.1074/jbc.M802663200
Source: PubMed

ABSTRACT The P2X7 receptor regulates cell growth through mediation of apoptosis. P2X7 levels are lower in cancer epithelial cells than in normal cells, and previous studies showed that expression of P2X7 was regulated post-transcriptionally. The objective of the study was to understand regulation of P2X7 mRNA stability. Overexpression of a reporter containing the full-length human P2X7 3'-untranslated region (3'-UTR) or reporters containing parts of the 3'-UTR-P2X7 were associated with increased abundance of the construct in normal cells and decreased abundance in cancer epithelial cells. Sequences within the 3'-UTR-P2X7, which are putative target sites for the microRNAs, miR-186 (middle segment) and miR-150 (distal segment), decreased the abundance of the P2X7 transcript. Overexpression in cancer cells of mutated miR-186 and miR-150 target sites was associated with lower levels of the reporter genes. In normal cells overexpression of the mutated miR-186 target site was associated with marked increased concentration, but overexpression of the miR-150 target site reporters, wild-type and mutant, did not change over time. Levels of miR-186 and miR-150 were higher in cancer than in normal cells, and treatment with miR-186 and miR-150 inhibitors increased P2X7 mRNA. In human embryonic kidney-293 cells heterologously expressing the full-length 3'-UTR-P2X7 luciferase reporter, miR-186 and miR-150 inhibitors increased luciferase activity, whereas miR-186 and miR-150 mimics decreased luciferase activity after actinomycin D treatment. These data suggest that increased expression of miR-186 and miR-150 in cancer epithelial cells decreases P2X7 mRNA by activation of miR-186 and miR-150 instability target sites located at the 3'-UTR-P2X7.

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    • ". We also detected upregulated miR- 181b-5p and −186-5p, which may suppress plasminogen activator inhibitor-1 in VSMCs (Chen et al. 2014) and promote apoptosis (Zhou et al. 2008), thus interfering with extracellular matrix degradation, and structural and functional changes in VSMCs. miR-155 is highly expressed in various cell types including VSMCs and endothelial cells (Faraoni et al. 2009). "
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    ABSTRACT: Atherosclerosis is an inflammatory disease with possible contributions from bacterial antigens. We aimed to investigate the role of oral bacteria as inducers of inflammatory cascades in smooth muscle cells from carotid endarterectomy patients (AthSMCs) and healthy controls (HSMCs). Inactivated Streptococcus mitis, S. sanguinis, S. gorgonii, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were used to stimulate inflammation in HSMCs and AthSMCs. Tumor necrosis factor-α (TNFα) was used as a positive control in all stimulations. Interleukin-6 (IL-6) levels were determined from cell culture supernatants and microRNA expression profiles from cells after 24 h of bacterial stimulation. Genome wide expression (GWE) analyses were performed after 5 h stimulation. All studied bacteria induced pro inflammatory IL-6 production in both SMCs. The most powerful inducer of IL-6 was A. actinomycetemcomitans (p < 0.001). Of the 84 studied miRNAs, expression of 9 miRNAs differed significantly (p ≤ 0.001) between HSMCs and AthSMCs stimulated with inactivated bacteria or TNFα. The data was divided into two groups: high IL-6 producers (A. actinomytectemcomititans and TNFα) and low IL-6 producers (streptococcal strains and P. gingivalis). The expression of 4 miRNAs (miR-181-5p, -186-5p, -28-5p and -155-5p) differed statistically significantly (p < 0.001) between healthy HSMCs and AthSMCs in the low IL-6 producer group. According to multidimensional scaling, two gene expression clusters were seen: one in HSMCs and one AthSMCs. Our results suggest that inactivated oral bacteria induce inflammation that is differently regulated in healthy and atherosclerotic SMCs.
