Role of microRNA-21 in uveal melanoma cell invasion and metastasis by regulating p53 and its downstream protein

AIM: To reveal the insight mechanism of liver metastasis in uveal melanoma, we investigated cell functions of microR NA-21 in three different uveal melanoma cell lines and analyze the relationship of target gene p53 and its downstream targets.METHODS: Quantitative reverse transcription polymerase chain reaction(qR T-PCR) was used to detect microR NA-21 expression in normal uveal tissue and uveal melanoma cell lines. Lenti-virus expression system was used to construct OCM-1, MuM-2 B and M619 cell line with stable overexpression and inhibition of microR NA-21. In vitro cell function tests such as cell proliferation, cell apoptosis, cell circle and abilities of migration and invasion were examined by MTT, Brd U assay, flow cytometry, transwell assay and Matrigel invasion assay respectively. The target gene was predicted by bioinformatics and confirmed by using a dual luciferase reporter assay. The expression of p53 and its suspected downstream targets LIM and SH3 protein 1(LASP1) and glutathione S transferase pi(GST-Pi) were determined by qR T-PCR in mR NA level and Western blotting analysis in protein level. Finally, the effect of microR NA-21 in a xenograft tumor model was assessed in four-week-old BALB/c nude mice. RESULTS: Compared to normal uveal melanoma, expressions of micro RNA-21 were significantly higher in uveal melanoma cell lines. Overexpression of microR NA-21 promoted proliferation, migration, and invasion of OCM-1, M619 and MuM-2 B cells, while inhibition of microR NA-21 reveal opposite effects. Wild type p53 was identified as a target gene of micro RNA-21-3 p, and proved by dual luciferase reporter assay. Up-regulated micro RNA-21 inhibited the expression of wild type p53 gene, and the increased expression of LASP1 in mR NA level and protein level, while down-regulated microR NA-21 presented opposite way. However, GST-pi showed the potential pattern as expected, but relative mR NA level showed no statistically significant difference in OCM-1 cells. Furthermore, the mR NA expression of GST-pi was decreased in microR NA-21 overexpressing Mu M-2 B, and increased in M619 cells with inhibition of microR NA-21. In vivo, inhibition of microR NA-21 reduced tumor growth with statistically significant difference.CONCLUSION: These findings provide novel insight into molecular etiology of microR NA-21 in uveal melanoma cell lines, and suggest that microR NA-21 might be a potential candidate for the diagnosis and prognostic factor of human uveal melanoma.