摘要
目的:探讨miR-34a在氯胺酮致发育期大鼠海马神经元凋亡中的作用及对Wnt/β-catenin通路的影响。方法:应用生物信息学软件分析预测并通过双荧光素酶报告实验验证miR-34a与Wnt1之间的靶向关系。取7 d龄雄性SD大鼠海马组织,分离培养获得神经元,加0.2 mg/L氯胺酮处理24 h,以未处理细胞为空白对照,采用qRT-PCR检测miR-34a的表达,Annexin V-FITC/PI双染法检测凋亡,Western blot检测Bax、Bcl-2蛋白的表达。另取大鼠海马神经元分为4组,以0.2 mg/L氯胺酮处理24 h后分为4组,miR-NC组、miR-34a组、anti-miR-NC组和anti-miR-34a组,转染48 h后采用Western blot检测Wnt/β-catenin通路相关蛋白Wnt1、β-catenin、TCF-4和Cyclin D1的表达。30只14 d龄SD雄性大鼠随机分为5组,空白对照组、氯胺酮组、anti-miR-NC+si-NC组、anti-miR-34a+si-NC组和anti-miR-34a+si-Wnt1组,每组6只。末次给药24 h后,采用Morris水迷宫实验检测大鼠空间学习能力,qRT-PCR检测miR-34a的表达,TUNEL染色检测大鼠海马神经元凋亡。结果:生物信息学软件分析和双荧光素酶报告实验证实Wnt1和miR-34a有靶向关系。氯胺酮处理会升高海马神经元中miR-34a表达,促进细胞凋亡和Bax的表达,降低Bcl-2的表达(P<0.05)。转染miR-34a模拟物可下调经氯胺酮处理的海马神经元中Wnt/β-catenin信号通路相关蛋白的表达,转染miR-34a抑制剂则可上调该信号通路相关蛋白的表达(P<0.05)。氯胺酮处理会升高SD大鼠海马组织中miR-34a表达,促进细胞凋亡,导致其空间学习能力障碍,miR-34a抑制剂可抑制氯胺酮致大鼠海马组织中细胞凋亡并改善大鼠空间学习能力,下调Wnt1的表达则可减弱miR-34a抑制剂的作用(P<0.05)。结论:miR-34a可促进氯胺酮致发育期大鼠海马神经元凋亡,其机制可能与靶向Wnt1调节Wnt/β-catenin通路有关。
Aim:To investigate the role of miR-34a in ketamine-induced apoptosis of hippocampal neurons in developmental rats and its effect on Wnt/β-catenin pathway.Methods:Bioinformatics software analysis and dual luciferase reporter assay were used to verify the targeting relationship between miR-34a and Wnt1.The hippocampal tissue of 7-day-old male SD rats was separated and cultured to obtain neurons,the obtain cells were treated with 0.2 mg/L ketamine for 24 hours,and untreated cells were used as a blank control.The expression of miR-34a was detected by qRT-PCR,Annexin V-FITC/PI double staining method was used to detect apoptosis,and Western blot was used to detect the expressions of Bax and Bcl-2 protein.In addition,rat hippocampal neurons were treated with 0.2 mg/L ketamine for 24 hours and allocated into 4 groups,miR-NC group,miR-34a group,anti-miR-NC group and anti-miR-34a group,and Western blot was used to detect expressions of Wnt/β-catenin pathway related proteins Wnt1,β-catenin,TCF-4 and Cyclin D1.Thirty 14-day-old SD male rats were randomly allocated into 5 groups,black control group,ketamine group,anti-miR-NC+si-NC group,anti-miR-34a+si-NC group,anti-miR-34a+si-Wnt1 group,6 in each group.24 hours after the last administration,Morris water maze test was used to detect the spatial learning ability of rats,qRT-PCR was used to detect the expression of miR-34a,and TUNEL staining was used to detect the apoptosis of rat hippocampal neurons.Results:Bioinformatics software analysis and dual luciferase reporter assay confirmed that there was targeting relationship between Wnt1 and miR-34a.Ketamine treatment increased the expression of miR-34a in hippocampal neurons,promoted the apoptosis and expression of Bax,and decreased the expression of Bcl-2(P<0.05).miR-34a could down-regulate the expression of Wnt/β-catenin signaling pathway related proteins in hippocampal neurons treated with ketamine,while anti-miR-34a could up-regulate the expression of the signaling pathway related proteins(P<0.05).Ketamine treatment could increase the expression of miR-34a in the hippocampus of SD rats and promote cell apoptosis,leading to impairment of their spatial learning ability,anti-miR-34a could inhibit ketamine-induced cell apoptosis and improve their spatial learning ability,while down-regulating the expression of Wnt1 could attenuate the anti-apoptotic effect of anti-miR-34a on ketamine treated rats(P<0.05).Conclusion:miR-34a can promote ketamine-induced apoptosis of hippocampal neurons in rats during the developmental stage,and its mechanism may be related to the regulation of Wnt/β-catenin pathway by targeting Wnt1.
作者
赵晨璐
赵以林
张雪
ZHAO Chenlu;ZHAO Yilin;ZHANG Xue(Department of Anesthesiology,Zhumadian Central Hospital,Zhumadian,Henan 463000;Department of Anesthesiology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430032)
出处
《郑州大学学报（医学版）》
CAS
北大核心
2021年第1期6-11,共6页
Journal of Zhengzhou University(Medical Sciences)
基金
河南省医学科技重大攻关项目(201806028)。
