lncRNA HOTAIR/miR-17-5p/Smad7通路调控激素性股骨头坏死的机制研究

Mechanism of LncRNA HOTAIR/miR-17-5p/Smad7 Pathway Regulating Steroid-induced Femoral Head Necrosis

目的研究lncRNA HOTAIR对激素性股骨头坏死(steroidinduced necrosis of femoral head,SONFH)的影响。方法收集激素性股骨头坏死患者骨髓组织25例,正常对照组为25例股骨颈骨折患者。培养人骨髓间充质干细胞(hBMSCs),用地塞米松(DEX)培养,CCK-8测定细胞增殖。用HOTAIR过表达载体或miR-17-5p模拟物转染细胞。hBMSCs分为hBMSCs组、hBMSCs+成骨诱导组、hBMSCs+成骨诱导+空载组、hBMSCs+成骨诱导+过表达HOTAIR组、hBMSCs+成脂诱导组、hBMSCs+成脂诱导+空载组和hBMSCs+成脂诱导+过表达HOTAIR组。RT-qPCR测HOTAIR、miR-17-5p、Smad7、RUNX2、RRAR-γ的mRNA表达。Western blot法检测Smad7蛋白的表达。双荧光素酶报告基因测法确认HOTAIR和miR-17-5p间的结合。碱性磷酸酶(ALP)活力检测试剂盒与茜素红染色评估细胞的成骨分化;油红O染色评估细胞的成脂分化。结果HOTAIR和Smad7在激素性股骨头坏死组织中表达上调,而miR-17-5p表达下调(P均<0.05)。双荧光素酶报告实验证实hBMSCs中HOTAIR的直接结合靶点是miR-17-5p,而Smad7是miR-17-5p的靶基因。HOTAIR的过表达诱导了hBMSCs的成脂分化和RRAR-γ表达,并抑制了成骨分化和RUNX2表达(P均<0.05)。结论过表达lncRNA HOTAIR直接靶向miR-17-5p并抑制hBMSCs的成骨分化,还能够促进成脂分化和Smad7的表达。

Objective To investigate the effect of LncRNA HOTAIR on steroid-induced necrosis femoral head(SONFH).Methods Bone marrow tissues of 25 patients with SONFH were collected,and 25 patients with femoral neck fractures were included in the control group.Human bone marrow mesenchymal stem cells(hBMSCs)were cultured with Dexamethasone(DEX),and cell proliferation were measured by CCK-8.Cells were transfected with HOTAIR overexpression vector or miR-17-5p mimics.HBMSCs were divided into 7groups,including hBMSCs group.HBMSCs+osteogenic induction solution group;HBMSCs+osteogenic induction solution+non-load group;HBMSCs+osteogenic induction solution+overexpression HOTAIR lentivirus group;hBMSCs+adipogenic induction solution group;hBMSCs+adipogenic induction solution+non-load group;hBMSCs+adipogenic induction solution+overexpression HOTAIR lentivirus group.The mRNA expressions of HOTAIR,miR-17-5p,Smad7,RUNX2 and RRAR-γwere detected by real-time quantitative polymerase chain reaction(RT-qPCR).The expression of Smad7 protein was detected by Western blot.Dual-luciferase reporter gene assay were performed to confirm the binding between HOTAIR and miR-17-5p.The osteogenic differentiation of cells was evaluated by alkaline phosphatase(ALP)activity assay kit and alizarin red staining.Adipogenic differentiation was evaluated by oil red O staining.Results The expressions of HOTAIR and Smad7 were up-regulated in SONFH tissues,while the expression of miR-17-5p were down-regulated(all P<0.05).Dual-luciferase reporter gene assay confirmed that the direct binding target of HOTAIR in hBMSCs was miR-17-5p,and Smad7 was the target gene of miR-17-5p.Overexpression of HOTAIR induced adipogenic differentiation and RRAR-γexpression of hBMSCs,and inhibited osteogenic differentiation and RUNX2 expression(all P<0.05).ConclusionOverexpression of HOTAIR directly targets miR-17-5p and inhibits osteogenic differentiation of hBMSCs,and also promotes adipogenic differentiation and expression of Smad7.