    SpringerPlus 12/2015; 4(1). DOI:10.1186/s40064-015-0993-8
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    • "October 2011 | Volume 4 | Article 28 | 12 Table A4 | Additional targets of ChE-targeting miRs (common to more than one ChE). miR ID Validated target common to ACHE-R and AChE-S Hsa-miR-186 Pro-apoptotic P2 × 7 purinergic receptor(Zhou et al., 2008) "
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    ABSTRACT: MicroRNAs (miRs) have emerged as important gene silencers affecting many target mRNAs. Here, we report the identification of 244 miRs that target the 3'-untranslated regions of different cholinesterase transcripts: 116 for butyrylcholinesterase (BChE), 47 for the synaptic acetylcholinesterase (AChE-S) splice variant, and 81 for the normally rare splice variant AChE-R. Of these, 11 and 6 miRs target both AChE-S and AChE-R, and AChE-R and BChE transcripts, respectively. BChE and AChE-S showed no overlapping miRs, attesting to their distinct modes of miR regulation. Generally, miRs can suppress a number of targets; thereby controlling an entire battery of functions. To evaluate the importance of the cholinesterase-targeted miRs in other specific biological processes we searched for their other experimentally validated target transcripts and analyzed the gene ontology enriched biological processes these transcripts are involved in. Interestingly, a number of the resulting categories are also related to cholinesterases. They include, for BChE, response to glucocorticoid stimulus, and for AChE, response to wounding and two child terms of neuron development: regulation of axonogenesis and regulation of dendrite morphogenesis. Importantly, all of the AChE-targeting miRs found to be related to these selected processes were directed against the normally rare AChE-R splice variant, with three of them, including the neurogenesis regulator miR-132, also directed against AChE-S. Our findings point at the AChE-R splice variant as particularly susceptible to miR regulation, highlight those biological functions of cholinesterases that are likely to be subject to miR post-transcriptional control, demonstrate the selectivity of miRs in regulating specific biological processes, and open new venues for targeted interference with these specific processes.
    Frontiers in Molecular Neuroscience 10/2011; 4:28. DOI:10.3389/fnmol.2011.00028 · 4.08 Impact Factor
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    ABSTRACT: ObjectiveTo investigate the effects of microRNA-15a (miR-15a) on the growth of the lymphoma Raji cell line. MethodsA eukaryotic expression vector of precursor miR-15a (pre-miR-15a) was constructed. Paired oligonucleotides for premiR-15a expression were designed and synthesized chemically. Annealed oligonucleotides were inserted into a pGCSILGFP vector under a U6 promoter to construct a pre-miR-15a expression plasmid. Oligonucleotides with a scrambled sequence which were used as a negative control were also constructed into the pGCSIL-GFP vector. A recombinant expression vector was identified through PCR and sequencing. A pre-miR-15a expression plasmid and a negative control plasmid were all transferred into Raji cells with lipofectamine 2000. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was employed to explore expression of miR-15a. The expression levels of the protein were assayed via immunofluorescence. The growth effect of Raji cells was measured by CCK8 assay. Apoptosis was determined using Hoechst dyeing and flow cytometry. The growth-inhibitory effect on Raji cells was measured using a CCK8 assay. ResultsThe results showed that the insertion sequence was correct. The expression level of miR-15a was obviously higher in Raji cells transferred with pre-miR-15a plasmid than in the blank group and in the negative control group. After Raji cells were transferred with the pre-miR-15a expression plasmid for 48 h, Bcl-2 protein expression levels were obviously decreased, which indicated that there was a significant diff erence as compared with the negative control group and blank group (P < 0.05). The CCK8 assay showed that transfection of pre-miR-15a expression plasmid decreased the cell growth at 24, 48 and 72 h post-transfection. After Raji cells were transferred with pre-miR-15a expression plasmid, apoptotic cells can be seen with Hoechst dyeing, and the cell apoptotic rate was obviously higher than that in blank group and negative control group. ConclusionPre-miR-15a can induce apoptosis and inhibit the growth of Raji cells. Key Wordshsa-mir-15 microRNA-lymphoma-cell line-Bcl-2 protein
    Clinical Oncology and Cancer Research 02/2010; 7(1):22-26. DOI:10.1007/s11805-010-0022-1
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