MicroRNAs (miRNAs) are endogenous short (∼22) nucleotide RNAs that regulate gene function by modification of target mRNAs. miRNA-1 (miR-1) and miRNA-206 (miR-206) are highly expressed in skeletal muscle. Due to the tissue-specific nature of miR-1/206 for skeletal muscles, we investigated the role of miR-1/206 in the development of rhabdomyosarcoma. Initially, we demonstrated that miR-1/206 expression was suppressed in rhabdomyosarcomas and found at very low levels in a rhabdomyosarcoma RD cell line. Transient transfection of miR-1/206 into cultured RD cells led to a significant decrease in cell growth and migration. Using bioinformatics, we identified two putative miR-1/206 binding sites within the 3′-untranslated region of the human c-Met mRNA. miR-1/206 was then shown to have activity on mRNA expression by targeting the c-Met 3′-untranslated region. The expression of c-Met protein was shown to be down-regulated by subsequent Western blot analysis. Conversely, up-regulation of c-Met was confirmed in tissue samples of human rhabdomyosarcoma, with its level inversely correlated with miR-1/206 expression. In vivo, miR-1/206-expressing tumor cells showed growth delay in comparison with negative control. Our results demonstrated that miR-1/206 suppressed c-Met expression in rhabdomyosarcoma and could function as a potent tumor suppressor in c-Met-overexpressing tumors. Inhibition of miR-1/206 function could contribute to aberrant cell proliferation and migration, leading to rhabdomyosarcoma development.

Purpose. 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. methods. 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. results. 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. conclusions. These results demonstrate that miR-34a acts as a tumor suppressor in uveal melanoma cell proliferation and migration through the downregulation of c-Met.

Purpose.: MicroRNAs (miRNAs) can contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. The authors' previous studies on miR-34a showed that miRNA can influence the growth of uveal melanoma cells. In this study, they investigated the role of miR-137 in the pathogenesis of uveal melanoma. Methods.: Real-time RT-PCR was used to screen the expression levels of miR-137 in uveal melanocytes and uveal melanoma cell lines. Cell proliferation was examined by MTS assay and cell cycle was analyzed by flow cytometry. The target genes of miR-137 were predicted by bioinformatics and confirmed using a luciferase reporter assay. The expression of MITF, CDK6, and cell cycle regulatory proteins was determined by Western blot analysis. The ability to increase miR-137 expression by epigenetic drugs was tested using real-time RT-PCR. Results.: miR-137 expression was lower in uveal melanoma cell lines than in uveal melanocytes. Ectopic transfection of miR-137 into uveal melanoma cells induced G1 cell cycle arrest, leading to a significant decrease in cell growth. Overexpression of miR-137 downregulated MITF, a transcription factor with oncogenic activity. Moreover, the introduction of miR-137 downregulated the oncogenic tyrosine kinase protein receptor c-Met and cell cycle–related proteins, including CDK6. One avenue to increase the expression levels of miR-137 was through treatment with a DNA hypomethylating agent, 5-aza-2′-deoxycytidine, and a histone deacetylase inhibitor, trichostatin A. Conclusions.: The results showed that miR-137 can act as a tumor suppressor in uveal melanoma cell proliferation through downregulation of the targets MITF and CDK6. miR-137 may be epigenetically silenced during uveal melanoma tumorigenesis.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play central roles in diverse pathological processes. In this study, we investigated the effect of microRNA-182 (miR-182) on the development of posterior uveal melanomas. Initially, we demonstrated that miR-182 expression was dependent on p53 induction in uveal melanoma cells. Interestingly, transient transfection of miR-182 into cultured uveal melanoma cells led to a significant decrease in cell growth, migration, and invasiveness. Cells transfected with miR-182 demonstrated cell cycle G1 arrest and increased apoptotic activity. Using bioinformatics, we identified three potential targets of miR-182, namely MITF, BCL2 and cyclin D2. miR-182 was shown to have activity on mRNA expression by targeting the 3′ untranslated region of MITF, BCL2 and cyclin D2. Subsequent Western blot analysis confirmed the downregulation of MITF, BCL2 and cyclin D2 protein expression. The expression of oncogene c-Met and its downstream Akt and ERK1/2 pathways was also downregulated by miR-182. Concordant with the findings that miR-182 was decreased in uveal melanoma tissue samples, overexpression of miR-182 also suppressed the in vivo growth of uveal melanoma cells. Our results demonstrated that miR-182, a p53 dependent miRNA, suppressed the expression of MITF, BCL2, cyclin D2 and functioned as a potent tumor suppressor in uveal melanoma cells.

