摘要
目的骨关节炎(osteoarthritis,OA)是一种常见的关节疾病,可由内源性和表观遗传因素引起。软骨退变可恶化为OA。本研究旨在探讨长链非编码RNA(long non-coding RNA,LncRNA)GAS5在OA中的基本机制。方法14例正常膝关节软骨组织取自2018年7月至2020年7月因胫骨平台骨折在复旦大学附属中山医院吴淞医院行骨折内固定的患者,男8例,女6例;年龄19~40岁,平均(31±5)岁。35例OA膝关节软骨组织取自2018年7月至2020年7月在复旦大学附属中山医院吴淞医院就诊,诊断为OA且有膝关节置换手术指征的患者,男17例,女18例;年龄62~86岁,平均年龄(70±4)岁。采用实时荧光定量聚合酶链式反应(real time-quantitative polymerase chain reaction,RT-qPCR)检测金属蛋白酶抑制剂(tissue inhibitor of metalloproteinase,TIMP)-3 mRNA的表达情况。使用白细胞介素(interleukin,IL)-1β(10 ng/mL)建立OA细胞模型,分析OA基因芯片GSE16464。RT-qPCR和western-blot检测软骨分解代谢抑制因子TIMP-3的表达。用芯片定量聚合酶链反应(quantitative polymerase chain reaction,qPCR)检测TIMP-3启动子区富集的CpG岛情况。用生物信息学方法预测GAS5与TIMP-3的靶向关系,并用双荧光素酶报告基因检测法进行验证。结果在OA患者组织中TIMP-3 mRNA表达显著低于正常软骨组织,OA患者组与正常软骨组织软骨细胞TIMP-3启动子区甲基化阳性率分别为72.1%和35.7%。LncRNAGAS5在OA中高表达,LncRNAGAS5在OA中表达较正常软骨组织中表达明显上调(P<0.05)。Blast序列比对GAS5序列与TIMP-3启动子序列具有结合位点。沉默LncRNA GAS5促进TIMP-3表达,抑制OA软骨细胞胶原降解。双荧光素酶报告基因实验验证GAS5与TIMP-3具有靶向调控关系。结论OA软骨组织中TIMP-3低表达,而LncRNA GAS5表达上调;LncRNA GAS5可富集DNA甲基化转移酶,抑制TIMP-3的表达,促进软骨细胞胶原蛋白降解。
Objective To investigate the basic mechanism of LncRNA GAS5 in osteoarthritis.Methods 14 normal knee cartilage tissues were collected from patients who received internal fixation for tibial plateau fracture in Zhongshan Hospital Wusong Branch,Fudan University from July 2018 to July 2020,including 8 males and 6 females.The age ranged from 19 to 40 years,with an average of(31±5)years.35 OA knee cartilage tissues were collected from patients diagnosed with OA and indicated for knee replacement surgery in Zhongshan Hospital Wusong Branch of Fudan University from July 2018 to July 2020,including 17 males and 18 females.The average age was(70±4)years.The expression of TIMP-3 mRNA was detected by real time quantitative polymerase chain reaction(RT-qPCR).OA cell model was established by treatment with interleukin-1β(10 ng/mL).GSE16464 OA gene chips were analyzed.The expression of TIMP-3 was detected by RT-qPCR and Western blot.CpG islands enriched in TIMP-3 promoter region were detected by chip quantitative polymerase chain reaction,qPCR(qPCR).The target relationship between GAS5 and TIMP-3 was predicted by bioinformatics method,and verified by double luciferase reporter gene detection.Results TIMP-3 mRNA expression in OA patients was significantly lower than that in normal cartilage tissues.The positive rates of TIMP-3 promoter methylation in OA patients and normal cartilage tissue controls were 72.1%and 35.7%,respectively.The expression of LncRNA GAS5 in OA was significantly higher than that in normal cartilage tissues(P<0.05).Blast sequence alignment has binding site between GAS5 sequence and TIMP-3 promoter sequence.Silencing LncRNA GAS5 promoted TIMP-3 expression and inhibited collagen degradation in OA chondrocytes.Double luciferase reporter gene assay confirmed that GAS5 and TIMP-3 have a targeted regulatory relationship.Conclusion TIMP-3 expression is low in OA cartilage,while LncRNA GAS5 expression was up-regulated;LncRNA GAS5 can enrich DNA methyltransferase,inhibit TIMP-3 expression and promote collagen deg
作者
何小文
丁徐
张东华
陈羿丞
宋登新
王健
He Xiaowen;Ding Xu;Zhang Donghua(Department of Orthopedics,Zhongshan Hospital Wusong Branch,Fudan University,Shanghai 200940,China)
出处
《实用骨科杂志》
2021年第11期999-1004,共6页
Journal of Practical Orthopaedics
基金
上海市卫生健康委员会科研课题(20204Y0365)
上海市自然科学基金(19ZR1429200)。
