微小RNA-145靶向Notch通路抑制人韧带成纤维细胞向成骨细胞分化  

MicroRNA - 145 targeting Notch pathway inhibits differentiation of human ligament fibroblasts into osteoblasts

作　　者：王亚寒[1] 罗建平[1] 王小刚[1] 杨彬[1] 崔力扬[1] 

机构地区：[1]河南省人民医院骨科,郑州450003

出　　处：《中华实验外科杂志》2017年第11期1828-1831,共4页Chinese Journal of Experimental Surgery

摘　　要：目的探讨微小RNA（miRNA，miR）-145对人韧带成纤维细胞（HLFs）向成骨细胞分化的影响及其可能的作用机制。方法用含地塞米松、抗坏血酸和β-磷酸甘油培养液诱导HLFs细胞向成骨细胞分化，过表达或抑制细胞中miR-145水平，碱性磷酸酶（ALP）试剂盒检测ALP活性，Western blot分析骨代谢相关细胞因子以及Notch通路相关蛋白表达，荧光素酶报告基因方法检测miR-145靶基因。结果强直性脊柱炎患者骨化韧带组织中miR-145表达水平显著下降（0.21±0.09比0.64±0.13；t＝7.543，P＝0.000）；与正常组比较，诱导液处理可显著抑制miR-145的表达（0.22±0.09比0.73±0.13；t＝4.812，P＝0.009），提高ALP、成骨细胞特异性转录因子（OSX）、Runt相关基因2（Runx2）水平（157.9±23.8比76.7±19.2，t＝－4.599，P＝0.010；1.01±0.21比0.32±0.10，t＝－5.138，P＝0.007；0.95±0.32比0.27±0.08，t＝－3.571，P＝0.023），同时抑制破骨细胞分化因子核因子κ B受体活化因子（RANK）及其配体（RANKL）表达（0.30±0.17比0.74±0.13，t＝3.561，P＝0.024；0.36±0.11比0.85±0.20，t＝3.718，P＝0.021），miR-145过表达可显著削弱诱导液预处理带来的上述改变，而miR-145敲减可增加诱导液的作用；同时miR-145敲减可显著增加诱导液预处理诱导的Notch1、Hes1蛋白表达上调（1.81±0.19比1.20±0.23，t＝－3.542，P＝0.024；1.66±0.20比1.13±0.22，t＝－3.088，P＝0.037），提高Notch通路活性，此种作用可被Notch通路抑制剂DAPT逆转；荧光素酶报告基因结果显示miR-145对Notch通路的作用可能通过直接靶向调控解整合素样金属蛋白酶17（ADAM17）蛋白表达实现。结论miR-145可通过直接靶向ADAM17调控Notch通路活性，进而抑制HLFs向成骨细胞分化。Objective To investigate the effect of microRNA （miRNA, miR） - 145 on human ligament fibroblasts （HLFs） differentiation and its underlying mechanism. Methods HLFs were induced to differentiate into osteoblasts by osmiasone, ascorbic acid and β- phosphoglycerin, miR - 145 was over- expressed or knockdown in the cells, alkaline phosphatase （ALP） activity was detected by ALP kit assay, the expression of bone metabolism related cytokines and Notch pathway - related proteins were analyzed by reverse transcription - polymerase chain reaction （ RT - PCR） and Western blotting, dual - luciferase assay was also performed to detect the target gene of miR - 145. Results miR - 145 expression was significantly decreased in the ossification ligament of AS patients （0. 21 ± 0. 09 vs. 0. 64 ± 0. 13, t = 7. 543, P = 0.000） ; Compared with the normal group, treating with dexamethasone, ascorbic acid and β- glycero- phosphate significantly decreased the expression of miR- 145 （0. 22 ± 0. 09 vs. 0. 73 ± 0. 13, t = 4. 812, P = 0.009） , consistent with an increased ALP, Osterix （OSX） and runt related transcription factor - 2 （RUNX2） expression （157.9 ±23.8 vs. 76. 7 ± 19. 2, t = -4. 599, P =0. 010; 1.01 ±0. 21 vs. 0. 32 ± 0. 10, t= -5. 138, P=0.007; 0.95 ±0.32 vs. 0.27 ±0.08, t= -3.571, P =0.023） , and decreased receptor activator of nuclear factor κB （RANK） and receptor activator of nuclear factor κB ligand （RANKL） expression （0.30±0.17 vs. 0.74±0.13, t=3.561, P=0.024; 0.36±0.11 vs. 0.85 ± 0. 20, t =3. 718, P=0. 021 ）. Conversely, overexpression of miR - 375 promoted the opposite effects. Moreover, miR - 145 knockdown can increase the activity of Notch signaling pathway by up - regulate the Notchl andHesl protein expression （1.81 ±0.19 vs. 1.20±0.23, t= -3. 542, P=0.024; 1.66±0. 20 vs. 1.13 ±0. 22, t = -3. 088, P =0. 037）. These effects can be attuned by DAPT or miR - 145 overexpression. We also identified that miR - 145 modulate the Notch signaling pathway by d

关 键 词：微小RNA-145 人韧带成纤维细胞 解整合素样金属蛋白酶17 NOTCH 韧带骨化 

分 类 号：R68[医药卫生—骨科学]