摘要
目的 研究miR-132-3p靶向调控第10号染色体缺失的磷酸酶基因(PTEN)/蛋白激酶B(Akt)信号通路对缺氧/复氧(H/R)诱导海马神经元HT22细胞损伤的影响.方法 体外培养HT22细胞并随机分为对照组、H/R组、阴性对照(miR-132-3p mimics 阴性对照+空载质粒)组、miR-132-3p mimics 组(miR-132-3p mimics)、PTEN 敲低组(PTEN siRNA 质粒)、miR-132-3p mimics+PTEN 过表达组(miR-132-3p mimics+PTEN 过表达质粒),除对照组外,其余各组进行H/R处理,同时进行分组转染,然后采用qRT-PCR实验、免疫印迹法检测各组细胞miR-132-3p与PTEN/Akt通路相关蛋白表达;采用MTT法、TUNEL染色分别检测各组细胞增殖与凋亡情况;采用试剂盒测量各组细胞活性氧(ROS)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、白细胞介素(IL)-6、IL-18水平;采用免疫印迹法检测各组细胞凋亡相关蛋白(Cleaved Caspase-3、Bax、Bcl-2)表达;采用双荧光素酶报告实验验证miR-132-3p对HT22细胞PTEN的靶向调控.结果 与对照组相比,H/R组细胞miR-132-3p表达、p-Akt/Akt、GSH-Px与SOD水平、Bcl-2蛋白表达、细胞活力降低(P＜0.05),细胞凋亡率、PTEN mRNA与蛋白表达、ROS相对水平、LDH产生水平、IL-6与IL-18水平、Bax与Cleaved Caspase-3蛋白表达升高(P＜0.05).与H/R组相比,miR-132-3p mimics组、PTEN敲低组p-Akt/Akt、GSH-Px与SOD水平、Bcl-2蛋白表达、细胞活力升高(P＜0.05),细胞凋亡率、PTEN mRNA与蛋白表达、ROS相对水平、LDH产生水平、IL-6与IL-18水平、Bax与Cleaved Caspase-3蛋白表达降低(P＜0.05);阴性对照组细胞各指标无明显变化(P＞0.05).与miR-132-3p mimics相比,miR-132-3p mimics+PTEN 过表达组 p-Akt/Akt、GSH-Px 与 SOD 水平、Bcl-2 蛋白表达、细胞活力降低(P＜0.05),细胞凋亡率、PTEN mRNA与蛋白表达、ROS相对水平、LDH产生水平、IL-6与IL-18水平、Bax与Cleaved Caspase-3蛋白表达升高(P＜0.05).miR-132-3p可靶向下调HT22细胞PTEN表达.结论 miR-132-3p可通过靶向下调PTEN表达而激活Akt信号,进而抑制H/R诱导的HT22细胞炎症与凋亡,缓解其细胞损伤.
Abstract
Objective To investigate the effect of miR-132-3p on hypoxia/reoxygenation(H/R)induced damage to hippocampal neurons HT22 cells by targeting the phosphatase and tensin homology deleted on chromosome ten(PTEN)/protein kinase B(Akt)signaling pathway.Methods HT22 cells were cultured in vitro and randomly separa-ted into control group,H/R group,negative control group(miR-132-3p mimics negative control+empty plasmid),miR-132-3p mimics group(miR-132-3p mimics),PTEN knockdown group(PTEN siRNA plasmid),and miR-132-3p mimics+PTEN overexpression group(miR-132-3p mimics+PTEN overexpression plasmid),except for the control group,all other groups were subjected to H/R treatment and transfection,qRT-PCR assay and immunoblotting were applied to detect the expression of miR-132-3p and PTEN/Akt pathway related proteins of cells in each group;MTT method and TUNEL staining were applied to detect cell proliferation and apoptosis in each group;kits were applied to measure the levels of reactive oxygen species(ROS),lactate dehydrogenase(LDH),superoxide dismutase(SOD),glutathione peroxi-dase(GSH-Px),interleukin-6(IL-6),and IL-18 of cells in each group;immunoblotting was applied to detect the expres-sion of apoptosis related proteins(Cleaved Caspase-3,Bax,Bcl-2)of cells in each group;dual luciferase reporter experi-ment was applied to verify the targeted regulation of miR-132-3p on PTEN in HT22 cells.Results Compared with the control group,the expression of miR-132-3p,the levels of p-Akt/Akt,GSH-Px and SOD,the expression of Bcl-2 pro-tein,and cell viability in the H/R group were reduced(P＜0.05),the apoptosis rate,the expression of PTEN mRNA and protein,the relative level of ROS,the production level of LDH,the levels of IL-6 and IL-18,and the expression of Bax and Cleaved Caspase-3 proteins were increased(P＜0.05).Compared with the H/R group,the levels of p-Akt/Akt,GSH-Px and SOD,the expression of Bcl-2 protein,and cell viability in the miR-132-3p mimics group and PTEN knockdown group were increased(P＜0.05),the apoptosis rate,the expression of PTEN mRNA and protein,the rela-tive level of ROS,the production level of LDH,the levels of IL-6 and IL-18,and the expression of Bax and Cleaved Caspase-3 proteins were decreased(P＜0.05);there was no significant change in all indicators of cells in the negative control group(P＞0.05).Compared with miR-132-3p mimics group,the levels of p-Akt/Akt,GSH-Px and SOD,the ex-pression of Bcl-2 protein,and cell viability in the miR-132-3p mimics+PTEN overexpression group were reduced(P＜0.05),the apoptosis rate,the expression of PTEN mRNA and protein,the relative level of ROS,the production level of LDH,the levels of IL-6 and IL-18,and the expression of Bax and Cleaved Caspase-3 proteins were increased(P＜0.05).MiR-132-3p was able to target downregulation of PTEN expression in HT22 cells.Conclusion MiR-132-3p can activate Akt signaling by targeting down regulation of PTEN expression,thereby inhibiting H/R-induced inflammation and apoptosis in HT22 cells,and alleviating their cellular damage.
基金项目
河南省医学科技攻关计划(LHGJ20210746)
NETL
NSTL
万方数据