title: MicroRNA-101 (miR-101) post-transcriptionally regulates the expression of EP4 receptor in colon cancers.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
authors.affiliation: The University of Arizona, Tucson, AZ, USA.
authors.forename: Anupama
authors.initials: A
authors.lastname: Chandramouli
authors.forename: Benjamin Chidi
authors.initials: BC
authors.lastname: Onyeagucha
authors.forename: Melania E
authors.initials: ME
authors.lastname: Mercado-Pimentel
authors.forename: Lenka
authors.initials: L
authors.lastname: Stankova
authors.forename: Nisreen Abu
authors.initials: NA
authors.lastname: Shahin
authors.forename: Bonnie J
authors.initials: BJ
authors.lastname: LaFleur
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Heimark
authors.forename: Achyut K
authors.initials: AK
authors.lastname: Bhattacharyya
authors.forename: Mark A
authors.initials: MA
authors.lastname: Nelson
mesh_terms: D020413:3' Untranslated Regions; D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000236:Adenoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D001483:Base Sequence; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D002465:Cell Movement; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D035683:MicroRNAs / Q000378:metabolism / Q000502:physiology*; D034622:RNA Interference; D058307:Receptors, Prostaglandin E, EP2 Subtype / Q000235:genetics / Q000378:metabolism; D058309:Receptors, Prostaglandin E, EP4 Subtype / Q000235:genetics / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; C534072:MIRN101 microRNA, human; D035683:MicroRNAs; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D049410:Luciferases, Renilla