Abstract: Construction of miR-223 Expression Vector and Its Regulation to Target Gene ArteminWenxinWU1, Yanliang CHEN2, Zengliang LIU2, Yuzheng HE3, Airong CUI1, Shujun LI3Correspondence to: Shujun LI, E-mail: lsj821@sina.com1Department of Pathology, The Second Hospital of Hebei Medical University, Shijiazhuang 050051, China2Shexian Hospital, Handan 056400, China3Department of Thoracic Surgery, The Second Hospital of Hebei Medical University, Shijiazhuang 050051, ChinaAbstract Objective: To construct a eukaryotic expression vector of hsa-miR-223 and to verify the regulatory effect ofhsa-miR-223 on target gene artemin in human esophageal carcinoma cell KYSE150. Methods: PCR primers were designed andmiR-223 precursor sequence was amplified from genomic DNA. PCR product was cloned to the linearized pMD18-T Simple vectorand then was subcloned into pCDNA3.1 ( + ) by BamHⅠand EcoRⅠdouble-ligated. The recombinant plasmid pCDNA3.1 (+ )-miR-223 was analyzed through restriction enzyme digestion and sequencing analysis. Mature miR-223 expression was higher inKYSE150 cells transfected with pCDNA3.1 ( + )-miR-223 than that in KYSE150 cells transfected with pCDNA3.1 ( + ) by real-timePCR. Software and websites of bioinformatics including TargetScan, PicTar and MirBase were used for target gene prediction. Recom-bined vector pMIR-ARTN was constructed to express artemin 3'UTR, and mutation expression vector pMIR-ARTN-Mut was also con-structed. Co-transfection of both recombined vectors were performed in HEK293 cells and dual-luciferase reporter assay was analyzed.Western blot was used to detect the expression of artemin protein in KYSE150 cell line transfected with pCDNA3.1(+)-miR-223 andpCDNA3.1 ( + ). Results: Eukaryotic expression vector pCDNA3.1(+)-miR-223 was successfully constructed and it expressed maturemiR-223 in human esophageal carcinoma KYSE150 cells transfected with pCDNA3.1 ( + )-miR-223. Artemin was selected as a candi-date of target genes which has 7 base pairs completely matched to miR-223 in the 3’ UTR seed zone. Recombined vector pMIR-ARTNfused ARTN 3'UTR and mutation expression vector pMIR-ARTN-Mut were constructed. Dual-luciferase reporter assay showed thatmiR-223 decreased the activity of luciferase co-transfected with pMIR-ARTN. Overexpression of exogenous miR-223 in KYSE150 cellline significantly suppressed the expression of artemin protein. Conclusion: MiR-223 can suppress artemin gene expression at thepost-transcriptional level by targeting the specific sequence of artemin 3'UTR.Keywords Hsa-miR-223; Eukaryotic expression vector; Reporter gene