Yan Dongsheng, Zhou Xiangtian, Chen Xiaoyan, Hu Dan-Ning, Dong Xiang Da, Wang Jiao, Lu Fan, Tu LiLi, Qu Jia
School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, People's Republic of China.
Invest Ophthalmol Vis Sci. 2009 Apr;50(4):1559-65. doi: 10.1167/iovs.08-2681. Epub 2008 Nov 21.
MicroRNAs (miRNAs) are endogenously expressed, noncoding, small RNAs that inhibit protein translation through binding to target mRNAs. Recent studies have demonstrated that miRNAs can regulate tumor cell proliferation and migration. MicroRNA-34a (miR-34a), a potential key effector of the p53 tumor-suppressor gene, was studied as a potential tumor suppressor in uveal melanoma.
Northern blot analysis was performed to detect the expression level of miR-34a in uveal melanoma cells and melanocytes. Subsequently, melanoma cell proliferation and migration were examined by MTS cell proliferation and transwell migration assays, respectively. The target of miR-34a was predicted by bioinformatics and confirmed using a luciferase assay. In addition, expression of c-Met and cell cycle-related proteins was determined by Western blotting and immunofluorescence after the introduction of miR-34a.
miR-34a is actively expressed in melanocytes but not in uveal melanoma cells based on Northern blot analysis. Transfection of miR-34a into uveal melanoma cells led to a significant decrease in cell growth and migration. After identification of two putative miR-34a binding sites within the 3' UTR of the human c-Met mRNA, miR-34a was shown to suppress luciferase activity using HEK293 cells with a luciferase reporter construct containing the binding sites. miR-34a was confirmed to downregulate the expression of c-Met protein by Western blotting and immunofluorescence. Furthermore, the introduction of miR-34a downregulated phosphorylated Akt and cell cycle-related proteins.
These results demonstrate that miR-34a acts as a tumor suppressor in uveal melanoma cell proliferation and migration through the downregulation of c-Met.
微小RNA（miRNA）是内源性表达的非编码小RNA，通过与靶mRNA结合来抑制蛋白质翻译。最近的研究表明，miRNA可调节肿瘤细胞的增殖和迁移。微小RNA-34a（miR-34a）作为p53肿瘤抑制基因的潜在关键效应物，被作为葡萄膜黑色素瘤中的一种潜在肿瘤抑制因子进行研究。
进行Northern印迹分析以检测葡萄膜黑色素瘤细胞和黑素细胞中miR-34a的表达水平。随后，分别通过MTS细胞增殖试验和Transwell迁移试验检测黑色素瘤细胞的增殖和迁移情况。通过生物信息学预测miR-34a的靶标，并使用荧光素酶测定法进行验证。此外，在导入miR-34a后，通过蛋白质免疫印迹法和免疫荧光法测定c-Met和细胞周期相关蛋白的表达。
基于Northern印迹分析，miR-34a在黑素细胞中活跃表达，但在葡萄膜黑色素瘤细胞中不表达。将miR-34a转染到葡萄膜黑色素瘤细胞中导致细胞生长和迁移显著减少。在鉴定出人c-Met mRNA的3'UTR内的两个假定miR-34a结合位点后，使用含有这些结合位点的荧光素酶报告构建体的HEK293细胞显示miR-34a可抑制荧光素酶活性。通过蛋白质免疫印迹法和免疫荧光法证实miR-34a可下调c-Met蛋白的表达。此外，导入miR-34a可下调磷酸化的Akt和细胞周期相关蛋白。
这些结果表明，miR-34a通过下调c-Met在葡萄膜黑色素瘤细胞增殖和迁移中起肿瘤抑制作用。
