miR-181a-5p靶向ATG5抑制雨蛙素诱导的大鼠胰腺腺泡细胞AR42J自噬的机制研究  

Study on the mechanism of miR-181a-5p targeting ATG5 to inhibit autophagy in rat pancreatic acinar AR42J cells induced by cerulein

作　　者：史清泉[1] 苗彬[2] 王烁 陶琳[4] 沈晨[4] Shi Qingquan;Miao Bin;Wang Shuo;Tao Lin;Shen Chen(Department of Critical Care Medicine,Beijing Hospital of Traditional Chinese Medicine,Capital Medical University,Beijing 100010,China;Department of Infectious Diseases,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China;Department of Infectious Diseases,y,Beijing Hospital of Traditional Chinese Medicine,Capital Medical University,Beijing 100010,China;Department of Gastroenterology,Beijing Hospital of Traditional Chinese Medicine,Capital Medical University,Beijing 100010,China)

机构地区：[1]首都医科大学附属北京中医医院重症医学科,100010 [2]首都医科大学附属北京友谊医院感染科,100050 [3]首都医科大学附属北京中医医院感染性疾病科,100010 [4]首都医科大学附属北京中医医院消化科,100010

出　　处：《中华消化病与影像杂志（电子版）》2024年第6期524-530,共7页Chinese Journal of Digestion and Medical Imageology(Electronic Edition)

基　　金：国家自然科学基金(82274259)。

摘　　要：目的探讨miR-181a-5p靶向ATG5抑制雨蛙素诱导的大鼠胰腺腺泡细胞AR42J自噬的机制。方法通过100 nmol/L雨蛙素处理AR42J细胞体外模拟急性胰腺炎(AP),并将miR-181a-5p mimics和oe-ATG5转染至AR42J细胞,分为Control组(未经任何处理)、AP组(模型组)、AP+mimic NC和AP+mimic miR组(转染mimic NC或miR-181a-5p mimic至AR42J细胞24 h,再经雨蛙素诱导建模)、AP+mimic miR+oe-NC和AP+mimic miR+oe-ATG5组(共转染miR-181a-5p mimic、oe-NC或oe-ATG5至AR42J细胞24 h,再经雨蛙素诱导建模)。使用ELISA检测TNF-α和IL-6的表达水平,RT-qPCR检测miR-181a-5p表达。采用MTT测定细胞活力,免疫荧光检测自噬标记物LC3。miRWalk数据库预测miR-181a-5p和ATG5的潜在结合位点,并通过双荧光素酶报告实验验证miR-181a-5p与ATG5的靶向关系。Western blot检测自噬相关蛋白(Beclin-1、LC3Ⅱ/Ⅰ)和ATG5蛋白的表达。结果成功构建AP体外模型,AP组中TNF-α和IL-6表达水平较Control组显著升高,且miR-181a-5p在雨蛙素诱导的AR42J细胞中显著下调。与AP+mimic NC组相比,AP+mimic miR组细胞活力显著提高,自噬相关蛋白Beclin-1和LC3Ⅱ/Ⅰ的表达显著下调,LC3阳性细胞数量减少。双荧光素酶实验结果显示,miR-181a-5p可靶向负调控ATG5的表达。进一步实验表明,过表达ATG5可部分逆转miR-181a-5p过表达对雨蛙素诱导自噬的抑制作用。结论miR-181a-5p通过靶向负调控ATG5,抑制雨蛙素诱导的AR42J细胞自噬。这一机制可能在AP的自噬调控中发挥重要作用。Objective To investigate the mechanism of miR-181a-5p targeting ATG5 to inhibit autophagy in rat pancreatic acinar AR42J cells induced by cerulein.Methods AR42J cells were treated with 100 nmol/L cerulein in vitro to simulate acute pancreatitis(AP).miR-181a-5p mimics and oe-ATG5 were transfected into AR42J cells.AR42J cells were divided into Control group(without any treatment),AP group(model group),AP+mimic NC and AP+mimic miR groups(transfected mimic NC or miR-181a-5p mimic into AR42J cells for 24 h,and then modeled induced by cerulein),and AP+mimic miR+oe-NC and AP+mimic miR+oe-ATG5 groups(transfected miR-181a-5p mimic,oe-NC or oe-ATG5 into AR42J cells for 24 h,and then modeled induced by cerulein).ELISA was used to determine the expression levels of TNF-αand IL-6,and RT-qPCR was performed to determine the expression of miR-181a-5p.MTT assay was used to assess cell viability,and immunofluorescence was conducted to detect the autophagy marker LC3.The miRWalk database predicted potential binding sites between miR-181a-5p and ATG5,and the dual-luciferase reporter assay was used to verify the targeting relationship between miR-181a-5p and ATG5.Western blot analysis was employed to measure the expression levels of autophagy-related proteins(Beclin-1,LC3Ⅱ/Ⅰ)and ATG5 protein.Results An in vitro AP model was successfully established.Significantly increased levels of TNF-αand IL-6 were noticed in the AP group compared to the Control group.miR-181a-5p expression was down-regulated in cerulein-induced AR42J cells.Compared to the AP+mimic NC group,the AP+mimic miR group showed increased cell viability,down-regulated expression patterns of autophagy-related proteins Beclin-1 and LC3Ⅱ/Ⅰ,and a reduced number of LC3-positive cells.Dual-luciferase reporter assay revealed that miR-181a-5p negatively regulated ATG5.Further experiments indicated that re-expression of ATG5 could partially reverse the inhibitory effect of miR-181a-5p overexpression on autophagy induced by cerulein in AR42J cells.Conclusion miR-181a-5p inh

关 键 词：急性胰腺炎 miR-181a-5p ATG5 自噬 AR42J细胞 

分 类 号：R576[医药卫生—消化系统]