微小RNA-125a-5p负性调控基质金属蛋白酶11对骨肉瘤细胞增殖和凋亡的影响

The effects of microRNA-125a-5p on proliferation and apoptosis of osteosarcoma cells by targeting matrix metalloproteinase 11

目的检测微小RNA-125a-5p(miR-125a-5p)在骨肉瘤细胞及正常成骨细胞(NHOst)中的表达特征,并探讨其通过负性调控基质金属蛋白酶11(MMP-11)对骨肉瘤细胞增殖和凋亡的影响。方法 采用荧光定量PCR分析骨肉瘤细胞及正常成骨细胞中miR-125a-5p的表达情况。体外转染miR-125a-5p模拟物(mimics)至骨肉瘤细胞系(HOS和Saos-2),并观察其对细胞增殖及凋亡的影响。通过双荧光素酶报告基因试验及Western blot验证miR-125a-5p对MMP-11的负性调控作用。结果 miR-125a-5p在HOS和Saos-2细胞中的表达均显著低于NHOst细胞( P <0.05)。HOS和Saos-2细胞中转染miR-125a-5p mimics可显著升高细胞内源性miR-125a-5p表达水平( P <0.05)。过表达miR-125a-5p抑制HOS和Saos-2细胞的增殖,并促进细胞凋亡( P < 0.05)。miR-125a-5p与MMP-11 信使RNA(mRNA)3′-非编码区(3′-UTR)存在互补结合位点,其通过与MMP-11结合而抑制骨肉瘤细胞中MMP-11蛋白的表达( P <0.05)。结论 miR-125a-5p在骨肉瘤细胞中呈现低表达,过表达miR-125a-5p可通过负性调控MMP-11抑制骨肉瘤细胞的增殖,并促进细胞凋亡,提示miR-125a-5p可作为骨肉瘤新的分子治疗靶点。

Objective To detect the expression characteristics of miR-125a-5p in osteosarcoma cells and normal osteoblasts (NHOst),and explore its effects on the proliferation and apoptosis of osteosarcoma cells by negative regulation of matrix metalloproteinase 11(MMP-11). Methods Quantitative RT-PCR analysis was used to detect the expression levels of miR-125a-5p in osteosarcoma cells (HOS and Saos-2) and NHOst cells. The miR-125a-5p mimics were transfected into osteosarcoma cell lines (HOS and Saos-2) in vitro , and the effects on cell proliferation and apoptosis were observed. The negative regulatory effect of miR-125a-5p on MMP-11 expression in osteosarcoma cells was verified by the dual luciferase reporter assay and Western blot. Results The miR-125a-5p expression was significantly down-regulated in HOS and Saos-2 cells than that in the NHOst cell ( P <0.05). Transfection of miR-125a-5p mimics in HOS and Saos-2 cells dramatically increased the expression levels of miR-125a-5p ( P <0.05). Restoration of miR-125a-5p markedly inhibited cell growth and induced cell apoptosis in HOS and Saos-2 cells ( P <0.05). miR-125a-5p could inhibit the expression of MMP-11 protein by targeting MMP-11 mRNA 3′-non coding region (3′-UTR)( P <0.05). Conclusions The miR-125a-5p is down-regulated in osteosarcoma cells,and over-expression of miR-125a-5p inhibits cell proliferation and promotes cell apoptosis in osteosarcoma cells by targeting MMP-11,suggesting miR-125a-5p may be used as a new molecular therapeutic target for osteosarcoma.