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

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胞的凋亡,过表达XIST可逆转miR-34a介导的细胞凋亡。由此,炎症反应可通过下调miR-34a促进XIST的表达,后者通过促进线粒体功能的维持减少细胞凋亡。

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 expression was down-regulated(t=2.220,P<0.001) at 4 h time point,and the difference disappeared at D14.LPS up-regulated mitochondrial volume and membrane potential,while over-expression of miR-34a led to down-regulation of both.And the effects of the two treatments compensated for each other.LPS stimulation significantly decreased cellular oxygen consumption(t=4.323,P=0.014),ATP production(t=4.323,P=0.014) and the maximum oxygen consumption(t=1.885,P=0.008),while miR-34a over-expression had the opposite effect(t=4.245,P=0.004).LPS reduced cell apoptosis whereas miR-34a over-expression promoted it.XIST over-expression can reverse the cell apoptosis mediated by miR-34.In conclusion,inflammation may promote the expression of XIST by down-regulating miR-34a and the former suppresses cell apoptosis by promoting the maintenance of mitochondrial function.