miR-192 Regulates dihydrofolate reductase and cellular proliferation through the p53-microRNA circuit.
miR-192 Regulates dihydrofolate reductase and cellular proliferation through the p53-microRNA circuit.
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DOI：
10.1158/1078-0432.ccr-08-1422
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发表时间：
2008-12-15
期刊：
影响因子：
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通讯作者：
Ju J
中科院分区：
文献类型：
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作者：
Song B;Wang Y;Kudo K;Gavin EJ;Xi Y;Ju J
本研究旨在探讨miR-192在结肠癌中的分子机制。以p53不同状态的人结肠癌细胞系为模型系统，研究miR-192对细胞增殖、细胞周期调控的影响及其调控机制。我们的研究结果表明，miR-192的关键靶基因之一是二氢叶酸还原酶（DHFR）。miR-192通过p53-miRNA通路影响细胞增殖。Western免疫印迹分析表明，miR-192显著降低了DHFR的表达。进一步的研究表明，这种抑制是由于翻译停滞，而不是mRNA降解。在含有野生型p53的结肠癌细胞系中，通过miR-192的异位表达观察到比含有突变型p53的细胞更深刻的细胞增殖抑制。因此，miR-192对细胞增殖的作用主要是p53依赖性的。miR-192的过表达在HCT-116（wt-p53）细胞中触发G1和G2停滞，但在HCT-116（null-p53）细胞中不触发。细胞周期检查点控制基因p53和p21在过表达miR-192的细胞中高度过表达。在用甲氨蝶呤处理的HCT-116（wt-p53）和RKO（wt-p53）细胞中，内源性miR-192表达增加，这引起p53表达的诱导。染色质免疫沉淀（ChIP）-qPCR分析显示p53蛋白与miR-192启动子序列相互作用。这些结果表明，miR-192可能是另一种参与p53肿瘤抑制网络的miRNA候选者，对细胞周期控制和细胞增殖具有显著影响。
The purpose of this study is to investigate the molecular mechanism of miR-192 in colon cancer. Human colon cancer cell lines with different p53 status were used as our model system to study the impact of miR-192 on cell proliferation, cell cycle control and mechanism of regulation. Our results show that one of the key miR-192 target genes is dihydrofolate reductase (DHFR). miR-192 impacts cellular proliferation through the p53-miRNA circuit. Western immunoblot analyses indicated that the expression of DHFR was significantly decreased by miR-192. Further investigation revealed that such suppression was due to translational arrest rather than mRNA degradation. More profound inhibition of cellular proliferation was observed by ectopic expression of miR-192 in colon cancer cell lines containing wild type p53 than cells containing mutant p53. Thus, the effect of miR-192 on cellular proliferation is mainly p53 dependent. Over-expression of miR-192 triggered both G1 and G2 arrest in HCT-116 (wt-p53) cells but not in HCT-116 (null-p53) cells. The cell cycle check point control genes p53 and p21 were highly over-expressed in cells that over-expressed miR-192. Endogenous miR-192 expression was increased in HCT-116 (wt-p53) and RKO (wt-p53) cells treated with methotrexate, which caused an induction of p53 expression. Chromatin immunoprecipitation (ChIP)-qPCR analysis revealed that the p53 protein interacted with the miR-192 promoter sequence. These results indicate that miR-192 may be another miRNA candidate that is involved in the p53 tumor suppressor network with significant impact on cell cycle control and cell proliferation.