长链非编码RNA-AF289591调控SOST表达对成骨细胞分化的影响

Effect of lncRNA-AF289591 on bone formation by modulating SOST gene expression

目的探讨长链非编码RNA(long noncoding RNA,lncRNA)-AF289591对间充质干细胞(human mesenchymal stem cells,hMSC)成骨分化的影响及其分子机制。方法收集骨质疏松症(osteoporosis,OP)患者以及健康人群的骨髓标本,通过lncRNA芯片分析hMSC中lncRNAs的表达,并通过实时荧光定量聚合酶链式反应(real-time polymerase chain reaction,qPCR)验证lncRNA-AF289591在两类人群中的表达,ELISA检测骨髓组织中硬骨抑素(sclerostin)水平。分离hMSC并通过流式细胞技术进行细胞鉴定,qPCR检测hMSC骨形成相关基因COL、OPN、OCN表达以及SOST基因表达水平,试剂盒检测碱性磷酸酶(alkaline phosphatase,ALP)活性,免疫印迹法(Western blot)检测硬骨抑素和RUNX2蛋白表达水平,荧光素酶报告基因检测Wnt-1启动子活性。结果相比健康人群,OP患者骨髓组织中lncRNA-AF289591表达显著升高,伴随着SOST基因表达水平增加(P<0.05)。分离hMSC并通过流式细胞技术确定为阳性细胞(CD105、CD44和CD29表达百分率分别为99.9%、100%和99.8%),过表达lncRNA-AF289591后,抑制骨形成相关分子COL、OPN、OCN基因表达(P<0.05),降低ALP活性(P<0.05),促进SOST基因表达(P<0.05),抑制Wnt-1启动子活性以及减少RUNX2蛋白表达水平(P<0.05)。干扰lncRNA-AF289591表达后,结果与上述情况相反(P<0.05)。结论lncRNA-AF289591表达通过调控SOST基因表达而抑制hMSC成骨分化。

Objective To investigate the role and mechanism of long noncoding RNA(lncRNA-AF289591 on bone formation from human mesenchymal stem cells(hMSC).Methods Bone marrow collected from osteoporosis patients and healthy subjects was analyzed with lncRNA array.The expression of lncRNA-AF289591 in bone marrow was tested by real-time polymerase chain reaction(qPCR).hMSC cells were isolated from healthy subjects and identified by flow cytometry with related antibodies.hMSC cells were transfected with lncRNA-AF289591 plasmid and siRNA.The expressions of COL,OPN,OCN,and SOST in hMSC were tested by qPCR.The activity of ALP was measured with commercial kit.The protein levels of sclerostin and RUNX2 were analyzed by Western blot.The activation of Wnt signal pathway was tested with luciferase reporter assay.Results Compared with healthy subjects,the expression of lncRNA-AF289591 was increased in bone marrow from osteoporosis patients,accompanied with the increasing of sclerostin level(P<0.05).hMSC cells were collected and identified by flow cytometry(the expression rate of CD105,CD44 and CD29 are 99.9%,100%,and 99.8%respectively).LncRNAAF289591 overexpression in hMSC reduced the expression of COL,OPN,and OCN genes(P<0.05).It also prompted the expression of SOST gene and increased the protein level of sclerostin(P<0.05),and reduced the activity of ALP(P<0.05).In addition,lncRNA-AF289591 inhibited Wnt signal pathway and reduced the protein level of RUNX2(P<0.05).Conclusion LncRNA-AF289591 can inhibit bone formation from hMSC by modulating SOST gene expression.