MicroRNA-145 Suppresses Uveal Melanoma Angiogenesis and Growth by Targeting Neuroblastoma RAS Viral Oncogene Homolog and Vascular Endothelial Growth Factor
Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, arising from melanocytes. It is associated with the development of systemic metastases in over half of the patients. Increased angiogenesis has been linked to higher metastasis and mortality rates in UM. However, the precise molecular mechanisms underlying these processes remain poorly understood. MicroRNAs (miRNAs) are a group of endogenous non-coding RNAs that regulate gene expression by repressing the stability of conventional mRNAs and/or inhibiting their translation into proteins. Among these, microRNA-145 (miR-145) has been shown to be downregulated in various tumors and may function as a tumor suppressor by controlling the expression of target genes involved in angiogenesis and proliferation.
In this study, we aimed to explore the potential target genes or pathways regulated by miR-145 in UM and the effect of miR-145 on invasion and angiogenesis. We collected 24 choroid samples, including 12 UM samples and 12 normal uveal tissues. The expression of neuroblastoma RAS viral oncogene homolog (N-RAS), phosphorylated protein kinase B (p-AKT), and vascular endothelial growth factor (VEGF) in UM tissues and normal uveal tissues was analyzed using Western blotting analysis. Lentivirus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145. Transwell and endothelial cell tube formation assays were used to measure the effects of miR-145 on the invasion and angiogenesis of UM in vitro. The downstream target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase assay. BALB/c nude mice models were established to investigate the mechanisms of miR-145 on tumor growth and angiogenesis in vivo.
The results of Western blotting analysis indicated that the expressions of N-RAS, p-AKT, and VEGF in UM tumor tissues were significantly higher than those in normal uveal tissue. Luciferase assay demonstrated N-RAS and VEGF as downstream targets of miR-145. Moreover, tube formation assay revealed that miR-145-transfected human microvascular endothelial cell line formed shorter tube length and less branch points as compared with controls. In addition, the numbers of invaded MUM-2B and OCM-1 cells with miR-145 overexpression were significantly lower than the controls. In vivo, xenografts expressing miR-145 had smaller sizes and lower weights.
Our results indicated that miR-145 is an important tumor suppressor and the inhibitory strategies against N-RAS/VEGF signaling pathway might be potential therapeutic applications for UM in the future. The findings of this study provide new insights into the molecular mechanisms of miR-145 in regulating UM angiogenesis and highlight its potential as a therapeutic target for UM treatment.
The expression of N-RAS, p-AKT, and VEGF in UM tissues was significantly higher than in normal uveal tissues, as confirmed by Western blotting analysis. This suggests that the N-RAS/VEGF signaling pathway plays a crucial role in UM progression. The overexpression of these proteins in UM tissues indicates their potential involvement in tumor angiogenesis and growth. The results are consistent with previous studies showing that VEGF-A is overexpressed in several UM cell lines, including MEL-270, OM-431, OMM-2.3, and OMM-2.5.
To further understand the molecular mechanism of miR-145 in inhibiting angiogenesis and cell invasion, we searched for potential targets of miR-145 using miRanda and TargetScan. We found that NRAS and VEGFA could be the potential targets of miR-145. Luciferase activity assays were performed in HEK 293T cells to validate these miR-145 targets. The luciferase activities of vectors carrying wild type VEGFA 3′-UTR and NRAS 3′-UTR were attenuated by co-transfection with miR-145-5p, while there was no significant difference in groups transfected with mutant 3′-UTR of VEGFA or NRAS and the negative control of miR-145-5p. These data demonstrated that VEGFA and NRAS might be the direct targets of miR-145-5p.
The effects of miR-145 on tumor angiogenesis and invasion were investigated in vitro using transwell invasion assays and endothelial cell tube formation assays. The results revealed that the overexpression of miR-145 could inhibit the invasion and angiogenesis of UM cells. The tube formation assay using endothelial cells showed that miR-145-transfected HMEC-1 cells formed shorter tube length and decreased branch points as compared with controls, indicating a decreased angiogenic activity. Transwell assays demonstrated that the numbers of invaded MUM-2B and OCM-1 cells with miR-145 overexpression were significantly lower than the controls, suggesting that cell invasion ability was significantly decreased by overexpression of miR-145 in MUM-2B and OCM-1 cells.
To investigate the effect of miR-145 on tumor growth and angiogenesis in vivo, we injected lentivirus-miR-scr- and lentivirus-miR-145-transduced OCM-1 cells into nude mice. We found that miR-145 expression in UM cells significantly reduced xenograft tumor growth and angiogenesis compared with those transfected with empty vector. At 30 days after subcutaneous injection, xenografts expressing miR-145 had smaller sizes and lower weights. In addition, the expression of N-RAS, p-AKT, m-TOR, and VEGF was also reduced by stable transfection of the miR-145 precursor.
The observed different effects of miR-145-5p in diverse tumors are very promising for application as diagnostic or prognostic biomarkers. To better understand the miR-145-mediated network on tumor angiogenesis, emphasis should be on searching for more target genes and exploring potential target therapies. When the results of our study are discussed, its limitations should be considered. First, although the ration of the tumor growth is higher in subcutaneous xenograft models and the lesions are easy to be removed, inoculated xenograft models (such as anterior chamber injection) could provide specific features of the ocular immune system leading to an intraocular tumor microenvironment. Further research on intraocular xenograft models is strongly needed, so that we can better understand the unique characteristics of UM. Second, since liver metastases occur in 95% of patients with metastatic UM, liver metastasis mouse models may offer a more detailed investigation of the biological behavior of metastatic UM cells. Such issues are expected to be addressed in our future work.
In conclusion, our results suggest that miR-145 suppresses tumor growth and invasion by directly targeting the N-RAS and VEGF signaling pathways in UM. The findings of our study also have identified miR-145 as an anti-angiogenic factor both in vitro and in vivo, which could have important implications for further understanding the signaling mechanisms involved in regulating UM angiogenesis. Accordingly, there is a good reason to speculate that miR-145 is an important tumor suppressor and the inhibitory strategies against the N-RAS/VEGF signaling pathway might be potential therapeutic applications for UM in the future.
doi.org/10.1097/CM9.0000000000000875
Was this helpful?
0 / 0