Regulation of Iron-Ion Transporter SLC11A2 by Three Identical miRNAs

• Published in: Biological & pharmaceutical bulletin Vol. 45; no. 9; pp. 1291 - 1299
• Main Authors: Sugino, Yuta, Uchiyama, Reina, Shibasaki, Chihiro, Kugawa, Fumihiko
• Format: Journal Article
• Language: English
• Published: Tokyo The Pharmaceutical Society of Japan 01.09.2022; Japan Science and Technology Agency
• Subjects: 3' Untranslated regions; Ammonium; Caco-2 cell; Gene expression; Intracellular; Iron; iron-ion; microRNA (miRNA); MicroRNAs; miRNA; Protein transport; SLC11A2 transporter; transcriptional regulation
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.b22-00214

Here, we searched for microRNAs (miRNAs) in silico that could interact with SLC11A2 mRNA, a solute carrier (SLC) iron-ion transporter, and investigated their effects on SLC11A2 gene expression using the cultured human colon carcinoma cell line, Caco-2. In silico analysis using the miRWalk2.0 database revealed that several types of miRNAs interact with the human SLC11A2 gene; we focused on three miRNAs, miR-149-5p, miR-362-5p, and miR-539-5p as candidates in this study. We first revealed that the three miRNAs interact with the SLC11A2 3′-untranslated region (3′-UTR) using a luciferase assay in a Caco-2 cell line. We then examined whether the expression of each miRNA affected the expression of SLC11A2 mRNAs and their transcribed transporter proteins. We found transiently expressed miRNAs significantly reduced the reporter activity of the SLC11A2 3′-UTR site in Caco-2 cells by significantly decreasing the SLC11A2 gene and protein expression in the miRNA-transfected Caco-2 cells. Subsequently, we investigated the effects of these miRNAs on SLC11A2′s iron-ion transporting activity by measuring iron-ion concentration in Caco-2 cells. Administration of ammonium iron (II) sulfate hexahydrate to Caco-2 cells significantly increased the intracellular iron-ion concentration. However, in iron-ion-pretreated cells, overexpression of each of the three miRNAs resulted in decreased intracellular iron-ion concentration. This indicated that overexpressed miRNAs inhibited iron-ion influx into Caco-2 cells by attenuating SLC11A2 transporting activity. Using in silico analysis, we predicted that three studied miRNAs could bind to the iron-ion influx transporter SLC11A2 and revealed that they regulate SLC11A2 gene expression and iron-ion transporting function in an in vitro system.