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

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 directly interacted with the 3＇- untranslated region of the suppressor of a disintegrin and metalloproteinase 17 （ADAM17）. Conclusion Taken together, our results suggest that miR - 145 may regulate the activity of Notch pathway by direct targeting ADAM17, thus inhibited the differentiation of HLFs into osteoblasts. Therefore, this study may provide novel strategies for the development of ankylosing spondylitis ligament os- sification.