miR-223-3p调控FOXO3a介导自噬在肝脏缺血再灌注损伤中的作用  

Effect of miR-223-3p regulates FOXO3a-mediated autophagy in hepatic ischemia-reperfusion injury

作　　者：王星星 宋虎[1] 杜晨阳[1] 王振[1] 张建军[2] Wang Xingxring;Song Hu;Du Chenyang;Wang Zhen;Zhang Jianjun(First Central Clinical College,Tianjin Medical University,Tianjin 300192,China;Organ Transplant Center,First Municipal Central Hospital,Tianjin 300192,China)

机构地区：[1]天津医科大学一中心临床学院,300192 [2]天津市第一中心医院器官移植中心,300192

出　　处：《中华器官移植杂志》2020年第7期423-429,共7页Chinese Journal of Organ Transplantation

基　　金：天津市自然科学基金(19JCZDJC36000)。

摘　　要：目的发现和探讨miR-223-3p可能通过调控FOXO3a介导自噬在肝缺血再灌注损伤中发挥作用。方法采用C57/BL6小鼠建立小鼠缺血再灌注(IR)模型,根据再灌注时间不同分为2 h组、6 h组、12 h组和24 h组,假手术组不进行钳夹肝蒂操作。培养小鼠肝AML12细胞,用miR-223-3p mimics、miR-223-3p inhibitor以及FOXO3a的干扰RNA处理细胞,并建立缺氧1 h复氧6 h的模型,分为miRNA对照组、miR-223-3p模拟物组、miR-223-3p抑制剂组以及siRNA对照组、FOXO3a siRNA组。HE染色、TUNEL检测不同时间点肝脏的损伤及细胞凋亡状况。免疫组化检测肝细胞内增殖细胞核抗原(PCNA)和Caspase-3的变化。RT-PCR和Western blot检测肝组织以及肝细胞内miR-223-3p、FOXO3a、LC3、p62和Caspase-3的表达变化。结果 HE染色、TUNEL结果显示12 h肝组织再灌注损伤以及细胞凋亡最严重。在肝组织IR模型中,RT-PCR结果显示IR各组miR-223-3p、FOXO3a的表达均高于假手术组(1.00±0),miR-223-3p在12 h时(9.13±2.12)达最高,FOXO3a在6 h时(5.23±0.90)达最高(P<0.05)。Western blot结果显示再灌注6 h时FOXO3a表达水平最高,12 h时LC3和Caspase-3表达水平最高(P<0.05)。在细胞缺氧复氧模型中,RT-PCR结果显示miRNA对照组(1.00±0)FOXO3a mRNA的表达较miR-223-3p模拟物组(0.45±0.21)降低,反之在miR-223-3p抑制剂组(2.73±0.53)升高(P<0.05)。Western blot检测显示FOXO3a蛋白表达在miR-223-3p抑制剂组最高,miR-223-3p模拟物组最低,反之LC3和Caspase-3均在miR-223-3p模拟物组表达最高(P<0.05)。siRNA对照组中FOXO3a表达较FOXO3a siRNA组高,同时FOXO3a siRNA组中Caspase-3和LC3表达更高。结论研究表明FOXO3a对肝缺血再灌注损伤具有保护作用,可能与其抑制细胞自噬以及凋亡有关,而miR-223-3p通过下调FOXO3a介导自噬促进损伤,提示miR-223-3p和FOXO3a成负相关且可能是肝脏损伤的潜在基因治疗靶点。Objective To explore the effect of miR-223-3p regulating FOXO3a-mediated autophagy in hepatic injury-reperfusion injury(LIRI).Methods The model of hepatic ischemia-reperfusion injury(IR)was established in C57BL6 mice.According to different reperfusion timepoints,mice were randomly divided into 2 h,6 h,12 h and 24 h group.For sham group,there was no intraoperative clamping of hepatic pedicle.Murine hepatic AML12 cells were treated with miR-223-3p mimics,miR-223-3p inhibitor and FOXO3a interfering RNA.A hypoxic 1 h reoxygenation 6 h model was established.And miRNA-NC,miR-223-3p mimics,miR-223-3p inhibitor and siRNA-NC and FOXO3a siRNA groups were assigned.Hepatic injury and apoptosis were detected by hematoxylin eosin or TdT-mediated nick end labeling(HE/TUNEL)at different timepoints.The changes of proliferating cell nuclear antigen(PCNA)and Caspase-3 in hepatocytes were detected by immunohistochemistry.Reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were employed for detecting the expressions of miR-223-3p,FOXO3a,LC3,p62 and Caspase-3 in hepatocytes.Results The results of HE/TUNEL indicated that reperfusion injury and apoptosis of hepatic tissue were most severe in 12 h group.In hepatic ischemia-reperfusion model,RT-PCR results showed that the expressions of miR-223-3p and FOXO3a were higher in IR group than those in sham group(1.00±0),the expression level of miR-223-3p mRNA peaked at 12 h(9.13±2.12)after reperfusion and FOXO3a was the highest at 6 h(5.23±0.90,P<0.05).Western blot showed that the expression of FOXO3a peaked at 6 h post-reperfusion and the expressions of LC3 and caspase-3 were the highest at 12 h.(P<0.05).In the model of cell hypoxia and reoxygenation,RT-PCR indicated that the expression of FOXO3a mRNA decreased in miR-223-3p mimics group(0.45±0.21)as compared with miRNA-NC group(1.00±0).In contrast,miR-223-3p inhibitor group increased(2.73±0.53,P<0.05).Western blot indicated that FOXO3a protein expression was highest in miR-223-3p inhibitor and miR-223-3p mimics group

关 键 词：肝移植 缺血再灌注 自噬 

分 类 号：R657.3[医药卫生—外科学]