Purpose. MicroRNA-124a (miR-124a), an abundant microRNA in the central neuron system, has been linked to tumor progression. Here, we investigated the role of miR-124a in uveal melanoma development. Methods. Expression of miR-124a in uveal melanoma cells was examined using real time RT-PCR. The effect of miR-124a on cell proliferation, migration, and invasion was analyzed using MTS assay, flow cytometry, and transwell experiments. The ability of miR-124a to repress tumor growth was tested in vivo. Target genes of miR-124a were first predicted by bioinformatics, confirmed using a luciferase assay, and their expression determined by Western blotting. DNA methylation and histone modification of miR-124a was analyzed by methylation-specific PCR and ChIP assay. Finally, epigenetic drugs were used to alter the expression of miR-124a. Results. miR-124a expression was downregulated in both uveal melanoma cells and clinical specimens. Transient transfection of miR-124a into uveal melanoma cells inhibited cell growth, migration, and invasion. Moreover, introduction of miR-124a suppressed in vivo growth of tumor. Potential targets of miR-124a were found to include CDK4, CDK6, cyclin D2, and EZH2. Knockdown of EZH2 by siRNA resulted in inhibition of uveal melanoma cell migration and invasion. In addition, miR-124a expression was found to be regulated via epigenetic mechanisms, with its expression restored when cells were treated with a DNA hypomethylating agent, 5-aza-2′-deoxycytidine, and a histone deacetylase inhibitor, trichostatin A. Conclusions. Our results demonstrated that miR-124a could function as a potent tumor suppressor by regulation of multiple targets, and was epigenetically silenced in the development of uveal melanoma.

Purpose.: MicroRNAs (miRNAs) are endogenous short chain (∼22-nucleotide) noncoding RNAs that inhibit protein translation through binding to target mRNAs. Recent studies have implied that miRNAs play a regulatory role in corneal development. Here we profile their involvement in corneal epithelial renewal, develop an miRNA-target network that affects wound healing outcome, and investigate the function of miR-204 in this response. Methods.: NanoString nCounter technology and bioinformatics analyzed miRNA expression levels and their targets during mouse corneal epithelial wound healing. Real-time RT-PCR was performed to detect miR-204 expression in mouse corneal epithelium. Human corneal epithelial cells (HCECs) were transfected with miR-204 using transfection reagent. MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) and a scratch wound-healing assay evaluated the effects of miR-204 expression on HCEC proliferation and migration, respectively. Cell cycle analysis was performed by flow cytometry. Expression of sirtuin 1 (SIRT1) was determined by Western blot analysis. Results.: Fifteen miRNAs were dramatically downregulated, whereas 14 other miRNAs were markedly upregulated during corneal wound healing. Expression of miR-204 fell the most during this process. Transfection of miR-204 into HCECs led to a significant decline in cell proliferation and induced cell cycle G1-arrest. Furthermore, in these cells, miR-204 also inhibited migration. Sirtuin 1 was confirmed as a target of miR-204. Conclusions.: During mouse corneal epithelial wound healing, a complex miRNA-gene network was resolved that is modulated by changes in miR-204 expression. Downregulation of this miRNA appears to be an essential response to injury since its decline promotes human corneal epithelial cell proliferation and migration. Therefore, miR-204 could be a biomarker of this process.