MicroRNA-101 (miR-101) post-transcriptionally regulates the expression of EP4 receptor in colon cancers
MicroRNA-101 (miR-101) post-transcriptionally regulates the expression of EP4 receptor in colon cancers
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DOI：
10.4161/cbt.13.3.18874
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发表时间：
2012-02-01
影响因子：
3.6
通讯作者：
Nelson, Mark A.
中科院分区：
文献类型：
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作者：
Chandramouli, Anupama;Onyeagucha, Benjamin Chidi;Nelson, Mark A.
目的：前列腺素E2受体EP 4在大肠癌发生过程中表达上调。然而，导致EP 4受体失调的机制尚不清楚。本研究旨在探讨miRNA对EP 4受体的调节。实验设计：我们通过免疫组织化学方法分析了26例结肠癌（即15例腺癌和9例腺瘤）和16例正常结肠标本的EP 4受体表达。生物信息学方法鉴定了与EP 4受体的3 '-UTR的推定microRNA结合位点。分别通过qRT-PCR和Western分析分析结肠癌细胞系和肿瘤标本的miR-101和EP 4表达，同时使用原位杂交来证实我们的结果。结果：miR-101和EP 4受体蛋白表达水平呈负相关，与正常对照组相比，差异有统计学意义（P < 0. 05）。用miR-101转染LS 174 T细胞显著抑制含有EP 4受体-3 '-UTR的荧光素酶报告基因。相反，突变型EP 4受体-3 '-UTR构建体不受影响。miR-101的异位表达显著降低细胞增殖和运动性。共转染EP 4受体可使结肠癌细胞免于miR-101的肿瘤抑制作用。此外，EP 4受体信号传导的药理学抑制或EP 4受体的沉默表型模仿了miR-101的作用。结论：本研究为miR-101在转录后水平调控EP-4受体表达提供了新的思路，并为针对miR-101的治疗提供了新的思路。
Purpose: Expression of the PGE2 receptor, EP4, is up-regulated during colorectal carcinogenesis. However the mechanism leading to deregulation of the EP4 receptor is not known. The present study was conducted to investigate the regulation of EP4 receptor by miRNAs.Experimental Design: We analyzed 26 colon cancers (i.e. 15 adenocarcinomas and 9 adenomas) and 16 normal colon specimens for EP4 receptor expression by immunohistochemistry. A bioinformatics approached identified putative microRNA binding sites with the 3'-UTR of the EP4 receptor. Both colon cancer cell lines and tumor specimens were analyzed for miR-101 and EP4 expression by qRT-PCR and Western analysis respectively and simultaneously in situ hybridizations was used to confirm our results. In vitro and in vivo assays were used to confirm our clinical findings.Results: We observed an inverse correlation between the levels of miR-101 and EP4 receptor protein. Transfection of LS174T cells with miR-101 significantly suppressed a luciferase reporter containing the EP4 receptor-3'-UTR. In contrast, a mutant EP4 receptor-3'-UTR construct was unaffected. Ectopic expression of miR-101 markedly reduced cell proliferation and motility. Co-transfection of EP4 receptor could rescue colon cancer cells from the tumor suppressive effects of miR-101. Moreover, the pharmacologic inhibition of EP4 receptor signaling or silencing of EP4 receptor phenocopied the effect of miR-101. This is the first study to show that the EP4 receptor is negatively regulated by miR-101.Conclusions: These data provide new insights in the modulation of EP-4 receptor expression at the post-transcriptional level by miR-101 and suggests therapeutic strategies against miR-101 targets may be warranted.