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

作　　者：陈蓝妮 张惠姣 徐雪姣 许珂[1] 朱岷[1] CHEN Lanni;ZHANG Huijiao;XU Xuejiao;XU Ke;ZHU Min(Department of Endocrinology,Children's Hospital of Chongqing Medical University,National Clinical Research Center for Child Health and Disorders,Key Laboratory of Child Development and Disorders of Ministry of Education,Chongqing Key Laboratory of Pediatrics,Chongqing,400014,China)

机构地区：[1]重庆医科大学附属儿童医院内分泌科,国家儿童健康与疾病临床医学研究中心,儿童发育疾病研究教育部重点实验室,儿科学重庆市重点实验室,重庆400014

出　　处：《第三军医大学学报》2021年第13期1212-1218,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81170723)。

摘　　要：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 mi

关 键 词：miR-370-3P JAK2/STAT5B 人关节软骨细胞 细胞增殖 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]