LncRNA UCA1靶向miR-582-5p调控缺血低氧性脑神经细胞存活的实验研究  

LncRNA UCA1 regulates the survival of hypoxic-ischemic brain neurons by targeting miR-582-5p

作　　者：王玉松[1] 李培[2] 饶国敏 孔祥慧 骆泓洁 王素洁[2] 张志杰 李健[2] Wang Yusong;Li Pei;Rao Guomin;Kong Xianghui;Luo Hongjie;Wang Sujie;Zhang Zhijie;Li Jian(Department of Neurology,No.982 Hospital,Joint Service Support Force of the Chinese People's Liberation Army,Hebei 063000,China)

机构地区：[1]中国人民解放军联勤保障部队第九八二医院神经内科,河北063000 [2]唐山市工人医院 [3]唐山南湖医院

出　　处：《脑与神经疾病杂志》2023年第9期581-586,共6页Journal of Brain and Nervous Diseases

基　　金：河北省科技计划项目(16397747D)。

摘　　要：目的 观察长链非编码RNA尿路上皮癌胚抗原1 (LncRNA UCA1)在大脑中动脉阻塞(MCAO)大鼠模型、氧糖剥夺/复氧(OGD/R) PC12细胞中的表达,探究其调控脑神经细胞增殖、凋亡的潜在机制。方法 MCAO法建立大鼠缺血再灌注损伤模型;OGD/R法制作PC12细胞损伤模型。shcon、sh-UCA1给予大鼠右脑室注射;脂质体法转染si-con、si-UCA1、agomiRNA、agomiR-582-5p、siUCA1+antagomiRNA、si-UCA1+antagomiR-582-5p至PC12细胞。荧光逆转录聚合酶链式反应(RT-qPCR)实验检测组织、细胞中LncRNA UCA1、miR-582-5p表达;DNA末端转移酶介导的dUTP缺口末端标记法(TUNEL)检测脑组织神经元凋亡;细胞计数(CCK8)实验、5-乙炔基-2’脱氧尿嘧啶核苷(EdU)染色、克隆形成实验检测细胞增殖能力;流式细胞术检测细胞凋亡;双荧光素酶报告基因检测实验检测细胞荧光活性。结果 与空白组相比,模型组大鼠脑组织LncRNA UCA1升高,TUNEL凋亡率升高,shUCA1给药组大鼠脑组织LncRNA UCA1降低,TUNEL凋亡率也降低(P<0.05)。与对照组相比,OGD/R组细胞LncRNA UCA1升高,细胞增殖率、EdU阳性率、克隆形成数均降低,凋亡率升高,转染si-UCA1的细胞LncRNA UCA1降低,细胞增殖率、EdU阳性率、克隆形成数均升高,凋亡率降低(P<0.05);LncRNA UCA1直接靶向负调控miR-582-5p,且在大鼠脑组织中miR-582-5p的水平与LncRNA UCA1呈负相关性(R^(2)=0.249,P<0.05)。miR-582-5p在模型组大鼠脑组织、OGD/R细胞中均低表达(P<0.05);与agomiRNA组相比,agomiR-582-5p组细胞miR-582-5p表达升高,细胞增殖率、EdU阳性率、克隆形成数均升高,凋亡率降低(P<0.05)。抑制miR-582-5p减弱敲减LncRNA UCA1对OG D/R细胞的增殖促进和凋亡抑制作用。结论LncRNA UCA1参与缺血低氧性脑神经细胞的损伤过程,抑制OGD/R PC12细胞增殖,促进凋亡,这种不利于神经细胞存活的作用与靶向miR-582-5p有关。Objective To observe the expression of long chain noncoding RNA urothelial carcinoembryonic antigen 1(LncRNA UCAI)in middle cerebral artery occlusion(MCAO)rat model and oxygen glucose deprivation/reoxygenation(OGD/R)PC12 cells,and to explore its potential mechanism in regulating the proliferation and apoptosis of brain neurons.Methods MCAO method was used to establish the model of ischemiareperfusion injury in rats;PC12 cell injury model was established by OGD/R method.Sh-con and sh-UCA1 were injected into the right ventricle of rats.si-con,si-UCA1,agomiRNA,agomiR-582-5p,si-UCA1+antigmiRNA,si-UCA1+antigmiRNA-582-5p were transfected into PC12 cells by liposome method.The expression of Incrna UCA1 and mir-582-5p in tissues and cells was detected by fluorescent reverse transcription polymerase chain reaction(RT-qPCR);DNA terminal transferase mediated dUTP nick end labeling(TUNEL)was used to detect neuronal apoptosis in brain tssue;Cell counting(CCK8),5-ethynyl-2'deoxyuridine(EDU)staining and clonogenic assay were used to detect cell proliferation;Apoptosis was detected by flow cytometry.Double luciferase reporter gene assay was used to detect cell fluorescence activity.Results compared with the blank group,the LncRNA UCA1 and TUNEL apoptosis rate in the model group increased,while the LncRNA UCA1 and TUNEL apoptosis rate in the sh-UCA1 treated group decreased(P<0.05);Compared with the control group,the LncRNA UCA1 of cells in ogd/r group increased,the cell proliferation rate,edu positive rate and clone formation number decreased,and the apoptosis rate increased.The LncRNA UCAI of cells transfected with si-UCAI decreased,the cell proliferation rate,edu positive rate and clone formation number increased,and the apoptosis rate decreased(P<0.05);Lncrna UCA1 directly targeted and negatively regulated miR-582-5p,and the level of miR-582-5p in rat brain was negatively correlated with LncRNA UCA1(R^(2)=0.249,P<0.05).MiR-582-5p was low expressed in the brain tissue and ogd/r cells of the model group(P<0.05);Compared with the

关 键 词：长链非编码RNA尿路上皮癌胚抗原1 缺血低氧性脑神经损伤 miR-582-5p 增殖 凋亡 

分 类 号：R743.31[医药卫生—神经病学与精神病学]