Background

Accumulating study indicated that microRNA (miRNA) played critical role in the osteosarcoma (OS). The role and mechanisms of miR-193b in OS cell lines were still unknown.

Objective

We resolved the miR-193b expression in OS cell line and normal cell by RT-PCR assay. The effects of upregulated miR-193b on OS cell proliferation, migration, invasion and apoptosis were evaluated using CCK-8 assay, transwell assay, would-healing assay and flow cytometric analysis in vitro, respectively. We investigated the effect of upregulated miR-193b on the mRNA level of cell cycle protein CCND1 and CCNE1 using RT-PCR assay and the protein level of epithelial to mesenchymal transition (EMT)-related protein E-cadherin, vimentin, and N-cadherin by western blotting assay in MG-63 and U2SO cells. Furthermore, luciferase reporter assays were employed to identify the candidate target gene RAB7A of miR-193b.

Results

The expression of miR-193b was downregulated in OS cells. In MG-63 and U2SO cells, ectopic miR-193b expression inhibited cell proliferation, migration, invasion, and induced apoptosis. We found that miR-193b reduced the mRNA expression of CCND1 and CCNE1, and regulated the associated proteins of EMT including E-cadherin, vimentin and N-cadherin in MG-63and U2SO cell lines. Moreover, the candidate target gene RAB7A was negatively regulated by miR-193b. In addition, upregulated RAB7A rescued the inhibitory effect of miR-193b mimics on the development of OS cell.

Conclusion

In conclusion, this study suggested that miR-193b overexpression inhibited cell proliferation, migration, invasion, and induced cell apoptosis by down-regulating RAB7A in OS cell lines.

中文翻译：

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MiR-193b通过靶向RAB7A促进骨肉瘤细胞增殖和迁移并抑制细胞凋亡

背景

越来越多的研究表明，microRNA（miRNA）在骨肉瘤（OS）中起关键作用。miR-193b在OS细胞系中的作用和机制尚不清楚。

目的

我们通过RT-PCR测定解析了miR-193b在OS细胞系和正常细胞中的表达。分别使用CCK-8检测，transwell检测，will-healing检测和流式细胞术分析了miR-193b上调对OS细胞增殖，迁移，侵袭和凋亡的影响。我们使用RT-PCR分析了上调的miR-193b对细胞周期蛋白CCND1和CCNE1 mRNA水平的影响以及上皮到间质转化（EMT）相关蛋白E-钙粘蛋白，波形蛋白和N-钙粘蛋白的蛋白水平免疫印迹法检测MG-63和U2SO细胞。此外，采用萤光素酶报告基因测定来鉴定miR-193b的候选靶基因RAB7A。

结果

miR-193b的表达在OS细胞中下调。在MG-63和U2SO细胞中，异位miR-193b表达抑制细胞增殖，迁移，侵袭并诱导凋亡。我们发现miR-193b降低了MG-63和U2SO细胞系中CCND1和CCNE1的mRNA表达，并调节了EMT的相关蛋白，包括E-钙粘蛋白，波形蛋白和N-钙粘蛋白。而且，候选靶基因RAB7A被miR-193b负调控。此外，上调的RAB7A挽救了miR-193b模拟物对OS细胞发育的抑制作用。

结论

总之，这项研究表明，miR-193b过表达通过下调OS细胞系中的RAB7A抑制细胞增殖，迁移，侵袭并诱导细胞凋亡。