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


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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    • "The results reveal a new possible target for regulating Ras/ERK activity in lung cancer, which can be accomplished by affecting the miR-203 level. As the downstream signal transduction pathway of SRC, activation of Ras/ERK pathway is associated with cell transformation and tumor progression [31]. Consistent with this, we lastly showed that miR-203 could suppress SRC expression and, in turn, inhibit proliferation and migration and promote apoptosis of lung cancer cells. "
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    ABSTRACT: SRC, also known as proto-oncogene c-Src, is a non-receptor tyrosine kinase that plays an important role in cancer progression by promoting survival, angiogenesis, proliferation, and invasion pathways. In this study, we found that SRC protein levels were consistently upregulated in lung cancer tissues, but that SRC mRNA levels varied randomly, suggesting that a post-transcriptional mechanism was involved in SRC regulation. Because microRNAs (miRNAs) are powerful post-transcriptional regulators of gene expression, we used bioinformatic analyses to search for miRNAs that potentially target SRC. We identified specific targeting sites for miR-203 in the 3'-untranslated region (3'-UTR) of SRC. We then experimentally validated miR-203 as a direct regulator of SRC using cell transfection and luciferase assays and showed that miR-203 inhibited SRC expression and consequently triggered suppression of the SRC/Ras/ERK pathway. Finally, we demonstrated that the repression of SRC by miR-203 suppressed the proliferation and migration and promoted the apoptosis of lung cancer cells. In summary, this study provides the first clues regarding the role of miR-203 as a tumor suppressor in lung cancer cells through the inhibition of SRC translation.
    PLoS ONE 08/2014; 9(8):e105570. DOI:10.1371/journal.pone.0105570 · 3.23 Impact Factor
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    • "Previous studies have shown the pro-apoptotic gene P2X7 is targeted by miR-150 instably in HeLa cells or E10 cells [8], [25]. Mechanisms that induce reduced expression of P2X7 receptor in cancer epithelial cells involve hypermethylation of the P2X7 gene and decreased transcription; enhanced degradation of the P2X7 transcript occurs through the action of microRNAs miR-186 and miR-150 [8], [11], [24]. "
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    ABSTRACT: The P2X7 receptor regulates cell growth through mediation of apoptosis. Low level expression of P2X7 has been linked to cancer development because tumor cells harboring a defective P2X7 mechanism can escape P2X7 pro-apoptotic control. microRNAs (miRNAs) function as negative regulators of post-transcriptional gene expression, playing major roles in cellular differentiation, proliferation, and metastasis. In this study, we found that miR-150 was over-expressed in breast cancer cell lines and tissues. In these breast cancer cell lines, blocking the action of miR-150 with inhibitors leads to cell death, while ectopic expression of the miR-150 results in increased cell proliferation. We deploy a microRNA sponge strategy to inhibit miR-150 in vitro, and the result demonstrates that the 3'-untranslated region (3'UTR) of P2X7 receptor contains a highly conserved miR-150-binding motif and its direct interaction with miR-150 down-regulates endogenous P2X7 protein levels. Furthermore, our findings demonstrate that miR-150 over-expression promotes growth, clonogenicity and reduces apoptosis in breast cancer cells. Meanwhile, these findings can be decapitated in nude mice with breast cancer xenografts. Finally, these observations strengthen our working hypothesis that up-regulation of miR-150 in breast cancer is inversely associated with P2X7 receptor expression level. Together, these findings establish miR-150 as a novel regulator of P2X7 and a potential therapeutic target for breast cancer.
    PLoS ONE 12/2013; 8(12):e80707. DOI:10.1371/journal.pone.0080707 · 3.23 Impact Factor
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