miR-374b-5p靶向调控PTEN抑制七氟醚诱导的小鼠海马神经元HT22细胞凋亡  

Inhibition of Sevoflurane-induced Apoptosis of HT22 Cells in Mouse Hippocampal Neurons by Targeted Regulation of PTEN via miR-374b-5p

作　　者：葛晓丹 康佳静 李瑜[1] 贾英萍 GE Xiaodan;KANG Jiajing;LI Yu;JIA Yingping(Children’s Hospital Affiliated to Zhengzhou University Henan Children’s Hospital,Zhengzhou Henan 450018,China)

机构地区：[1]郑州大学附属儿童医院河南省儿童医院麻醉与围术期医学科,郑州450018

出　　处：《河南医学高等专科学校学报》2024年第4期425-432,共8页Journal of Henan Medical College

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20200621)。

摘　　要：目的 探究miR-374b-5p对七氟醚(sevoflurane,Sev)引起的小鼠海马神经元HT22细胞损伤的保护作用。方法 将HT22细胞并分为6组:对照组、Sev组、miR-con+Sev组、miR-374b-5p+Sev组、miR-374b-5p+pcDNA+Sev组、miR-374b-5p+pcDNA-PTEN+Sev组。RT-qPCR检测各组miR-374b-5p和PTEN mRNA的表达水平,蛋白质免疫印迹(Western blot)检测各组PTEN、Bax、Bcl-2、Cleaved caspase-3的表达水平,双荧光素酶活性实验验证miR-374b-5p对PTEN的靶向作用,CCK8检测HT22细胞活性;流式细胞术检测HT22细胞凋亡;DCFH-DA探针检测HT22细胞ROS水平;酶联免疫吸附试验(ELISA)检测HT22细胞中丙二醛(MDA)和谷胱甘肽(GSH)含量。结果 与对照组比较,在Sev处理组miR-374b-5p的表达水平下降,PTEN mRNA和PTEN蛋白表达水平增加,差异有统计学意义(P<0.05)。miR-374b-5p通过与HT22细胞中PTEN mRNA的3’UTR结合而抑制PTEN的表达。与对照组比较,Sev组HT22细胞中PTEN mRNA、PTEN、ROS、MDA含量、Cleaved caspase-3、Bax表达量和细胞凋亡率升高,而miR-374b-5p、GSH含量、Bcl-2表达量和细胞存活率降低,差异有统计学意义(P<0.05);与miR-con+Sev组比较,miR-374b-5p+Sev组的HT22细胞中PTEN mRNA、PTEN、ROS、MDA含量Cleaved caspase-3、Bax表达量和细胞凋亡率降低,而miR-374b-5p、GSH含量、Bcl-2表达量和细胞存活率升高,差异有统计学意义(P<0.05);与miR-374b-5p+pcDNA+Sev组比较,miR-374b-5p+pcDNA-PTEN+Sev组的HT22细胞中PTEN mRNA、PTEN、ROS、MDA含量、Cleaved caspase-3、Bax表达量和细胞凋亡率升高,而GSH含量、Bcl-2表达量和细胞存活率含量降低,差异有统计学意义(P<0.05)。结论 预处理miR-374b-5p可能通过抑制PTEN抑制Sev处理HT22细胞凋亡,从而发挥对神经细胞的保护作用。Objective To investigate the protective effect of miR-374b-5p against sevoflurane(Sev) induced cell damage in mouse hippocampal neuron HT22 cells.Methods HT22 cells were divided into 6 groups:control group,Sev group,miR-con+Sev group,miR-374b-5p+Sev group,miR-374b-5p+pcDNA+Sev group,and miR-374b-5p+pcDNA-PTEN+Sev group.RT-qPCR was used to detect the expression levels of miR-374b-5p and PTEN mRNA in each group.Western blot was used to detect the expression levels of PTEN,Bax,Bcl-2 and Cleaved caspase-3 in each group.Double Luciferase activity experiment was used to verify the targeting effect of miR-374b-5p on PTEN,and CCK8 was used to detect the activity of HT22 cells;Flow cytometry was used to detect HT22 cell apoptosis;DCFH-DA probe was used to detect ROS levels in HT22 cells.Enzyme-linked immunosorbent assay(ELISA) was used to detect malondialdehyde(MDA) and glutathione(GSH) levels in HT22 cells.Results Compared with the control group,miR-374b-5p expression in the Sev treatment group decreased,while the levels of PTEN mRNA and PTEN protein increased(P<0.05).And miR-374b-5p inhibited PTEN expression by binding to the 3'UTR of PTEN mRNA in HT22 cells.Compared with the control group,both the levels of PTEN mRNA,PTEN,ROS,MDA,Cleaved caspase-3,Bax,and apoptosis rate in HT22 cells of hippocampal neurons in Sev group increased,while levels of miR-374b-5p,Bcl-2,GSH content,and cell survival rate decreased(P<0.05).Compared with the miR-con+Sev group,the levels of PTEN mRNA,PTEN,ROS,MDA,Cleaved caspase-3,Bax expression and apoptosis were decreased,while levels of miR-374b-5p,GSH content,Bcl-2 expression and cell survival were elevated in HT22 cells in miR-374b-5p+Sev group(P<0.05).Compared with the miR-374b-5p+pcDNA+Sev group,the miR-374b-5p+pcDNA PTEN+Sev group showed an increase in levels of PTEN mRNA,PTEN,ROS,MDA content,Cleaved caspase-3,Bax,and cell apoptosis rate in HT22 cells of mouse hippocampal neurons,while showed a decrease in levels of GSH content,Bcl-2 expression level,and cell survival rate decreased(P<0.0

关 键 词：七氟醚 miR-374b-5p 氧化应激 细胞凋亡 

分 类 号：R614[医药卫生—麻醉学]