lncRNA HOTAIR调节miR-20a-5p/TGF-β1轴改善新生癫痫大鼠认知能力  

lncRNA HOTAIR improves the cognitive ability of neonatal epileptic rats by regulating the miR-20a-5p/TGF-β1 axis

作　　者：李妍 文亚娜 费容 LI Yan;WEN Ya-na;FEI Rong(Department of Pediatrics,Changshou District People's Hospital of Chongqing,Chongqing 401220,China;Department of Neurology,Changshou District People's Hospital of Chongqing,Chongqing 401220,China)

机构地区：[1]重庆市长寿区人民医院儿科,重庆401220 [2]重庆市长寿区人民医院神经内科,重庆401220

出　　处：《现代免疫学》2024年第3期197-205,共9页Current Immunology

摘　　要：为探讨长链非编码RNA(long non-coding RNA,lncRNA)同源异型盒基因转录反义基因间RNA(HOX transcript antisense RNA,HOTAIR)对miR-20a-5p/TGF-β1信号轴的调控作用及对新生癫痫大鼠认知能力的影响,采集20例癫痫患者及20例健康对照者肘静脉血,分离血清,qRT-PCR法检测lncRNA HOTAIR的表达;予新生大鼠腹腔注射海人酸构建癫痫模型,并随机分为正常对照组、模型组、lncRNA HOTAIR过表达(Lv-HOTAIR)组、阴性对照(Lv-NC)组、TGF-β1抑制剂组和Lv-HOTAIR+TGF-β1抑制剂组,每组20只。Racine分级标准评估大鼠的癫痫发作程度;水迷宫试验评估大鼠的认知功能;Timm染色观察大鼠海马组织苔藓纤维的发芽情况;免疫组化法检测M2型小胶质细胞特异性标志蛋白精氨酸酶1(Arginase 1,Arg-1)的阳性表达情况;TUNEL法评估大鼠海马神经元的凋亡情况;qRT-PCR法检测海马组织lncRNA HOTAIR、miR-20a-5p的表达;Western blotting检测海马组织TGF-β1、M1型小胶质细胞极化标志蛋白CD11、M2型小胶质细胞极化标志蛋白CD206、IL-6和IL-1β的表达;双荧光素酶报告基因实验验证lncRNA HOTAIR对miR-20a-5p的靶向调控作用以及miR-20a-5p对TGF-β1的靶向调控作用。建立癫痫体外细胞模型,同时上调lncRNA HOTAIR及miR-20a-5p表达,验证miR-20a-5p对lncRNA HOTAIR抗癫痫作用的逆转。结果显示,lncRNA HOTAIR在癫痫患者血清中的表达显著低于健康对照者(P<0.05);lncRNA HOTAIR可靶向负调控miR-20a-5p表达,miR-20a-5p可靶向负调控TGF-β1表达。与正常对照组相比,模型组大鼠癫痫发作等级评分升高,认知功能降低,苔藓纤维发芽及神经元凋亡严重,miR-20a-5p表达升高,lncRNA HOTAIR及TGF-β1表达降低,小胶质细胞M2型抑炎表型活性降低(均P<0.05)。上调lncRNA HOTAIR表达可降低癫痫大鼠疾病发作等级,提高认知功能,改善苔藓纤维发芽及减少神经元凋亡,抑制miR-20a-5p表达并增加TGF-β1介导的小胶质细胞M2型抑炎表型活化,发挥�This study proposed to explore the regulation of long non-coding RNA(lncRNA)HOX transcript antisense RNA(HOTAIR)on miR-20a-5p/TGF-β1 signaling axis,and its effect on the cognitive ability of neonatal rats with epilepsy.The elbow venous blood was collected from twenty epilepsy patients and twenty healthy controls,the serum was isolated and the expression of lncRNA HOTAIR was detected by qRT-PCR.Neonatal rats were intraperitoneally injected with kainic acid to establish the epilepsy models,and were randomly divided into normal control group,model group,lncRNA HOTAIR over-expression(Lv-HOTAIR)group,negative control(Lv-NC)group,TGF-β1 inhibitor group,and Lv-HOTAIR+TGF-β1 inhibitor group,with twenty rats per group.Racine grading standard was referred to to assess the degree of epileptic seizures.Water maze test was performed to evaluate the rats cognitive function.Timm staining was used to observe the sprouting status of mossy fibers.Immunohistochemistry assay was used to detect the positive expression of M2-type microglia-specific protein marker Arginase 1(Arg-1).TUNEL staining was set out to detect the apoptosis rate of rat hippocampal neurons.qRT-PCR was used to detect the expression of lncRNA HOTAIR and miR-20a-5p in hippocampus.Western blotting was run to detect the expressions of TGF-β1,M1 type microglia polarization protein marker CD11,M2 type microglia polarization protein marker CD206,IL-6,and IL-1βin hippocampus.Dual-luciferase report gene experiment was performed to verify the targeting regulation of lncRNA HOTAIR on miR-20a-5p and that of miR-20a-5p on TGF-β1.An in vitro epilepsy cell model was established,and lncRNA HOTAIR and miR-20a-5p were simultaneously over-expressed to verify the reversal of miR-20a-5p on the anti-epileptic effect of lncRNA HOTAIR.The results showed that the expression of lncRNA HOTAIR in the serum of epilepsy patients was significantly lower than that of healthy controls(P<0.05).lncRNA HOTAIR negatively regulates the miR-20a-5p expression,which in turn negatively regulates th

关 键 词：癫痫 长链非编码核糖核酸同源异型盒基因转录反义基因间核糖核酸 微小核糖核酸20a-5p 转化生长因子Β1 认知能力 

分 类 号：R742.1[医药卫生—神经病学与精神病学]