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

The P2X(7) receptor regulates cell growth through mediation of apoptosis. P2X(7) levels are lower in cancer epithelial cells than in normal cells, and previous studies showed that expression of P2X(7) was regulated post-transcriptionally. The objective of the study was to understand regulation of P2X(7) mRNA stability. Overexpression of a reporter containing the full-length human P2X(7) 3'-untranslated region (3'-UTR) or reporters containing parts of the 3'-UTR-P2X(7) were associated with increased abundance of the construct in normal cells and decreased abundance in cancer epithelial cells. Sequences within the 3'-UTR-P2X(7), which are putative target sites for the microRNAs, miR-186 (middle segment) and miR-150 (distal segment), decreased the abundance of the P2X(7) 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 P2X(7) mRNA. In human embryonic kidney-293 cells heterologously expressing the full-length 3'-UTR-P2X(7) 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 P2X(7) mRNA by activation of miR-186 and miR-150 instability target sites located at the 3'-UTR-P2X(7).