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Published on: May 21, 2019
MicroRNAs miR-186 and miR-150 down-regulate expression of the pro-apoptotic purinergic P2X7 receptor by activation of
Lingyin Zhou1, Xiaoping Qi, Judith A Potashkin
1Department of Reproductive Biology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
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.
Insights
MicroRNAs miR-186 and miR-150 reduce P2X7 receptor mRNA stability in cancer cells. Inhibiting these microRNAs increases P2X7 mRNA levels, suggesting a role in cancer progression.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Regulation
Background:
- The P2X7 receptor is crucial for regulating cell growth and apoptosis.
- Lower P2X7 receptor levels are observed in cancer epithelial cells compared to normal cells.
- Previous research indicated post-transcriptional regulation of P2X7 expression.
Purpose of the Study:
- To investigate the post-transcriptional regulation of P2X7 messenger RNA (mRNA) stability.
- To identify specific sequences within the P2X7 3'-untranslated region (3'-UTR) that influence mRNA stability.
- To elucidate the role of microRNAs (miRNAs) in controlling P2X7 mRNA levels in cancer.
Main Methods:
- Utilized reporter gene assays with full-length and truncated human P2X7 3'-UTR constructs.
- Investigated the impact of specific microRNA target sites (miR-186 and miR-150) within the P2X7 3'-UTR.
- Assessed the effects of miRNA mimics and inhibitors on P2X7 mRNA and reporter gene expression in normal and cancer cells.
- Quantified miRNA levels in normal versus cancer cells.
Main Results:
- The P2X7 3'-UTR significantly influenced reporter gene abundance, with decreased expression in cancer cells versus normal cells.
- Specific sequences within the P2X7 3'-UTR, targeted by miR-186 and miR-150, were found to decrease P2X7 transcript abundance.
- Overexpression of miR-186 and miR-150 was higher in cancer cells, and their inhibition led to increased P2X7 mRNA levels.
- Inhibition of miR-186 and miR-150 increased reporter gene activity, while mimics decreased it, confirming their regulatory role.
Conclusions:
- Increased expression of miR-186 and miR-150 in cancer epithelial cells contributes to reduced P2X7 mRNA levels.
- These microRNAs likely decrease P2X7 mRNA stability by targeting specific sites within the P2X7 3'-UTR.
- The findings suggest a mechanism by which miR-186 and miR-150 may promote cancer progression through P2X7 downregulation.
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