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
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
复制标题
DOI：
10.1074/jbc.m802663200
复制
发表时间：
2008-10-17
影响因子：
4.8
通讯作者：
Gorodeski, George I.
中科院分区：
文献类型：
--
作者：
Zhou, Lingyin;Qi, Xiaoping;Gorodeski, George I.
P2 X（7）受体通过介导细胞凋亡来调节细胞生长。P2 X（7）在癌上皮细胞中的水平低于正常细胞，先前的研究表明P2 X（7）的表达是在转录后调节的。本研究的目的是了解P2 X（7）mRNA稳定性的调节。含有全长人P2 X（7）3 '-非翻译区（3'-UTR）的报告基因或含有部分3 '-UTR-P2 X（7）的报告基因的过表达与正常细胞中构建体丰度的增加和癌上皮细胞中丰度的降低相关。3 '-UTR-P2 X（7）内的序列是microRNA、miR-186（中间片段）和miR-150（远端片段）的假定靶位点，降低了P2 X（7）转录物的丰度。突变的miR-186和miR-150靶位点在癌细胞中的过表达与较低水平的报告基因相关。在正常细胞中，突变的miR-186靶位点的过表达与浓度显著增加相关，但miR-150靶位点报告基因（野生型和突变型）的过表达并不随时间而改变。miR-186和miR-150在癌症中的水平高于正常细胞，并且用miR-186和miR-150抑制剂治疗增加了P2 X（7）mRNA。在异源表达全长3 '-UTR-P2 X（7）荧光素酶报告基因的人胚肾-293细胞中，miR-186和miR-150抑制剂增加荧光素酶活性，而miR-186和miR-150模拟物在放线菌素D处理后降低荧光素酶活性。这些数据表明，癌上皮细胞中miR-186和miR-150的表达增加通过激活位于3 '-UTR-P2 X的miR-186和miR-150不稳定性靶位点来降低P2 X（7）mRNA（7）。
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).