微小218-5p调控Wnt家族成员2B抑制骨肉瘤细胞143B的增殖、迁移和侵袭

MiR-218-5p inhibits the proliferation,migration and invasion of osteosarcoma cells 143B via regulating Wnt family member 2B

目的探讨微小RNA-218-5p(miR-218-5p)对骨肉瘤细胞增殖,侵袭和迁移能力的影响及其作用机制。方法2016年1月至2019年1月,实时荧光定量PCR检测miR-218-5p在骨肉瘤与正常骨组织及正常成骨细胞株与骨肉瘤细胞株的表达差异。应用CCK-8法检测过表达miR-218-5p对人骨肉瘤细胞株143B增殖的影响;细胞划痕实验检测和Transwell小室实验检测过表达miR-218-5p对骨肉瘤细胞143B迁移能力的影响。同时,应用在线miRNA靶基因预测软件预测miR-218-5p的靶基因,并应用荧光素酶报告实验进行结合位点验证。蛋白质印迹法验证靶基因的蛋白表达水平。结果miR-218-5p在骨肉瘤组织中的表达水平(0.59±0.21)%明显低于正常骨组织,骨肉瘤细胞系中的表达水平(0.712±0.015)%、(0.685±0.016)%、(0.542±0.021)%、(0.524±0.011)%明显低于正常成骨细胞,P<0.05。同时,与对照组相比,过表达miR-218-5p(miR-218-5p mimic)的骨肉瘤细胞143B的细胞活力t6h(0.274±0.011),t24h(0.514±0.04),t48h(0.828±0.16),t72h(1.212±0.013),侵袭(164±12)个和迁移距离(26±2.4)%明显减弱,P<0.05。应用TargetScan网站进行预测,结果显示Wnt家族成员2B(WNT2B)为miR-218-5p的潜在靶基因之一;荧光素酶报告实验结果表明miR-218-5p直接靶向WNT2B;蛋白质印迹法证实miR-218-5p过表达组细胞中WNT2B蛋白的表达水平较对照组明显减少。结论miR-218-5p在人骨肉瘤组织和癌细胞中低表达,其可能通过靶向WNT2B抑制骨肉瘤细胞143B的增殖、迁移和侵袭。

Objective To investigate the effect of miR-218-5 p on proliferation,invasion and migration of osteosarcoma cells and its mechanism.Methods From January 2016 to January 2019,the expression differences of miR-218-5 p in osteosarcoma and normal bone tissue and normal osteoblasts cell lines and osteosarcoma cell lines were detected by real-time fluorescence quantitative PCR.While the effect of miR-218-5 p overexpression in osteosarcoma cells 143 B were investigated using CCK-8.The effects of miR-218-5 p overexpression on osteosarcoma cells 143 B migration and invasion was tested using wound healing assay and transwell assay.Meanwhile,the target genes of miR-218-5 p were predicted by online miRNA target gene prediction software,and the binding sites were verified by luciferase reporting experiment.Results The expression level of miR-218-5 p in osteosarcoma tissues(0.59±0.21)%was significantly lower than that in adjacent tissues,and the expression level in osteosarcoma cell lines(0.712±0.015)%,(0.685±0.016)%,(0.542±0.021)%,(0.524±0.011)%was significantly lower than that in normal osteoblast cells(P<0.05).Meanwhile,compared with the control group,143 B cells of osteosarcoma cells overexpressing miR-218-5 p mimic showed significantly reduced cell activity t6h(0.274±0.011),t24h(0.514±0.04),t48h(0.828±0.16),t72h(1.212±0.013),invasion(164±12),and migration distance(26±2.4)%,P<0.05.TargetScan was used for prediction,and the results showed that Wnt family member 2B(WNT2B)was one of the potential target genes of miR-218-5 p.Luciferase reporting results showed that miR-218-5 p directly targeted WNT2B.Western blot confirmed that the expression level of WNT2 B protein in the miR-218-5 P overexpression group was significantly lower than that in the control group.Conclusion miR-218-5 p is low expressed in human osteosarcoma tissues and cancer cells,miR-218-5 p plays as suppressor in osteosarcoma,which inhibiting proliferation,migration and invasion of osteosarcoma cells 143B via targeting WNT2B.