MiR-30c对骨肉瘤细胞生物学功能的影响研究

The research on the biological functions of miR-30c in osteosarcoma cells

目的探究微小RNA(microRNA,miR)-30c在骨肉瘤细胞中可能发挥的生物学功能。方法荧光定量PCR检测6种骨肉瘤细胞系及人正常成骨细胞中miR-30c的表达水平,筛选目的细胞系;将细胞分为空白组、对照组及miR-30c过表达组,克隆形成和CCK-8实验检测miR-30c对骨肉瘤细胞体外增殖能力的影响;流式细胞术检测miR-30c对骨肉瘤细胞凋亡的影响;Transwell小室实验分析骨肉瘤细胞体外侵袭和迁移能力的改变;裸鼠皮下成瘤实验验证miR-30c在体内对骨肉瘤细胞增殖的影响。结果U2OS、Saos2、Mg63、HuO9、KIKU、143B 6种OS细胞系中miR-30c表达水平均明显低于hFOB1.19细胞,U2OS细胞中miR-30c表达水平最低(P<0.05)。miR-30c过表达组克隆数目明显少于对照组和空白组(P<0.05)。随时间延长,各组细胞增殖能力呈升高趋势,但miR-30c过表达组细胞增殖能力明显低于空白组和对照组,组间、时点间、组间·时点间交互作用差异有统计学意义(P<0.05)。过表达miR-30c组U2OS细胞凋亡率明显高于空白组和对照组(P<0.05)。miR-30c过表达组细胞转染miR-30c mimic后侵袭细胞数和迁移细胞数明显低于空白组和对照组(P<0.05)。裸鼠皮下接瘤4周后,miR-30c过表达组小鼠体内移植瘤体积和重量明显小于空白组和对照组(P<0.05)。结论miR-30c可能作为抑癌基因参与骨肉瘤细胞U2OS的增殖及转移。

Objective To investigate the potential biological functions of miR-30c in osteosarcoma(OS)cells.Methods RT-PCR was used to detect the expression of miR-30c in seven cell lines.The candidate cells were divided into 3 groups:the mock group,the negative group(NC)and miR-30c overexpression group(miR-30c mimic).Colony formation and CCK-8 assays were used to the detect the changes in cell proliferation after transfection.Flow cytometry was used to detect the effect of miR-30c on cell apoptosis in candidate OS cell.Transwell invasion and migration assays were conducted to assess the effects of miR-30c on cell metastasis capabilities in vitro after transfection.Subcutaneous tumor formation assay in nude mice was used to confirm the effect of miR-30c on OS cell proliferation in vivo.Results The expression of miR-30c in U2OS,Saos2,Mg63,HuO9,KIKU,143B cell lines were significantly lower than in hFOB1.19 while U2OS cells have the biggest fold changes with hFOB1.19(P<0.05).The miR-30c mimic group had fewer cell clones than the mock group and NC group(P<0.05).The cell proliferation ability of each group showed an increasing trend,the cell proliferation ability of miR-30c mimic group was significantly lower than the other groups,which had statistically significant differences in interactions between groups,time points and group·time points(P<0.05).The apoptosis rate of U2OS cells in the miR-30c mimic was significantly higher than that in the mock group and NC group(P<0.05).The capacity of cell invasion and migration were significantly suppressed in U2OS after transfection with miR-30c mimics when compared with NC group(P<0.05).After 4 weeks of subcutaneous tumor implantation in nude mice,the volume and weight of transplanted tumors in miR-30c mimic group were significantly smaller than those in mock group and NC group(P<0.05).Conclusion miR-30c may be involved in the proliferation and metastasis of U2OS as a tumor suppressor gene.