Salvi Alessandro, Sabelli Cristiano, Moncini Silvia, Venturin Marco, Arici Bruna, Riva Paola, Portolani Nazario, Giulini Stefano M, De Petro Giuseppina, Barlati Sergio
Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, IDET Centre of Excellence, University of Brescia, Italy.
FEBS J. 2009 Jun;276(11):2966-82. doi: 10.1111/j.1742-4658.2009.07014.x. Epub 2009 Apr 16.
Urokinase-type plasminogen activator (uPA) and c-met play a major role in cancer invasion and metastasis. Evidence has suggested that uPA and c-met overexpression may be coordinated in human hepatocellular carcinoma (HCC). In the present study, to understand whether the expression of these genes might be coregulated by specific microRNAs (miRs) in human cells, we predicted that Homo sapiens microRNA-23b could recognize two sites in the 3'-UTR of uPA and four sites in the c-met 3'-UTR by the algorithm pictar. The miR-23b expression analysis in human tumor and normal cells revealed an inverse trend with uPA and c-met expression, indicating that uPA and c-met negative regulation might depend on miR-23b expression. Transfection of miR-23b molecules in HCC cells (SKHep1C3) led to inhibition of protein expression of the target genes and caused a decrease in cell migration and proliferation capabilities. Furthermore, anti-miR-23b transfection in human normal AB2 dermal fibroblasts upregulated the expression of endogenous uPA and c-met. Cotransfection experiments in HCC cells of the miR-23b with pGL4.71 Renilla luciferase reporter gene constructs, containing the putative uPA and c-met 3'-UTR target sites, and with the pGL3 firefly luciferase-expressing vector showed a decrease in the relative luciferase activity. This would indicate that miR-23b can recognize target sites in the 3'-UTR of uPA and of c-met mRNAs and translationally repress the expression of uPA and c-met in HCC cells. The evidence obtained shows that overexpression of miR-23b leads to uPA and c-met downregulation and to decreased migration and proliferation abilities of HCC cells.
尿激酶型纤溶酶原激活剂（uPA）和c-met在癌症侵袭和转移中起主要作用。有证据表明，uPA和c-met的过表达在人类肝细胞癌（HCC）中可能是协同的。在本研究中，为了解这些基因的表达是否可能在人类细胞中受特定微小RNA（miR）的共同调控，我们通过pictar算法预测人类微小RNA-23b可以识别uPA 3'-UTR中的两个位点以及c-met 3'-UTR中的四个位点。对人类肿瘤细胞和正常细胞中miR-23b的表达分析显示，其与uPA和c-met的表达呈相反趋势，表明uPA和c-met的负调控可能依赖于miR-23b的表达。在肝癌细胞（SKHep1C3）中转染miR-23b分子导致靶基因的蛋白表达受到抑制，并使细胞迁移和增殖能力下降。此外，在人类正常AB2真皮成纤维细胞中转染抗miR-23b可上调内源性uPA和c-met的表达。在肝癌细胞中，将miR-23b与含有假定的uPA和c-met 3'-UTR靶位点的pGL4.71海肾荧光素酶报告基因构建体以及pGL3萤火虫荧光素酶表达载体共转染实验显示相对荧光素酶活性降低。这表明miR-23b可以识别uPA和c-met mRNA的3'-UTR中的靶位点，并在翻译水平上抑制肝癌细胞中uPA和c-met的表达。获得的证据表明，miR-23b的过表达导致uPA和c-met下调以及肝癌细胞迁移和增殖能力下降。
