title: miR-135b- and miR-146b-dependent silencing of calcium-sensing receptor expression in colorectal tumors.
abstract: Studies have shown that the calcium-sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco-2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain- and loss-of-function studies with the top candidates: miR-9, miR-27a, miR-135b, and miR-146b. Modulation of miR-135b or miR-146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR-135b and miR-146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR-135b and miR-146b, and this correlated inversely with CaSR expression (miR-135b: r = -0.684, p < 0.001 and miR-146b: r = -0.448, p < 0.001), supporting our in vitro findings. We demonstrate that miR-135b and miR-146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC.
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Irfete S
authors.initials: IS
authors.lastname: Fetahu
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Samawansha
authors.initials: S
authors.lastname: Tennakoon
authors.affiliation: Academic Endocrine Unit, Oxford Center for Diabetes, Endocrinology, and Metabolism, Radcliffe Department of Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom.
authors.forename: Kate E
authors.initials: KE
authors.lastname: Lines
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Gröschel
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Aggarwal
authors.affiliation: Department of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Ildiko
authors.initials: I
authors.lastname: Mesteri
authors.affiliation: Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria.
authors.forename: Sabina
authors.initials: S
authors.lastname: Baumgartner-Parzer
authors.affiliation: Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Mader
authors.affiliation: Academic Endocrine Unit, Oxford Center for Diabetes, Endocrinology, and Metabolism, Radcliffe Department of Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom.
authors.forename: Rajesh V
authors.initials: RV
authors.lastname: Thakker
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Enikő
authors.initials: E
authors.lastname: Kállay
mesh_terms: D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D044169:Receptors, Calcium-Sensing / Q000235:genetics* / Q000378:metabolism
chemical_list: C544965:MIRN135 microRNA, human; C542542:MIRN146 microRNA, human; D035683:MicroRNAs; D044169:Receptors, Calcium-Sensing
keywords: calcium-sensing receptor; colorectal cancer; miR-135b; miR-146b; microRNAs