摘要
目的探讨miR-375靶向锌指蛋白470(ZNF470)对人牙周膜干细胞(hPDLSCs)成软骨分化的调控作用。方法以诱导成软骨分化培养基诱导hPDLSCs成软骨分化,实时荧光定量PCR法检测成软骨分化诱导1、3、7、14、21 d时miR-375及ZNF470 mRNA的表达,双荧光素酶报告基因检测法分析miR-375与ZNF470的靶向调控关系。将hPDLSCs分为空白对照组、阴性对照组、miR-375过表达组、对照+空载组、miR-375过表达+空载组及miR-375过表达+ZNF470组,分别转染对照激动剂(NC-ago)、miR-375突变激动剂(miR-375-mut-ago)、miR-375激动剂(miR-375-ago)、NC-ago+空载、miR-375-ago+空载以及miR-375-ago+ZNF470载体,转染后对各组细胞进行成软骨分化诱导。采用阿尔新蓝染色观察各组成软骨分化情况,实时荧光定量PCR与Western blotting分别检测软骨标志性基因Ⅱ型胶原α1(COL2A1)、性别决定基因9(SOX9)、软骨可聚蛋白多糖(ACAN)以及透明质酸合酶2(HAS2)的mRNA与蛋白表达水平。结果诱导hPDLSCs成软骨分化过程中miR-375 mRNA表达呈时间依赖性下调,ZNF470 mRNA表达呈时间依赖性上调,miR-375与ZNF470表达呈负相关(r=-0.47,P<0.05)。双荧光素酶报告基因显示,miR-375可靶向负调控ZNF470。阿尔新蓝染色显示,成软骨分化程度miR-375过表达组<空白对照组、阴性对照组,miR-375过表达+空载组<miR-375过表达+ZNF470组<对照+空载组(P均<0.05)。实时荧光定量PCR与Western blotting显示,COL2A1、SOX9、ACAN、HAS2 mRNA与蛋白表达miR-375过表达组<空白对照组、阴性对照组,miR-375过表达+空载组<miR-375过表达+ZNF470组<对照+空载组(P均<0.05),空白对照组与阴性对照组各软骨标志性基因表达差异无统计学意义。结论miR-375能够靶向负调控ZNF470从而抑制hPDLSCs成软骨分化,miR-375可能是调控hPDLSCs成软骨分化的重要分子靶点。
Objective To investigate the regulatory effect of miR-375 on the chondrogenic differentiation of human periodontal ligament stem cells(hPDLSCs)by targeting zinc finger protein 470(ZNF470).Methods The chondrogenic differentiation medium was used to induce the chondrogenic differentiation of hPDLSCs.The expression levels of miR-375 and ZNF470 mRNA at 1,3,7,14,and 21 d after chondrogenic differentiation induction were detected by real-time quantitative PCR.The regulatory relationship between miR-375 and ZNF470 was analyzed by dual luciferase reporter gene assay.The hPDLSCs were divided into the blank control group,negative control group,miR-375 overexpression group,control+blank vector group,miR-375 overexpression+blank vector group,and miR-375 overexpression+ZNF470 group,and transfected with control agonist(NC-ago),miR-375 mutant agonist(miR-375-mut-ago),miR-375 agonist(miR-375-ago),NC-ago+blank vector,miR-375-ago+blank vector,and miR-375-ago+ZNF470 vector,respectively,and the cells of each group were induced to chondrogenic differentiation after transfection.The degree of chondrogenic differentiation in each group was detected by Alcian blue staining.The mRNA and protein expression levels of cartilage markers including type Ⅱ collagenα1(COL2A1),sex-determining region Y-box 9(SOX9),aggrecan(ACAN),and hyaluronan synthase 2(HAS2)were detected by real-time quantitative PCR and Western blotting.Results The mRNA expression levels of miR-375 and ZNF470 were down-regulated and up-regulated in a time-dependent manner during the chondrogenic differentiation of hPDLSCs,respectively.The expression of miR-375 was negatively correlated with the expression of ZNF470(r=-0.47,P<0.05).The results of dual luciferase reporter gene assay showed that miR-375 negatively regulated ZNF470 expression by targeting ZNF470.The results of Alcian blue staining showed that the degree of chondrogenic differentiation in the miR-375 overexpression group was lower than those of the blank control group and negative control group,while the degree of chondrogenic differentiation was as follows:the miR-375 overexpression+empty vector group<miR-375 overexpression+ZNF470 vector group<control+empty vector group(all P<0.05).The results of real-time quantitative PCR and Western blotting showed that the mRNA and protein expression levels of COL2A1,SOX9,ACAN,and HAS2 were lower in the miR-375 overexpression group than in the blank control group and negative control group,while the mRNA and protein expression levels of COL2A1,SOX9,ACAN and HAS2 were in the following order:the miR-375 overexpression+empty vector group<miR-375 overexpression+ZNF470 vector group<control+empty vector group(all P<0.05).There were no significant differences in the mRNA and protein expression levels of COL2A1,SOX9,ACAN or HAS2 between the blank control group and negative control group.Conclusion MiR-375 could target and negatively regulate ZNF470 to inhibit the chondrogenic differentiation of hPDLSCs,and miR-375 might be an important molecular target for regulating the chondrogenic differentiation of hPDLSCs.
作者
王艳华
袁淑静
潘裕兴
华烨
李世英
王天
WANG Yanhua;YUAN Shujing;PAN Yuxing;HUA Ye;LI Shiying;WANG Tian(Department of Stomatology,Tianjin Union Medical Center,Tianjin 300121,China)
出处
《山东医药》
CAS
2022年第17期28-32,共5页
Shandong Medical Journal
基金
天津市人民医院基金项目(2019YJ015)。
关键词
miR-375
人牙周膜干细胞
成软骨分化
锌指蛋白470
靶点
miR-375
human periodontal ligament stem cells
chondrogenic differentiation
zinc finger protein 470
target
