Molecular Mechanism Study Of MicroRNA-33a Through Targeted HIF-1α Inhibit The Invasion And Metastasis Of Melanoma

Object:Discuss miR-33a/b expressing features and significance in different transfer potential melanoma cell lines.Interfere miR-33a/b expression in different melanoma cell lines, observe the malignant biological function changes in vitro such as proliferation, adhesion, movement, apoptosis and epithelial-mesenchymal transition. Interfere miR-33a expression in A375melanoma cell line, observe the size, volume, quality of subcutaneous planting tumor in nude mice, the numbers of lung metastases in vivo. Discuss the possible mechanism of miR-33a/b affect the malignant biological function of melanoma cell.Methods:Through the real-time PCR method to detect miR-33a/b expression in low transfer aggressivity skin melanoma cell line WM35and high transfer aggressivity cell lines WM451, A375and SK-MEL-1. Explicit miR-33a/b expression in the melanoma cell lines, screening miR-33a/b high expression and low expression melanoma cell lines for further experimental study.Build pYr-LVX-pri-mir33a slow virus vector, and packaged into slow virus, and infect A375cells, develop a hsa-miR-33a stable expression cell line A375-pYr-LVX-pir-miR-33a; Build pYr-LVX-miR-33a-sponge slow virus vector, and packaged into slow virus, and infect WM35, WM451cells respectively, establish hsa-miR-33a stable inhibited cell lines WM35-pYr-LVX-miR-33a-sponge and WM451-pYr-LVX-miR-33a-sponge.Use miR-33a stable downregula-tion/up-regulation expression cell lines as the research object, using MTT method to detect cell proliferation ability; Plate clone formation test detect cell numbers of cloning; Flow cytometry to detect cell apoptosis rate; Cell scratch experiment, Transwell detection cell migration force; Western Blot method related to the transformation of epithelial-mesenchymal phenotype of E-Cadherin, N-Cadherin protein expression. More levels to explore miR-33a expression on cell proliferation, apoptosis, invasion and metastasis, and transformation of epithelial mesenchymal phenotype.By subcutaneous inoculation and tail vein injection of A375and A375-pYr-LVX-pri-miR-33a cells establish nude mice plant tumor and transplantation tumor model, observe the size, volume, quality of subcutaneous planting tumor in nude mice, the numbers of live and lung metastases in vivo.Bioinformatics analysis predict HIF-la may be an downstream target genes of miR-33a; Western Blot to detect HIF-lα protein expression level change with miR-33a expression; Application of dual luciferase report gene method to further verify whether HIF-lα is the direct target genes of miR-33a.Results:The quantity of miR-33a and miR-33b express in low transfer aggressivity skin melanoma cell line WM35is high, and the quantity in high transfer aggressivity melanoma cell lines WM451, A375and SK-MEL-1is relatively low (WM35> WM451> A375> SK-MEL-1);Raise miR-33a expression in A375cell line can inhibit cell proliferation, invasion and metastasis and epithelial-mesenchymal transition; Cut miR-33a expression in WM451and WM35cell lines can promote cell proliferation, invasion and metastasis and epithelial-mesenchymal transition, but has no significant differences in affecting cell apoptosis. MiR-33a can inhibit planting tumor and the formation of metastases in animal experiments. MiR-33a can inhibit tumor formation rate, reduce the numbers of lung metastases. Bioinformatics analysis predict HIF-la may be an downstream target genes of miR-33; Down-regulation WM35and WM451miR-33a expression, the expression of HIF-1α up; Up-regulation A375miR-33a expression, the expression of HIF-1α down. Dual luciferase report gene method further confirms that HIF-1α a direct target gene for miR-33a.Conclusion:The expression quantity of miR-33a and miR-33b on the in site skin melanoma is higher than the metastatic skin melanoma.MiR-33a can inhibit human melanoma cells proliferation, invasion metastasis and epithelial-mesenchymal transition, but has no significant differences in affecting cell apoptosis in vitro. MiR-33a can inhibit planting tumor in animal experiments, and the formation of metastases, it sugest that miR-33a is the tumor suppressor genes of melanoma.MiR-33a may directly target regulat HIF-la, thus influence melanoma cells malignant biological phenotype.