摘要
目的探讨miR-485-5p靶向调控WNT7B在调节骨髓间质干细胞(bone marrow mesenchymal stem cell,BMSC)成骨分化中的作用和机制。方法收集2023年1至10月于天津市天津医院因髋部骨折行髋关节置换的骨质疏松患者15例,采集股骨头中经双能X线吸收法检测骨密度降低区域的骨组织(骨质疏松组);根据年龄和体质指数匹配因骨关节炎行关节置换的患者15例,采集股骨头中骨密度正常区域的骨组织(无骨质疏松组)。使用qRT-PCR技术检测两组miR-485-5p和WNT7B的表达;利用生物信息学技术找出miR-485-5p的潜在靶基因,通过基因本体论富集分析和KEGG信号通路分析相关的生物过程和信号通路,分析miR-485-5p与WNT7B的靶向关系。构建miR-485-5p过表达和抑制模型,分为对照组、miR-485-5p组、miR-485-5p抑制组,分别行碱性磷酸酶染色、茜素红染色,并采用Western blot检测成骨相关蛋白(ALP、BMP-2、Runx2、OPN、OCN);通过转染miR-485-5p抑制剂和WNT7B siRNA,观察成骨相关蛋白的表达,分析加入WNT7B siRNA后miR-485-5p对BMSC成骨分化的影响。结果骨质疏松组miR-485-5p相对表达量为7.54±0.49,较无骨质疏松组的3.06±0.27高,差异有统计学意义(t=4.110,P<0.001);骨质疏松组WNT7B相对表达量为3.64±0.28,较无骨质疏松组的6.58±0.37低,差异有统计学意义(t=3.382,P<0.001);骨质疏松组miR-485-5p和WNT7B相对表达量相关性分析为负相关(r=-0.732,P<0.001)。富集分析结果发现miR-485-5p靶基因主要参与蛋白质合成和修饰、基因表达调控等生物过程;KEGG分析发现其参与TGF-β信号通路、Wnt信号通路、MAPK信号通路等,且miR-485-5p与WNT7B 3'UTR相结合,即miR-485-5p与WNT7B mRNA存在靶向关系。ALP染色和茜素红染色结果发现miR-485-5p组较对照组颜色浅,钙沉积物减少;而miR-485-5p抑制组染色加深,成骨细胞活性增强,骨矿化效果加强。miR-485-5p组ALP、BMP-2、Runx2、OPN、OCN、WNT7B和β-catenin蛋白的相对表达量分别为0.78±0.13、0.68±0.16、0.59±0.19、0.54±0.14、0.74±0.12、0.49±0.17、0.52±0.19,较对照组降低(P<0.05);miR-485-5p抑制组相对表达量分别为1.29±0.21、1.24±0.19、1.16±0.24、1.31±0.27、1.45±0.25、1.05±0.19、1.41±0.26,较对照组升高(P<0.05)。miR-485-5p抑制组OPN、WNT7B和β-catenin蛋白相对表达量分别为1.42±0.21、1.38±0.32、1.16±0.2,miR-485-5p抑制+WNT7Bi组分别降低至1.08±0.19、0.71±0.22、0.84±0.25,差异有统计学意义(P<0.05)。结论MiR-485-5p与WNT7B存在靶向关系,miR-485-5p过表达和抑制会调控WNT7B表达,从而影响成骨分化过程;其作用机制为miR-485-5p靶向调控WNT7B介导的Wnt/β-catenin信号通路影响BMSC成骨分化。
ObjectiveTo explore the role and mechanism of miR-485-5p targeted regulation of WNT7B in regulating osteogenic differentiation of bone marrow mesenchymal stem cell(BMSC).Methods15 osteoporotic patients who underwent hip replacement due to hip fracture in Tianjin Hospital from January to October 2023 were collected,and bone tissues in the femoral head in the area of reduced bone density detected by the dual-energy X-ray absorptiometry(DXA)method were collected(osteoporosis group);15 patients who underwent joint replacement due to osteoarthritis were matched according to their age and body mass index,and bone tissues in the femoral head in the area of normal bone density were collected(no osteoporosis group).MiR-485-5p and WNT7B were detected using qRT-PCR technology;the target genes and potential mechanisms of miR-485-5p were predicted using bioinformatics technology,and the relationship between miR-485-5p and WNT7B was analyzed by dual luciferase reporter system.The miR-485-5p overexpression(mimic)and inhibitor(inhibitor)were constructed and divided into control,miR-485-5p group and miR-485-5p inhibitor group.After alkaline phosphatase staining(ALP)and alizarin red staining(ARS),osteogenesis-related proteins were detected by Western blot(ALP,BMP-2,Runx2,OPN,OCN);expression of osteogenic proteins was detected by transfection of miR-485-5p inhibitor and WNT7B