miR-370-3P通过调节JAK2/STAT5B信号通路抑制人关节软骨细胞增殖

MiR-370-3P inhibits proliferation of human articular chondrocytes by regulating JAK2/STAT5B signaling pathway

目的研究非编码单链RNA(micro RNA,miRNA)miR-370-3P是否通过调控JAK2/STAT5B信号通路调节人关节软骨细胞(human chondrocytes-articular,HC-a)的增殖,并探讨其潜在作用机制。方法 (1)通过脂质体转染HC-a细胞构建miR-370-3P过表达/低表达人关节软骨细胞模型,分为5组:miR-370-3P模拟物mimic组(miR-370-3P过表达组)、miR-370-3P遏制物inhibitor组(miR-370-3P低表达组)、正常对照组、mimic空载体对照组(mimic normal control,mNC)、inhibitor空载体对照组(inhibitor normal control,iNC)。(2)miRNA实时荧光定量PCR检测各组HC-a细胞miR-370-3P的表达。(3)qRT-PCR检测各组HC-a细胞JAK2、STAT5B mRNA表达。(4)Western blot检测各组HC-a细胞中JAK2、STAT5B蛋白表达。(5)MTT比色法检测各组HC-a细胞相对增殖情况。(6)双荧光素酶实验:构建JAK2、STAT5B野生型和突变型双荧光素酶报告基因载体,分别与miR-370-3P模拟物和阴性对照序列共转染293T细胞,检测各组荧光素酶活性。结果 miRNA实时荧光定量PCR结果显示:与正常对照组相比,mimic组miR-370-3P表达上调(P<0.05),inhibitor组miR-370-3P表达下调(P<0.05),且空载体对照组和正常对照组之间的差异无统计学意义,表明miR-370-3P过表达/低表达软骨细胞模型构建成功。qRT-PCR结果显示:相比于正常对照组,JAK2和STAT5B在mimic组的mRNA相对表达量下调,在inhibitor组JAK2 mRNA和STAT5B mRNA相对表达量上调(P<0.05)。Western blot检测结果显示:JAK2和STAT5B蛋白表达在mimic组也有显著下调,在inhibitor组上调。MTT检测结果显示:miR-370-3P的过表达会降低HC-a的存活率(P<0.05),提示miR-370-3P的过表达会抑制人关节软骨细胞(HC-a)的增殖。双荧光素酶实验结果显示:miR-370-3P和JAK2-WT(野生型)、STAT5B-WT(野生型)质粒共转染后,荧光表达较对照组有显著下调(P<0.05),提示miR-370-3P可与JAK2-3′UTR、STAT5B-3′UTR直接结合发挥作用。结论 miR-370-3P可通过直接靶向调控JAK2/STAT5B信号通路进而抑制人关节软骨细胞(HC-a)的增殖。

Objective To investigate whether the non-coding single chain RNA miR-370-3P regulates the proliferation of human articular chondrocytes(HC-a)through the JAK2/STAT5B signaling pathway and to explore its potential mechanism.Methods The over-/under-expression of miR-370-3P models were constructed by transfecting HC-a cells,and then the cells were divided into 5 groups:miR-370-3P mimic group(over-expression group),miR-370-3P inhibitor group(under-expression group),mimic normal control group(mNC group),inhibitor normal control group(iNC group),and normal control group(NC group).The mRNA levels of miR-370-3P,JAK2 and STAT5B in each group were detected by qRT-PCR,and the protein levels of JAK2 and STAT5B were determined using Western blotting.In addition,MTT assay was adopted to measure the proliferation of HC-a cells in each group,and the binding of miR-370-3P with JAK2 and STAT5B was detected by dual luciferase assay.Results As compared with the NC group,the mRNA expression of miR-370-3P was upregulated in the mimic group and down-regulated in the inhibitor group(P<0.05),indicating successful construction of miR-370-3P over-expression/under-expression models.By contrast,the mRNA expression of JAK2 and STAT5B was decreased in the mimic group and increased in the inhibitor group(P<0.05),with no significant difference between the empty vector control groups(mNC and iNC groups)and the NC group.Western blotting results also confirmed the down-regulation of JAK2 and STAT5B in the mimic group and upregulation in the inhibitor group(P<0.05).MTT assay showed that miR-370-3P overexpression reduced the survival rate of HC-a cells(P<0.05),suggesting an inhibitory effect on HC-a cells proliferation.The results of dual luciferase assay displayed that after miR-370-3P was co-transfected with JAK2(wild type)as well as STAT5B(wild type),the fluorescence intensity was significantly down-regulated compared with the control group(P<0.05),indicating that miR-370-3P can directly bind to JAK2-3'UTR and STAT5B-3'UTR to function.Conclusion miR-370-3P can inhibit the proliferation of HC-a cells by direct regulation of JAK2/STAT5B signaling pathway.