miR-27b-3p通过靶向抑制HIF1AN调控颈脊髓损伤后骨质疏松症大鼠的成骨分化

miR-27b-3p regulates the osteogenic differentiation of rats with cervical spinal cord injury caused osteoporosis by targeted inhibition of HIF1AN

目的 探讨miR-27b-3p在颈脊髓损伤后骨质疏松症(CSCI-OP)发展中的作用与机制。方法 定量PCR检测30例CSCI-OP患者及50例健康体检者外周血中miR-27b-3p的表达。BMP2诱导骨髓间充质干细胞(MSCs)成骨,采用定量PCR检测MSCs中miR-27b-3p和成骨相关基因的mRNA水平。使用miR-27b-3p mimic过表达或使用miR-27b-3p inhibitor下调miR-27b-3p后检测miR-27b-3p的mRNA水平,探讨miR-27b-3p对MSCs成骨相关基因的调控作用。用茜素红染色检测过表达miR-27b-3p后MSCs的成骨能力。双荧光素酶报告基因检测验证miR-27b-3p与HIF1AN的结合情况。通过挽救实验验证miR-27b-3p是否可以通过靶向HIF1AN调控骨质疏松症的发展。建立CSCI-OP大鼠模型验证miR-27b-3p和HIF1AN的表达,micro-CT观察miR-27b-3p mimic对CSCI-OP大鼠股骨组织骨量的影响。结果 CSCI-OP组患者外周血中miR-27b-3p的表达低于对照组(P<0.05)。miR-27b-3p的表达随着成骨诱导时间的延长而上调(P<0.05)。过表达miR-27b-3p可促进MSCs成骨相关基因ALP、Runx2、OPN的mRNA表达(P<0.05)。MSCs过表达miR-27b-3p后,钙化结节数量增加(P<0.05)。双荧光素酶报告基因检测证实HIF1AN与miR-27b-3p具有多个结合位点。HIF1AN的mRNA和蛋白水平均受到miR-27b-3p的负调控。挽救实验表明,过表达HIF1AN抑制了miR-27b-3p对MSCs成骨的促进作用(P<0.05)。CSCI-OP大鼠中HIF1AN的表达上调(P<0.05),体内micro-CT观察到miR-27b-3p mimic促进了CSCI-OP大鼠ALP、Runx2和OPN的表达,增加了BV/TV(P<0.05),而这些作用都被HIF1AN过表达所抑制(P<0.05)。结论 miR-27b-3p在骨质疏松症患者外周血中高表达,其可通过靶向抑制HIF1AN促进成骨分化并抑制CSCI-OP的发生和进展。

Objective To explore the role and mechanism of miR-27b-3p in the development of cervical spinal cord injury caused osteoporosis(CSCI-OP). Methods Quantitative PCR(qPCR) was used to detect the expression of miR-27b-3p in peripheral blood of 30 patients with CSCI-OP and 50 healthy individuals. BMP2 induced bone formation of bone marrow mesenchymal stem cells(MSCs),and qPCR was used to detect the mRNA levels of miR-27b-3p and osteogenic related genes in MSCs. miR-27b-3p mimic or miR-27b-3p inhibitor was used to regulate miR-27b-3p, the mRNA level of miR-27b-3p was detected to explore the regulation effect of miR-27b-3p on MSCs osteogenic genes.Alizarin red staining was used to detect the osteogenic ability of MSCs after overexpression of miR-27b-3p.Dual luciferase reporter gene test was used to verify the binding of miR-27b-3p and HIF1AN. Rescue experiments was used to verify whether miR-27b-3p could regulate the development of osteoporosis by targeting HIF1AN. The CSCI-OP rats model was established to verify the expression of miR-27b-3p and HIF1AN. The effect of miR-27b-3p mimic on the bone mass of femur in CSCI-OP rats was observed by micro-CT. Results The expression of miR-27b-3p in peripheral blood of patients in the CSCI-OP group was lower than that in the control group(P<0.05). The expression of miR-27b-3p increased with the prolongation of osteogenic induction time(P<0.05). Overexpression of miR-27b-3p could promote the mRNA expression of bone formation related genes such as ALP,Runx2 and OPN of MSCs(P<0.05). Overexpression of miR-27b-3p in MSCs increased the number of calcified nodules(P<0.05). Dual luciferase reporter gene detection confirmed that there were multiple binding sites between HIF1AN and miR-27b-3p. The mRNA and protein levels of HIF1AN were negatively regulated by miR-27b-3p. Rescue experiments showed that overexpression of HIF1AN inhibited the promotion of miR-27b-3p for the osteogenesis of MSCs(P<0.05).The expression of HIF1AN was up-regulated in CSCI-OP rats(P<0.05).micro-CT in vivo observed that miR-27b-3p mimic promoted the expression of ALP,Runx2,and OPN in CSCI-OP rats, and increased BV/TV(P<0.05),however, these effects were inhibited by HIF1AN overexpression(P<0.05). Conclusion miR-27b-3p was highly expressed in peripheral blood of CSCI-OP patients, which promotes osteogenic differentiation and inhibits the occurrence and progression of CSCI-OP by targeted inhibition of HIF1AN.