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

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。...

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

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 itsnegative 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 inhibitedosteoblast 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.