炎性疼痛相关miR-34a通过XIST维持线粒体功能减少细胞凋亡  

Inflammatory pain-related miR-34a mediates mitochondrial homeostasis via XIST and attenuates cell apoptosis

作　　者：方静[1] 龚诚 项灵云 孙文 刘中原 冯觉平[1] FANG Jing;GONG Cheng;XIANG Ling-yun;SUN Wen;LIU Zhong-yuan;FEN Jue-ping(Department of Oncology,Puai Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology/Wuhan Fourth Hospital,Wuhan 430030,China;Department of General Surgery,Zhongnan Hospital of Wuhan University,Wuhan 430071,China)

机构地区：[1]华中科技大学同济医学院附属普爱医院/武汉市第四医院肿瘤科,武汉430030 [2]武汉大学中南医院普外科,武汉430071

出　　处：《现代免疫学》2024年第2期96-103,共8页Current Immunology

基　　金：湖北省自然科学基金(2016CF340);武汉市卫健委基金项目(WX19D65)。

摘　　要：为探究炎性疼痛通过下调miR-34a,介导X特异性失活转录本(X-inactive specific transcript,XIST)表达上调及通过维持巨噬细胞线粒体功能抗凋亡的机制,在雌性小鼠来源的细胞系J774A.1、女性SH-SY5Y细胞系中过表达miR-34a,或采用LPS处理后检测XIST表达,并分析miR-34a与XIST在患者体内的浓度相关性。采用MitoTracker、四甲基罗丹明乙酯(tetramethylrhodamine ethyl ester,TMRE)染色分析J774A.1细胞系的线粒体体积及线粒体膜电位变化;海马(Seahorse)呼吸实验检测线粒体压力,分析正常及过表达miR-34a的J774A.1细胞线粒体功能;TUNEL实验评估细胞的凋亡情况。采用完全弗氏佐剂(complete Freund’s adjuvant,CFA)诱导急性炎症疼痛模型,分别检测炎症急慢性期小鼠的疼痛反应阈值与XIST及miR-34a表达量的关系;Real-time PCR检测健康对照组与复杂区域性疼痛综合征(complex regional pain syndrome,CRPS)患者的外周血XIST mRNA表达,比较两者间表达的差异。结果显示,不区分性别比较对照组与CRPS组XIST表达差异无统计学意义(t=1.528,P=0.131),仅分析男性患者,两组间差异无统计学意义(t=0.945,P=0.353),单独分析女性患者可发现CRPS组的XIST mRNA表达量显著高于对照组(t=2.764,P=0.008)。过表达miR-34a可降低J774A.1、SH-SY5Y细胞系中XIST的表达量,患者在体数据提示miR-34a与XIST的表达呈负相关(r^(2)=0.681,P<0.001)。LPS刺激J774A.1细胞可抑制miR-34a表达,上调XIST表达量。小鼠疼痛模型建模4 h即可观察到XIST的表达上调(t=1.810,P<0.001)及miR-34a的表达下调(t=2.220,P<0.001),而上述差异在第14天消失。LPS上调线粒体体积及膜电位,而过表达miR-34a会导致二者下调,且2种处理的作用会发生相互抵消;LPS刺激可显著降低细胞的耗氧量(t=3.255,P=0.020)、ATP产生(t=4.323,P=0.014)及最大耗氧量(t=1.885,P=0.008),而miR-34a过表达的影响则相反(t=4.245,P=0.004);LPS可使细胞凋亡减少,miR-34a过表达可促进巨噬细The goal of this study is to elucidate the mechanism by which inflammatory pain regulated mitochondrial function and apoptosis of macrophages via down-regulation of miR-34a and up-regulation of X-inactive specific transcript(XIST).For this purpose,the expressions of XIST were measured in miR-34a over-expressed or LPS-treated J774A.1(female mouse cell line) and the SH-SY5Y(female human cell line) cells;And the in vivo correlation between the expressions of miR-34a and XIST was also analyzed.MitoTracker and TMRE(tetramethylrhodamine ethyl ester) staining were used to analyze the mitochondrial volume and membrane potential in J774A.1 cell line.The mitochondrial stress assay(Seahorse) was conducted to test the mitochondrial function in J774A.1 cells after over-expressing miR-34a and/or stimulated with LPS.TUNEL assay was used to assess cell line apoptosis.CFA(complete Freund's adjuvant)-induced acute inflammatory pain model was established to detect the pain reaction threshold and the expression of XIST and miR-34a in acute and chronic phases and healthy controls,respectively.Real-time PCR was used to detect the expression of XIST in the blood of complex regional pain syndrome(CRPS) patients and healthy controls.The results showed that there was no significant difference in XIST expression between the control group and the CRPS group in total human samples(t=1.528,P=0.131).And there was no statistical difference between the two groups(t=0.945,P=0.353) when only male patients were analyzed.However,in female patients,the XIST expression of the CRPS group was significantly higher than that of the control group(t=2.764,P=0.008).Over-expression of miR-34a reduced XIST expression in J774A.1 and SH-SY5Y cells.The datum from patients showed that the expression of miR-34a and XIST exhibited a negative correlation(r^(2)=0.681,P<0.001).LPS treatment inhibited the expression of miR-34a and up-regulated XIST expression in J774A.1 cell line.In mouse pain model,XIST expression was up-regulated(t=1.810,P<0.001) and miR-34a expressio

关 键 词：X特异性失活转录本 微小核糖核酸34a 疼痛 能量代谢 凋亡 

分 类 号：R34[医药卫生—基础医学]