摘要
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Western 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m RNA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m RNA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。
Objective： To explore the effects of miR-186 on the cell proliferation, apoptosis and invasion of human osteosarcoma cells, and identify its putative mechanisms. Methods： The expression of miR-186 in osteosarcoma 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 m RNA and protein expression of PTTG1（pituitary tumor transforming gene1） were explored using the Western blotting and RT-PCR assays. Results： miR-186 was low expressed in OS cell lines; Transfection with artificially synthesized miR-186 mimic significantly up-regulated the expression of miR-186 in HOS and U2-OS cells; the proliferation 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 m RNA level were both suppressed in cells transfected with miR-186mimic（P〈0.01）; however, scramble transfection group showed no statistical difference（P〈0.05）. Conclusion： Overexpression of miR-186 significantly inhibited cell proliferation and invasion, and promoted the apoptosis of OS cells, which might be related with PTTG1 suppression.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2017年第8期864-869,共6页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金资助项目(No.81502329)
重庆医科大学附属永川医院院内课题资助项目(No.YCZQN201514)~~
