miR-199a-3p通过靶向CABLES-1调控流体剪切力介导的成骨细胞增殖

Mir-199a-3p Mediates Fluid Shear Stress-Induced Osteoblast Proliferation by Targeting CABLES-1

目的探讨miR-199a-3p在流体剪切力(fluid shear stress,FSS)诱导成骨细胞增殖中的作用及其可能的分子机制。方法对成骨细胞MC3T3-E1加载1.2 Pa FSS,时间分别为0、15、30、45、60、75、90 min。使用miR-199a-3p模拟物或miR-199a-3p抑制物转染MC3T3-E1细胞。使用将过表达的miR-199a-3p以及其阴性对照分别转染MC3T3-E1细胞,并以1.2 Pa FSS处理45 min。将pcDNA NC、pcDNA-CABLES-1、si RNA NC、si RNA CABLES-1转染至MC3T3-E1细胞中。分别共转染pc DNA-CABLES-1与miR-199a-3p mimic以及si RNA-CABLES-1与miR-199a-3p inhibitor。CCK-8实验检测细胞活性;RT-qPCR检测CABLES-1、miR-199a-3p、CDK 6、Cyclin D1、PCNA表达水平;荧光素酶报告实验检测CABLES-1和miR-199a-3p的靶向关系。免疫荧光检测CABLES-1蛋白表达。Western blot检测CABLES-1、CDK 6、PCNA、Cyclin D1的蛋白表达。结果FSS作用下MC3T3-E1细胞中的miR-199a-3p出现显著下调。过表达的miR-199a-3p抑制成骨细胞增殖,下调miR-199a-3p表达促进成骨细胞增殖。miR-199a-3p可以逆转FSS诱导的成骨细胞增殖。双荧光素酶实验表明,miR-199a-3p靶向作用于CABLES-1,过表达的miR-199a-3p可抑制CBALES-1蛋白表达。CABLES-1能够促进成骨细胞增殖。miR-199a-3p通过CABLES-1抑制FSS诱导的成骨细胞增殖。结论FSS诱导的成骨细胞增殖通过下调miR-199a-3p并通过靶向作用于CABLES-1实现。研究结果为FSS诱导成骨细胞增殖机制的研究提供新方向,也为未来机械刺激在骨关节疾病治疗中的临床应用研究提供新思路。

Objective To explore the role of miR-199a-3p in osteoblast proliferation induced by fluid shear stress(FSS)and the potential molecular mechanism.Methods Osteoblast MC3T3-E1 was treated with 1.2 Pa FSS with time gradients of 0,15,30,45,60,75 and 90 min,respectively.MC3T3-E1 cells were transfected with miR-199a-3p mimic or miR-199a-3p inhibitor.MC3T3-E1 cells were transfected with miR-199a-3p mimic and its negative control and then treated with 1.2 Pa FSS for 45 min.The pc DNA NC,pc DNA-CABLES-1,si RNA NC and si RNA CABLES-1 were transfected into MC3T3-E1 cells.The pc DNA-CABLES-1 and miR-199a-3p mimic and SI NA-cables-1 and miR-199a-3p inhibitor were co-transfected,respectively.Cell activity was detected by CCK-8 assay.Real-time quantitative PCR(RT-qPCR)was used to detect expression levels of CABLES-1,miR-199a-3p,CDK 6,Cyclin D1 and PCNA.Luciferase reporting assay was used to detect targeting relationship between CABLES-1 and miR-199a-3p.Immunofluorescence was used to detect protein expression of CABLES-1.Western blot was used to detect protein expression of CABLES-1,CDK 6,PCNA and Cyclin D1.Results Mir-199a-3p in MC3T3-E1 cells was significantly down-regulated by FSS.Over-expressed miR-199a-3p inhibited osteoblast proliferation,and down-regulated miR-199a-3p expression promoted osteoblast proliferation.miR-199a-3p could reverse the FSS-induced proliferation in osteoblasts.Dual luciferase assay showed that miR-199a-3p targeted to CABLES-1 and over-expressed miR-199a-3p inhibited expression of CBALES-1 protein.CABLES-1 could promote proliferation of osteoblasts.miR-199a-3p inhibited osteoblast proliferation induced by FSS through CABLES-1.Conclusions FSS-induced osteoblast proliferation can be realized by down-regulated miR-199a-3p expression via targeting CABLES-1.The findings in this study provide new direction for researches on mechanism of FSS-induced osteoblast proliferation,as well as new ideas for future research on clinical application of mechanical loading in the treatment of bone and joint diseases.