miR-486-5p/PINK1通路调控线粒体自噬拮抗SK-N-SH细胞氧糖剥夺/复糖复氧损伤  

The miR-486-5p/PINK1 pathway regulates mitochondrial autophagy that antagonizes OGD/R damage in SK-N-SH cells

作　　者：蔡利 兰蕾 徐胜波 陈湘闻 杨鸿 黄东振 CAI Li;LAN Lei;XU Shengbo;CHEN Xiangwen;YANG Hong;HUANG Dongzhen(Department of Craniocerebral Surgery,Guangxi International Zhuang Medicine Hospital,Nanning,Guangxi 530001,China;Department of Acupuncture and Moxibustion,Guangxi International Zhuang Medicine Hospital,Nanning,Guangxi 530001,China)

机构地区：[1]广西国际壮医医院颅脑外科,南宁530001 [2]广西国际壮医医院针灸科,南宁530001

出　　处：《重庆医学》2023年第10期1448-1453,1459,共7页Chongqing medicine

基　　金：广西壮族自治区卫生健康委员会自筹经费科研课题(Z20211426)。

摘　　要：目的探究微RNA-486-5p/PTEN诱导激酶1(miR-486-5p/PINK1)通路调控线粒体自噬对SK-N-SH细胞糖氧剥夺/复糖复氧(OGD/R)损伤的影响及机制。方法实时荧光定量逆转录PCR(RT-qPCR)检测45例缺血性卒中(IS)患者和45例健康体检者血清miR-486-5p表达。双荧光素酶报告基因验证SK-N-SH细胞中miR-486-5p对PINK1的靶向关系。取对数生长期SK-N-SH细胞分为:Control、OGD/R、OGD/R+miR-486-5p mimic、OGD/R+miR-NC、OGD/R+miR-486-5p mimic+OE-NC、OGD/R+miR-486-5p mimic+OE-PINK1组。噻唑蓝(MTT)法检测细胞存活率,RT-qPCR检测miR-486-5p、PINK1、帕金蛋白(Parkin)mRNA表达,Western blot检测PINK1、Parkin、微管相关蛋白1轻链3Ⅱ(LC3-Ⅱ)、p62、Beclin1、半胱氨酰天冬氨酸蛋白酶(caspase)-3、caspase-9、细胞色素C(CytC)蛋白表达,流式细胞术检测细胞活性氧(ROS)含量及细胞凋亡率。结果IS组患者血清miR-486-5p表达较健康体检者明显降低(P<0.01)。miR-486-5p可以结合PINK1的3′端非编码区(P<0.01)。与Control组比较,OGD/R组细胞miR-486-5p、p62的表达及细胞存活率明显降低(P<0.001),PINK1、Parkin、LC3-Ⅱ、Beclin1、ROS、caspase-3、caspase-9、CytC的表达及细胞凋亡率明显升高(P<0.001)。与OGD/R组比较,OGD/R+miR-486-5p mimic组细胞miR-486-5p、p62的表达及细胞存活率明显升高(P<0.001),PINK1、Parkin、LC3-Ⅱ、Beclin1、ROS、caspase-3、caspase-9、CytC的表达及细胞凋亡率明显降低(P<0.001)。与OGD/R+miR-486-5p mimic+OE-NC组比较,OGD/R+miR-486-5p mimic+OE-PINK1组细胞miR-486-5p、p62的表达及细胞存活率明显降低(P<0.001),PINK1、Parkin、LC3-Ⅱ、Beclin1、RO S、caspase-3、caspase-9、CytC的表达及细胞凋亡率明显升高(P<0.001)。结论miR-486-5p可拮抗SK-N-SH细胞OGD/R损伤,可能与抑制PINK1/Parkin通路进而抑制线粒体自噬有关。Objective To investigate the effect and mechanism of microRNA-486-5p/PTEN induced kinase 1(miR-486-5p/PINK1)pathway regulating mitochondrial autophagy on glucose oxygen deprivation/resugar reoxygenation(OGD/R)damage in SK-N-SH cells.Methods Real-time quantitative PCR(RT-qPCR)was used to detect the expression of miR-486-5p in serum of 45 patients with ischemic stroke(IS)and 45 healthy subjects.Dual luciferase reporter gene was used to verified the targeting relationship of miR-486-5p to PINK1 in SK-N-SH cells.SK-N-SH cells at logarithmic growth stage were divided into the Control,OGD/R,OGD/R+miR-486-5p mimic,OGD/R+miR-NC,OGD/R+miR-486-5p mimic+OE-NC,OGD/R+miR-486-5p mimic+OE-PINK1 groups.Thiazole blue(MTT)was used to detect cell survival rate,the mRNA expressions of miR-486-5p,PINK1 and Parkin were detected by RT-qPCR.Western blot was used to detect protein expressions of PINK1,Parkin,microtubule-associated protein 1 light chain 3Ⅱ(LC3-Ⅱ),p62,Beclin1,caspase-3,caspase-9,and cytochrome C(CytC),ROS content and apoptosis rate were determined by flow cytometry.Results The expression of serum miR-486-5p in patients of the IS group was significantly lower than that in the healthy subjects(P<0.001).The miR-486-5p could bind to the PINK13′UTR(P<0.01).Compared with the Control group,the expressions of miR-486-5p,p62 and survival rate of the OGD/R group were significantly decreased(P<0.001),while the expressions of PINK1,Parkin,LC3-Ⅱ,Beclin1,ROS,caspase-3,caspase-9,CytC and apoptosis rate were significantly increased(P<0.001).Compared with the OGD/R group,the expressions of miR-486-5p,p62 and survival rate of the ODG/R+miR-486-5p mimic group were significantly increased(P<0.001),while the expressions of PINK1,Parkin,LC3-Ⅱ,Beclin1,ROS,caspase-3,caspase-9,CytC and apoptosis rate were significantly decreased(P<0.001).Compared with the OGD/R+miR-486-5p mimic+OE-NC group,the expressions of miR-486-5p,p62 and survival rate of the OGD/R+miR-486-5p mimic+OE-PINK1 group were significantly decreased(P<0.001),while the expre

关 键 词：卒中 微RNA-486-5p PINK1 线粒体自噬 氧糖剥夺/复糖复氧 缺血-再灌注损伤 

分 类 号：R743.3[医药卫生—神经病学与精神病学]