miR-20b-5p靶向MAPK1调控脑出血过程中脑微血管内皮细胞铁死亡

miR-20b-5p Targets MAPK1 to Regulate Ferroptosis of Brain Microvascular Endothelial Cells during Intracerebral Hemorrhage

目的:探讨miR-20b-5p对氧糖剥夺(OGD)/Hemin处理的脑微血管内皮细胞(BMVEC)功能的影响及机制。方法:将BMVEC分为Control组、agomir-NC组、agomir-miR-20b-5p组、antagomir-NC组和antagomir-miR-20b-5p组。使用Lipofectamine2000试剂对细胞进行相应的转染处理。BMVEC转染后,将BMVEC再分为Control组、OGD/Hemin组(O/H组)、OGD/Hemin+agomir-NC组(O/H+agomir-NC组)、OGD/Hemin+agomir-miR-20b-5p组(O/H+agomir-miR-20b-5p组)、OGD/Hemin+antagomir-NC组(O/H+antagomir-NC组)和OGD/Hemin+antagomir-miR-20b-5p组(O/H+antagomir-miR-20b-5p组)。Control组BMVEC正常培养,其他组BMVEC进行OGD/Hemin处理。MTT法检测BMVEC增殖,TUNEL染色检测BMVEC凋亡,Transwell检测BMVEC迁移。使用试剂盒检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)水平。使用Iron Assay试剂盒检测Fe2+含量。通过qRT-PCR检测miR-20b-5p和MAPK1 mRNA水平。通过Western blot检测MAPK1、Bax、Bcl-2、谷胱甘肽过氧化物酶4(GPX4)和前列腺素内过氧化物合酶2(PTGS2)蛋白表达水平。通过免疫荧光染色检测MAPK1的荧光强度水平。结果:与Control组和agomir-NC组比较,agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高(P<0.05)。与Control组和antagomir-NC组比较,antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低(P<0.05)。与Control组比较,O/H组BMVEC中的miR-20b-5p水平降低,细胞活力降低,TUNEL阳性率和Bax蛋白表达水平升高,Bcl-2蛋白表达水平降低,迁移数量降低,SOD和GSH-Px活性降低,MDA含量升高,Fe2+含量和PTGS2的蛋白表达水平升高,GPX4的蛋白表达水平降低,MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高(P<0.05)。与O/H组和O/H+agomir-NC组比较,O/H+agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高,细胞活力升高,TUNEL阳性率和Bax蛋白表达水平降低,Bcl-2蛋白表达水平升高,迁移数量升高,SOD和GSH-Px活性升高,MDA含量降低,Fe2+含量和PTGS2的蛋白表达水平降低,GPX4的蛋白表达水平升高,MAPK1的mRNA和蛋白表达水平以及相对荧光强度降低(P<0.05)。与O/H组和O/H+antagomir-NC组比较,O/H+antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低,细胞活力降低,TUNEL阳性率和Bax蛋白表达水平升高,Bcl-2蛋白表达水平降低,迁移数量降低,SOD和GSH-Px活性降低,MDA含量升高,Fe2+含量和PTGS2的蛋白表达水平升高,GPX4的蛋白表达水平降低,MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高(P<0.05)。结论:本研究表明上调miR-20b-5p通过抑制OGD/Hemin处理的BMVEC中MAPK1的表达从而抑制了铁死亡途径。

Objective:To investigate the effects and mechanism of miR-20b-5p on the function of brain microvascular endothelial cells(BMVEC)treated with oxygen glucose deprivation(OGD)/Hemin.Methods:BMVEC were divided into Control group,agomir-NC group,agomir-miR-20b-5p group,antagomir-NC group and antagomir-miR-20b-5p group.The cells were transfected with Lipofectamine 2000 reagent.After BMVEC transfection,BMVEC was divided into Control group,OGD/Hemin group(O/H),OGD/Hemin+agomir-NC group(O/H+agomir-NC),OGD/Hemin+agomir-miR-20b-5p group(O/H+agomir-miR-20b-5p),OGD/Hemin+antagomir-NC group(O/H+antagomir-NC)and OGD/Hemin+antagomir-miR-20b-5p group(O/H+antagomir-miR-20b-5p).BMVEC in Control group were cultured normally,and BMVEC in other groups were treated with OGD/Hemin.BMVEC proliferation was detected by MTT assay.BMVEC apoptosis was detected by TUNEL staining.BMVEC migration was detected by Transwell.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)were measured using a commercial kit.Fe2+content was determined using Iron Assay kit.The levels of miR-20b-5p and MAPK1 mRNA were detected by qRT-PCR.The protein expression levels of MAPK1,Bax,Bcl-2,glutathione peroxidase 4(GPX4)and prostaglandin peroxidase synthase 2(PTGS2)were detected by Western blot.Fluorescence intensity of MAPK1 was detected by immunofluorescence staining.Results:Compared with Control group and agomir-NC group,the level of miR-20b-5p in BMVEC of agomir-miR-20b-5p group increased(P<0.05).Compared with Control group and antagomir-NC group,the level of miR-20b-5p in BMVEC of antagomir-miR-20b-5p group decreased(P<0.05).Compared with Control group,miR-20b-5p level in BMVEC of O/H group decreased,cell viability decreased,TUNEL positive rate and Bax protein expression level increased,Bcl-2 protein expression level decreased,the number of migration decreased,SOD and GSH-Px activity decreased,MDA content increased,Fe2+content and PTGS2 protein expression level increased,GPX4 protein expression level decreased,the mRNA and protein expression level and the relative fluorescence intensity of MAPK1 increas(P<0.05).Compared with O/H group and O/H+agomir-NC group,the miR-20b-5p level in BMVEC of O/H+agomir-miR-20b-5p group increased,the cell viability increased,the positive rate of TUNEL and the expression level of Bax protein decreased,the expression level of Bcl-2 protein increased,the number of migration increased,the activity of SOD and GSH-Px increased,the content of MDA decreased,the content of Fe2+and the protein expression level of PTGS2 decreased,the protein expression level of GPX4 increased,the mRNA and protein expression level and the relative fluorescence intensity of MAPK1 decreased(P<0.05).Compared with O/H group and O/H+antagomir-NC group,miR-20b-5p level in BMVEC of O/H+antagomir-miR-20b-5p group decreased,cell viability decreased,TUNEL positive rate and Bax protein expression level increased,Bcl-2 protein expression level decreased,the number of migration decreased,SOD and GSH-Px activity decreased,MDA content increased,Fe2+content and PTGS2 protein expression level increased,GPX4 protein expression level decreased,the mRNA and protein expression level and the relative fluorescence intensity of MAPK1 increased(P<0.05).Conclusion:This study suggests that upregulation of miR-20b-5p inhibits the ferroptosis pathway by inhibiting the expression of MAPK1 in OGD/Hemin-treated BMVEC.