微RNA-23a通过缝隙连接蛋白43对颈椎后纵韧带细胞骨向分化的作用机制

Mechanism of microRNA-23a through connexin 43 on bone differentiation of cervical posterior longitudinal ligament cells

目的探讨微RNA(miR)-23a通过缝隙连接蛋白43(Cx43)对颈椎后纵韧带细胞骨向分化的作用及相关机制。方法选取本院收治的50例间断型颈椎后纵韧带骨化症(OPLL)患者(OPLL组)、50例颈椎外伤非OPLL患者(对照A组)和50例颈椎病非OPLL患者(对照B组)颈椎后纵韧带组织,培养后纵韧带细胞,实时荧光定量聚合酶链反应(qRT-PCR)检测3组细胞miR-23a、Cx43 mRNA的表达,采用Western blotingt检测Cx43蛋白的表达。取OPLL组对数期后纵韧带细胞,分别转染miR-23a agomir质粒(miR-23a过表达组)、miR-23a antagomir质粒(miR-23a低表达组)、agomir NC质粒(转染对照组),未经处理的后纵韧带细胞为空白组。采用双荧光素酶实验验证miR-23a与Cx43的靶向关系;采用碱性磷酸酶(ALP)染色检测矿化情况;采用Western blotingt检测p38丝裂原活化蛋白激酶(p38 MAPK)、磷酸化p38 MAPK(p-p38 MAPK)、细胞外信号调节激酶(ERK1/2)、磷酸化ERK1/2(p-ERK1/2)的蛋白表达。结果与对照A、B组比较,OPLL组miR-23a表达降低,Cx43 mRNA及蛋白表达升高,差异均有统计学意义(P<0.05)。与空白组、转染对照组比较,miR-23a过表达组miR-23a表达量升高、Cx43 mRNA表达量降低,miR-23a低表达组miR-23a表达量降低、Cx43 mRNA表达量升高,差异均有统计学意义(P<0.05)。双荧光素酶实验显示,miR-23a可有效抑制野生型质粒荧光素酶活性;与空白组、转染对照组比较,miR-23a过表达组矿化面积百分比、p-p38 MAPK/p38 MAPK蛋白表达、p-ERK1/2/ERK1/2蛋白表达降低,miR-23a低表达组矿化面积百分比、p-p38 MAPK/p38 MAPK蛋白表达、p-ERK1/2/ERK1/2蛋白表达升高,差异均有统计学意义(P<0.05)。结论OPLL病理条件下,颈椎后纵韧带细胞中miR-23a异常低表达,通过靶向上调Cx43激活MAPK信号通路,促进成骨分化。

Objective To investigate the effect of microRNA(miR)-23a on the bone differentiation of cervical posterior longitudinal ligament cells through connexin 43(Cx43)and its related mechanisms.Methods The posterior longitudinal ligament tissues of 50 patients with intermittent cervical ossfiication of posterior longitudinal ligamen(tOPLL group),50 cervical trauma without OPL(L control group A)and 50 cervical spondylosis without OPL(Lcontrol group B)were collected under aseptic conditions,and the cells were cultured and identified.Quantitative rea-tlime polymerase chain reactio(nqRT-PCR)was used to detect the expression of mi-R23a and Cx43 mRNA in 3 groups.Western blotting was used to deectt the expression of Cx43 protein.The OPLL grou’ps posterior longitudinal ligament cells in logarithmic phase wree transfected with miR-23a agomir plasmid(miR-23a overexpression group),miR-23a antagomir plasmid(miR-23a low expression group),and agomir NC plasmi(d transfection control group);the untreated posterior longitudinal ligament cells were set a sthe blank group.Dual luciferase experiment was used to verify the targeting relationship between miR-23a and Cx43.Alkaline phosphatase(ALP)staining was used to detect the percentage of mineralized are aW.estern blotting was used to detect the protein expression opf38 mitogen-activated protein kinase(MAPK),phosphorylated p38 MAPK(p-p38 MAPK),extracellular signal-regulated kinase(ERK1/2),and phosphorylated ERK1/(2 p-ERK1/2).Results Compared with control groups A and B,the expression of miR-23a decreased and the expressions of Cx43 mRNA and protein increasde in OPLL group,with a statistically significant difference(P<0.05).Compared with the blank group and transfection control group,the expression of miR-23a increased and the expression of Cx43 mRNA decreased in the mi-R23a overexpression group,while the expression of miR-23a decreased and the expression of Cx43 mRNA increased in the mi-2R3a low expression group,and the differences were statistically significant(P<0.05).Dual luciferase experiments showed that mi-2R3a could effectively inhibit wil-dtype plasmid luciferase activity.Compared with the blank group and the transfection control groupt,he percentage of mineralized area,p-p38 MAPK/p38 MAPK and p-ERK1/2/ERK1/2 in the miR-23a overexpression group were all decreased,which of the miR-23a low expression group were all increased,all with a statistical significanc(eP<0.05).Conclusion Under the pathological condition of OPLL,miR-23a is abnormally low expressed in cervical posterior longitudinal ligament cell,swhich can activate MAPK signal pathway through targeted up regulation of Cx43 to promote osteoblastic differentiation.