微小RNA-125a-3p介导Notch1信号通路对骨质疏松性骨折大鼠愈合过程的影响

Effects of mir-125a-3p mediated Notch1 signaling pathway on the healing process of osteoporotic fractures in rats

目的探讨微小核糖核酸(miR)-125a-3p靶向介导Notch1信号通路对Wistar大鼠骨质疏松性骨折愈合的影响。方法6月龄Wistar大鼠58只,假手术组8只(假手术组),其余50只鼠切除卵巢建立骨质疏松大鼠模型,测骨密度验证模型成立,再手术建立一侧股骨中点骨折模型,克氏针固定。去除模型不成功大鼠后分组转染,每组8只,包括:模型组、inhibitor NC组(转染抑制剂阴性对照组)、miR-125a-3p inhibitor组(转染miR-125a-3p抑制剂)、si-Notch1组(转染Notch si-RNA干扰)、miR-125a-3p inhibitor+si-Notch1组(miR-125a-3p抑制剂及Notch1 si-RNA联用组)。大鼠饲养8周后取断端组织,实时定量逆转录聚合酶链反应(qRT-PCR)检测miR-125a-3p及Notch1的表达,过生物信息学网站预测及双荧光素酶报告实验检测miR-125a-3p与Notch1的靶向作用,病理组织学及免疫组化观察、骨密度及生物力学测量共同分析各组骨折愈合情况。结果模型组较假手术组骨密度下降(P<0.05)。与假手术组相比,模型组中miR-125a-3p表达量(1.48±0.11)均上调,Notch1表达(0.75±0.02)下调(P<0.05)。miR-125a-3p mimic与野生型Notch1-WT共转染组中荧光素酶活性强度[萤火虫荧光素酶(0.58±0.02)]下降(P<0.05)。与inhibitor NC组相比,miR-125a-3p inhibitor组(0.62±0.29)及miR-125a-3p inhibitor+si-Notc1组(0.73±0.21)表达下调(P<0.05)。与inhibitor NC组相比,miR-125a-3p inhibitor组Notch1表达(0.93±0.35)提高,si-Notch1组(0.40±0.15)表达降低(P<0.05)。与si-Notch1组相比,miR-125a-3p inhibitor+si-Notc1组(0.79±0.33)Notch1表达提高(P<0.05)。转染8周后,假手术组骨小梁开始大量形成,厚度均匀,排列紧密、方向一致。模型组、inhibitor NC组骨细胞数大量减少,骨小梁较细,间隙较大且排列紊乱。miR-125a-3p inhibitor组骨小梁厚度增加,骨细胞数增多,骨质改善,与假手术组显微形态较为相似。si-Notch组骨细胞数大量缺失,骨小梁间隙极大,显微结构较模型组病理形态更明显。miR-125a-3p inhibitor+si-Notc1组介于miR-125a-3p inhibi⁃tor组与si-Notch1组间,骨小梁厚度有所增加。同inhibitor NC组相比,miR-125a-3p inhibitor组(120.60±6.65)阳性表达增加,si-Notch1组(60.60±2.84)减少(P<0.05)。同miR-125a-3p inhibitor组相比,miR-125a-3p inhibitor+si-Notc1组阳性细胞表达(78.00±2.27)降低,与si-Notch1组相比增多(P<0.05)。第4、6、8周,同假手术组相比,模型组(104.25±10.52、110.25±13.03、116.75±10.59)骨密度下降(P<0.05)。同inhibitor NC组相比,miR-125a-3p inhibitor组(119.02±9.55、131.25±10.57、146.75±12.47)骨密度上升,si-Notch1组降低(P<0.05)。同miR-125a-3p inhibitor组相比,miR-125a-3p inhibitor+si-Notc1组各项骨生物力学指标下降(P<0.05)。同假手术组相比,模型组各项骨生物力学指标,包括最大负载、极限应力及剪切应力下降(P<0.05);同inhibitor NC组相比,miR-125a-3p inhibitor组[最大负载:(125.01±12.05)N;极限应力:(86.75±5.11)Mpa;剪切应力:(1.62±0.34)N/mm2]各项骨生物力学指标上升,si-Notch1组降低(P<0.05)。同miR-125a-3p inhibitor组相比,miR-125a-3p inhibitor+si-Notc1组[最大负载:(95.02±8.41)N;极限应力:(56.52±4.95)Mpa;剪切应力:(1.30±0.17)N/mm2]各项骨生物力学指标下降;与si-Notch1组相比上升(均P<0.05)。结论抑制miR-125a-3p可以促进Notch1表达,增加BMP-2的阳性表达、增强骨密度及生物力学强度,进而促进骨质疏松性Wistar大鼠骨折愈合。

