miR-186通过下调垂体瘤转化基因1的表达抑制骨肉瘤细胞的增殖侵袭并诱导其凋亡

目的:探讨miR-186对骨肉瘤细胞增殖、凋亡及侵袭能力的影响,并探讨其可能机制.方法:实时荧光定量PCR检测骨肉瘤细胞HOS、U2-OS、Saos-2及成骨细胞NHOst中miR-186表达,并运用人工合成的miR-186模拟片段及对照scramble mimic转染至人骨肉瘤HOS及U2-OS细胞内,运用实时荧光定量PCR检测转染后细胞中miR-186的表达水平;分别运用CCK-8法、流式细胞检测技术及Transwall体外侵袭实验检测过表达miR-186对HOS及U2-OS细胞增殖、凋亡及侵袭能力的影响;运用Westem blotting及实时荧光定量PCR检测miR-186过表达对细胞中垂体瘤转化基因1(pituitary tumor transforming gene 1,PTTG1)的蛋白及mRNA表达水平的影响.结果:骨肉瘤细胞中miR-186呈现低表达;转染人工合成的miR-186片段可上调HOS及U2-OS细胞中miR-186的表达;miR-186过表达组细胞的增殖能力较scramble组明显下降(P＜0.01),转染组HOS(16.9±2.1)％ vs(10.4±1.6)％,P＜0.05]及U2-OS(22.6±2.9)％ vs (14.1±2.2)％,P＜0.05]细胞的凋亡比例明显高于scramble组,转染组HOS及U2-OS细胞的穿膜数明显低于scramble组(P＜0.01);转染组细胞中PTTG1的蛋白及mRNA表达水平均明显下降(P＜0.01),而scramble组无明显变化(P＞0.05);结论:miR-186能够抑制骨肉瘤细胞的增殖及侵袭并促进细胞凋亡,其作用机制可能与抑制PTTG1表达相关.

miR-186 suppresses the proliferation and invasion and promotes apoptosis of os-teosarcoma cells partially through targeting PTTG1

Objective: To explore the effects of miR-186 on the cell proliferation, apoptosis and invasion of hu-man osteosarcoma cells, and identify its putative mechanisms. Methods: The expression of miR-186 in osteosarco-ma cell lines (HOS, U2-OS and Saos-2) and osteoblast NHOst cells was detected using RT-PCR assays. Artificially synthesized miR-186 mimic and relative control scramble mimic was transfected into osteosarcoma HOS and U2-OS cell lines, and the expression of miR-186 in OS cells was detected using the RT-PCR assays upon transfection.Then, the effects of miR-186 over-expression on cell proliferation, apoptosis and invasion of OS cells were explored using the CCK-8, FCSE and transwell invasion assays, respectively. The effects of miR-186 over-expression on mRNAand protein expression of PTTG1 (pituitary tumor transforming gene1) were explored using the Western blot-ting and RT-PCR assays. Results: miR-186 was low expressed in OS cell lines; Transfection with artificially synthe-sized miR-186 mimic significantly up-regulated the expression of miR-186 in HOS and U2-OS cells; the prolifera-tion rate of cells transfected with miR-186 mimic was much lower than those transfected with scramble mimic HOS: (16.9±2.1)% vs (10.4±1.6)%; U2-OS: (22.6±2.9)% vs (14.1±2.2)%; (P<0.01); the apoptotic rates of HOS and U2-OS cells transfected with miR-186 mimic were higher than those transfected with scramble mimic HOS: (16.9±2.1)% vs (10.4±1.6)%; U2-OS: (22.6±2.9)% vs (14.1±2.2)%; all P<0.05], and the number of cells passing through the chambers in miR-186 mimic transfection group was less than those of scramble transfection group (P<0.01).The expression levels of PTTG1 at protein and mRNA level were both suppressed in cells transfected with miR-186 mimic (P<0.01); however, scramble transfection group showed no statistical difference (P>0.05). Conclusion: Over-expression of miR-186 significantly inhibited cell proliferation and invasion, and promoted the apoptosis of OS cells, which might be related with PTTG1 suppression.