siRNA into BMSC.ResultsThe relative expression of miR-485-5p in the osteoporosis group was 7.54±0.49,which was higher than that in the no-osteoporosis group with significant difference(t=4.11,P<0.001),while the relative expression of WNT7B was significantly lower(t=3.38,P<0.001),which was negatively correlated with miR-485-5p;bioinformatics analysis found that miR-485-5p targeted 666 genes,miR-485-5p could bind the 3'UTR of WNT7B,and the main mechanism was related to the Wnt/β-catenin signaling pathway;ALP activity and calcium deposition were reduced in the miR-485-5p group compared with the control group,and ALP,BMP-2,Runx2,OPN,OCN,WNT7B andβ-catenin proteins were 0.78±0.13,0.68±0.16,0.59±0.19,0.54±0.14,0.74±0.12,0.49±0.17,0.52±0.19,respectively,which were significantly reduced compared with the control group(t=3.214,P<0.001;t=3.637,P<0.001;t=3.479,P<0.001;t=4.062,P<0.001;t=4.271,P<0.001;t=4.164,P<0.001;t=4.621,P<0.001),and ALP activity,calcium deposition were reduced;ALP,BMP-2,Runx2,OPN,OCN in miR-485-5p inhibition group,WNT7B andβ-catenin protein relative expression were 1.29±0.21,1.24±0.19,1.16±0.24,1.31±0.27,1.45±0.25,1.05±0.19,1.41±0.26,respectively,which were significantly higher compared with the control group(t=3.156,P<0.001;t=3.645,P<0.001;t=3.473,P<0.001;t=3.954,P<0.001;t=4.006,P<0.001;t=3.889,P<0.001;t=4.513,P<0.001).The relative expression of OPN,WNT7B andβ-catenin proteins in the miR-485-5p inhibition group were 1.42±0.21,1.38±0.32,1.16±0.2.ALP activity was significantly lower in the miR-485-5p inhibition+WNT7Bi group,with lighter ARS staining,fewer bone deposits,and reduced bone-forming related proteins OPN,WNT7B andβ-catenin relative expression of 1.08±0.19,0.71±0.22,and 0.84±0.25,which were all significantly reduced(t=3.675,P<0.001;t=3.401,P<0.001;t=3.354,P<0.001).ConclusionMiR-485-5p overexpression slowed down the process of osteogenic differentiation and caused down-regulation of the expression of related proteins,whereas miR-485-5p inhibition promoted osteogenic differentiation and was negatively correlated with WNT7B in the bone tissues of osteoporosis patients.MiR-485-5p binds to the WNT7B mRNA target,which in turn influences the expression of related proteins of WNT7B,and the mechanism of its action is that miR-485-5p targeted to regulate WNT7B-mediated Wnt/β-catenin signalling pathway inhibits BMSC osteogenic differentiation.
作者
王湛
田爱现
马信龙
刘天盛
王毅
Wang Zhan;Tian Aixian;Ma Xinlong;Liu Tiansheng;Wang Yi(Department of Laboratory,Tianjin Hospital,Tianjin 300211,China;Institute of Orthopaedics,Tianjin Hospital(Tianjin Hospital of Tianjin University),Tianjin 300211,China;Three Wards of Hip and Joint,Tianjin Hospital,Tianjin 300211,China)
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2024年第16期1104-1113,共10页
Chinese Journal of Orthopaedics
基金
天津市科技计划重点项目(22JCZDJC00340,20JCZDJC00570)
