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Handan, China

Wu W.,Hebei Medical University | Chen Y.,Shexian Hospital | Liu Z.,Shexian Hospital | He Y.,Hebei Medical University | And 2 more authors.
Chinese Journal of Clinical Oncology | Year: 2011

Objective: To construct a eukaryotic expression vector of hsa-miR-223 and to verify the regulatory effect of hsa-miR-223 on target gene artemin in human esophageal carcinoma cell KYSE150. Methods: PCR primers were designed and miR-223 precursor sequence was amplified from genomic DNA. PCR product was cloned to the linearized pMD18-T Simple vector and then was subcloned into pCDNA3.1 (+) by BamH I and EcoR I double-ligated. The recombinant plasmid pCDNA3.1 (+)-miR-223 was analyzed through restriction enzyme digestion and sequencing analysis. Mature miR-223 expression was higher in KYSE150 cells transfected with pCDNA3.1 (+)-miR-223 than that in KYSE150 cells transfected with pCDNA3.1 (+) by real-time PCR. Software and websites of bioinformatics including TargetScan, PicTar and MirBase were used for target gene prediction. Recombined vector pMIR-ARTN was constructed to express artemin 3′UTR, and mutation expression vector pMIR-ARTN-Mut was also constructed. 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 and pCDNA3.1 (+). Results: Eukaryotic expression vector pCDNA3.1(+)-miR-223 was successfully constructed and it expressed mature miR-223 in human esophageal carcinoma KYSE150 cells transfected with pCDNA3.1 (+)-miR-223. Artemin was selected as a candidate of target genes which has 7 base pairs completely matched to miR-223 in the 3′UTR seed zone. Recombined vector pMIR-ARTN fused ARTN 3′UTR and mutation expression vector pMIR-ARTN-Mut were constructed. Dual-luciferase reporter assay showed that miR-223 decreased the activity of luciferase co-transfected with pMIR-ARTN. Overexpression of exogenous miR-223 in KYSE150 cell line significantly suppressed the expression of artemin protein. Conclusion: MiR-223 can suppress artemin gene expression at the post-transcriptional level by targeting the specific sequence of artemin 3′UTR. Source


Li S.,Hebei Medical University | Song X.,Shexian Hospital | Hou J.,Shexian Hospital | Cui A.,Hebei Medical University | And 3 more authors.
Chinese Journal of Clinical Oncology | Year: 2011

Objective: To investigate the effects of hsa-miR-223 on the migration and invasion capability of human esophageal cells, and to further explore its mechanism of action, which alters the biological function of esophageal carcinoma cells. Methods: The expression of miR-223 and Artemin (ARTN) in human esophageal carcinoma cell lines were determined using real-time polymerase chain reaction (PCR) and Western blot analysis. The stable cell line KYSE150, which overexpressed miR-223, was established through G418 screening. Wound healing and Boyden chamber assays were performed to analyze the migration and the invasion capabilities of the cells. Based on the changes in biological function, the mechanism of hsa-miR-223 involvement in migration and invasion was further studied using a dual-luciferase reporter assay and Western blot. Results: The results of real-time PCR indicated that hsa-miR-223 was downregulated in the esophageal carcinoma cell line KYSE150 with high metastatic potential, whereas ARTN was overexpressed in the KYSE150 cell line. The stable cell line KYSE-150 was successfully established. The wound healing assay and the Boyden chamber assay showed that the migration and invasion capability of KYSE-150 cell line were significantly suppressed. Moreover, the dual-luciferase reporter assay showed that miR-223 decreased the activity of luciferase in the stable KYSE150 cell line transfected with pMIR-ARTN. This was confirmed by the Western blot analysis, which indicated that miR-223 suppressed ARTN expression. Conclusion: Hsa-miR-223 is downregulated in the human esophageal carcinoma cell line KYSE150 with high metastatic potential. Acting as a tumor suppressor gene, hsa-miR-223 suppressed migration and invasion by targeting ARTN. ARTN is a potential therapeutic target for the biological treatment of tumors. Source

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