Objective This study aims to investigate the roles of miR-125a-3p in the osteoporotic fracture recovery in Wistar rats via regulating the Notch1 signaling pathway.Methods Fifty-five 6-month old Wistar rats were included in this study.8 rats were subject⁃ed to sham-surgery group,while the remaining 50 rats were ovariectomized for osteoporotic rat model establishment.The bone mineral density was measured for model identification.The model rats were further used for fracture inducement at the midpoint of one-side fe⁃mur and fixed with K-wire.After removing the unsuccessful model rats,the rats were transfected in groups,each with 8 rats,including:Sham,Model,inhibitor NC,miR-125a-3p inhibitor,si-Notch1 and miR-125-3p+si-Notch-1 groups.The tissues from the broken ends were collected 8weeks later,and then the expression of miR-125a-3p and Notch was determined using RT-qPCR.Target relation be⁃tween miR-125a-3p and Notch1 was predicted via bio-information analysis and dual luciferase reporter gene assay.The bone recovery from fracture was detected using histopathology analysis,Immunohistochemistry,bone mineral density and biomechanics measure⁃ments.Results The BMD of model group was lower than that of sham operation group(P<0.05).Compared with sham group,the ex⁃pression of mir-125a-3p was up-regulated(1.48±0.11)and Notch1 was down regulated(0.75±0.02)in model group(P<0.05).The lu⁃ciferase activity intensity[firefly luciferase(0.58±0.02)]decreased in the co transfection group of mir-125a-3p mimic and wild-type Notch1 WT(P<0.05).Compared with the inhibitor NC group,the expression of mir-125a-3p inhibitor group(0.62±0.29)and mir-125a-3p inhibitor+Si notc1 group(0.73±0.21)were down regulated(P<0.05).Compared with the inhibitor NC group,the expression of Notch1 in mir-125a-3p inhibitor group(0.93±0.35)was increased,while that in Si Notch1 group(0.40±0.15)was decreased(P<0.05).Compared with si-notch1 group,the expression of Notch1 in mir-125a-3p inhibitor+si-notchc1 group was(0.79±0.33)(P<0.05).After 8 weeks of transfection,a large number of trabeculae began to form in sham group,with uniform thickness,close arrange⁃ment and consistent direction.In model group and inhibitor NC group,the number of osteocytes decreased significantly,the trabecular bone was thinner,the gap was larger and the arrangement was disordered.In mir-125a-3p inhibitor group,the thickness of trabecular bone increased,the number of osteocytes increased,and the bone improved,which was similar to that in sham group.In Si notch group,the number of osteoblasts was largely absent,and the trabecular space was extremely large,and the microstructure was more obvious than that in model group.Mir-125a-3p inhibitor+si-notchc1 group was between mir-125a-3p inhibitor group and si-notch1 group,and trabecular thickness increased.Compared with the inhibitor NC group,the positive expression of mir-125a-3p inhibitor group(120.60±6.65)increased,while that of Si Notch1 group(60.60±2.84)decreased(P<0.05).Compared with the mir-125a-3p inhibitor group,the expression of positive cells in the mir-125a-3p inhibitor+si-notchc1 group was(78.00±2.27)lower,and increased compared with the si-notch1 group(P<0.05).Compared with sham group,BMD of model group[(104.25±10.52),(110.25±13.03),(116.75±10.59)]de⁃creased at 4,6 and 8 weeks(P<0.05).Compared with the inhibitor NC group,the bone mineral density of mir-125a-3p inhibitor group[(119.02±9.55),(131.25±10.57),(146.75±12.47)]increased,while that of si-notch1 group decreased(P<0.05).Compared with mir-125a-3p inhibitor group,the bone biomechanical indexes of mir-125a-3p inhibitor+Si notc1 group were decreased(P<0.05).Com⁃pared with sham group,the biomechanical parameters of model group,including maximum load,ultimate stress and shear stress,de⁃creased(P<0.05);compared with inhibitor NC group,mir-125a-3p decreased(P<0.05)In the inhibitor group[maximum load:(125.01±12.05)N;ultimate stress:(86.75±5.11)MPA;shear stress:(1.62±0.34)N/mm2]all bone biomechanical indexes increased,but decreased in the si-notch1 group(P<0.05).Compared with the mir-125a-3p inhibitor group,the bone biomechanical indexes of the mir-125a-3p inhibitor+si-notc1 group[maximum load:(95.02±8.41)N;ultimate stress:(56.52±4.95)MPA;shear stress:(1.30±0.17)N/mm2]decreased,and increased compared with the si-notch1 group(P<0.05).Conclusion MiR-125a-3p inhibition could promote Wistar rats recovered from osteoporotic fracture via promoting the expression of Notch1,increasing the positive expression of BMP-2,enhancing bone density and biomechanical strength.