miR-27b-3p调控SMAD1对骨肉瘤细胞增殖、迁移和侵袭作用的影响

Effect of miR-27b-3p regulating SMAD1 on osteosarcoma cell proliferation, migration and invasion

目的寻找并验证miR-27b-3p和SMAD1的关系,探讨miR-27b-3p对骨肉瘤细胞增殖、侵袭、迁移作用的影响,为骨肉瘤靶向治疗提供理论依据。方法采用生物信息学方法预测候选靶基因;应用双荧光素酶报告实验确定miR-27b-3p和SMAD1靶向关系;采用qRT-PCR法选择SAOS-2骨肉瘤细胞系。采用miR-27b-3p转染SAOS-2细胞后,分为miR-27b-3p inhibitor组、空白对照组和阴性对照组。采用MTT、Transwell迁移和侵袭实验检测miR-27b-3p对SAOS-2细胞的影响;采用Western blotting法检测沉默miR-27b-3p后SMAD1的表达量。结果生物信息学检测显示,miR-27b-3p与SMAD1-UTR存在结合位点;双荧光素酶报告实验显示,miR-27b-3p inhibitor组SMAD1的表达量高于阴性对照组,差异有统计学意义(P<0.05),SMAD1是miR-27b-3p的靶基因。SAOS-2细胞MTT、Transwell迁移实验和侵袭实验结果均显示,miR-27b-3p inhibitor组与空白对照组和阴性对照组细胞数相比降低,差异有统计学意义(P<0.05),miR-27b-3p inhibitor组细胞的增殖、迁移和侵袭能力降低。miR-27b-3p inhibitor组与阴性对照组SMAD1蛋白表达量相比明显降低(P<0.05)。结论miR-27b-3p可以通过调控SMAD1的表达促进骨肉瘤细胞增殖、迁移和侵袭作用。miR-27b-3p可能在骨肉瘤细胞中起着促癌基因作用。

Objective To assess the relationship between miR-27 b-3 p and SMAD1 and to explore the effects of MiR-27 b-3 p on osteosarcoma cell proliferation,invasion and migration,so as to provide theoretical basis for targeted therapy of osteosarcoma.Methods Candidate target genes were predicted with bioinformatics methods.The targeting relationship between miR-27 b-3 p and SMAD1 was determined with dual luciferase report experiments.The SAOS-2 osteosarcoma cell line was selected with qRT-PCR.After SAOS-2 cells were transfected with miR-27 b-3 p,they were divided into 3 groups:miR-27 b-3 p inhibitor group,blank control group and negative control group.The effects of miR-27 b-3 p on SAOS-2 cells were detected with MTT,Transwell migration and invasion assay.The expression of SMAD1 after miR-27 b-3 p silencing was determined with Western blotting.Results Bioinformatics analysis showed there were binding sites of miR-27 b-3 p and SMAD1-UTR.Dual luciferase experiments showed the miR-27 b-3 p inhibitor group had higher expression of SMAD1 than the negative control group(P<0.05),and SMAD1 was the target gene of miR-27 b-3 p.MTT,Transwell migration and invasion assay showed the miR-27 b-3 p inhibitor group had reduced cell proliferation,migration and migration than the blank control group and negative control group(P<0.05).The miR-27 b-3 p inhibitor group had significantly lower protein expression of SMAD1 than negative control group(P<0.05).Conclusion miR-27 b-3 p can promote the proliferation,migration and invasion of osteosarcoma cells by regulating the expression of SMAD1,and may play a role as an oncogene in osteosarcoma cells.