Qu Lin, Li Gang, Bi Yunlong, Cao Yang
Department of Orthopedics, the First Affiliated Hospital of Jinzhou Medical University, Jinzhou 121001, China.
Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai 200072, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018 Aug;34(8):725-731.
Objective To detect the effects of microRNA 125a-5p (miR-125a-5p) in heme oxygenase 1 (HO-1) truncation (HO-1Cδ23) regulating the blood-spinal barrier (BSCB) after hypoxic injury. Methods HCMEC/D3 and astrocytes were co-cultured in Transwell chamber to establish BSCB model in vitro, and then prepared with cobalt chloride to establish BSCB hypoxic injury model. The experiment was divided into blank group, non-virus loading group, HO-1Cδ23 group (Lv-HO-1Cδ23), miRNA negative control group (Lv-HO-1Cδ23 combined with miR-NC treatment), miRNA mimic group (Lv-HO-1Cδ23 combined with miR-125a-5p mimics treatment) and miRNA inhibitor group (Lv-HO-1Cδ23 combined with miR-125a-5p inhibitor treatment). The expression levels of miR-125a-5p and zonula occludens-1 (ZO-1), occludin and vascular endothelial adhesion protein (VE-cadherin) were detected by reverse transcription PCR and real-time quantitative PCR. Western blot was used to detect the expressions of HO-1, ZO-1, occludin and VE-cadherin proteins. The permeability of BSCB in vitro was evaluated by the spillage rate of horseradish peroxidase (HRP). Results The success rate of HO-1Cδ23 transfection was about 70%. After transfection, the content of HO-1Cδ23 was significantly increased, and the expression of miR-125a-5p up-regulated compared with the blank group. Compared with the blank group, the mRNA and protein levels of ZO-1, occludin, and VE-cadherin in the HO-1Cδ23 group, the miRNA negative control group, and miRNA mimic group increased, but the HRP spillover rate decreased; on the contrary, the mRNA and protein levels of ZO-1 occludin and VE-cadherin in the miRNA inhibitor group decreased, while the HRP spillover rate increased significantly. Compared with the HO-1Cδ23 group, the mRNA and protein levels of ZO-1, occludin and VE-cadherin increased significantly in the miRNA mimic group, and the HRP spillover rate decreased significantly, while the mRNA and protein levels of ZO-1, occludin, and VE-cadherin as well as the HRP spillover rate in the miRNA inhibitor group showed the opposite trend. Conclusion Under hypoxic injury, HO-1Cδ23 may promote the transcription and translation of the genes involved in junction and adhesion and reduce the permeability of BSCB by up-regulating the expression of miR-125a-5p.
目的 检测微小RNA 125a - 5p（miR - 125a - 5p）在血红素加氧酶1（HO - 1）截短体（HO - 1Cδ23）调控缺氧损伤后血脊髓屏障（BSCB）中的作用。方法 将人脑血管内皮细胞系HCMEC/D3与星形胶质细胞在Transwell小室中共培养以建立体外BSCB模型，然后用氯化钴处理建立BSCB缺氧损伤模型。实验分为空白组、非病毒转染组、HO - 1Cδ23组（Lv - HO - 1Cδ23）、微小RNA阴性对照组（Lv - HO - 1Cδ23联合miR - NC处理）、微小RNA模拟物组（Lv - HO - 1Cδ23联合miR - 125a - 5p模拟物处理）和微小RNA抑制剂组（Lv - HO - 1Cδ23联合miR - 125a - 5p抑制剂处理）。采用逆转录PCR和实时定量PCR检测miR - 125a - 5p、紧密连接蛋白1（ZO - 1）、闭合蛋白和血管内皮钙黏蛋白（VE - cadherin）的表达水平。采用蛋白质印迹法检测HO - 1、ZO - 1、闭合蛋白和VE - cadherin蛋白的表达。通过辣根过氧化物酶（HRP）泄漏率评估体外BSCB的通透性。结果 HO - 1Cδ23转染成功率约为70%。转染后，HO - 1Cδ23含量显著增加，与空白组相比miR - 125a - 5p表达上调。与空白组相比，HO - 1Cδ23组、微小RNA阴性对照组和微小RNA模拟物组中ZO - 1、闭合蛋白和VE - cadherin的mRNA和蛋白水平升高，但HRP泄漏率降低；相反，微小RNA抑制剂组中ZO - 1、闭合蛋白和VE - cadherin的mRNA和蛋白水平降低，而HRP泄漏率显著升高。与HO - 1Cδ23组相比，微小RNA模拟物组中ZO - 1、闭合蛋白和VE - cadherin的mRNA和蛋白水平显著升高，HRP泄漏率显著降低，而微小RNA抑制剂组中ZO - 1、闭合蛋白和VE - cadherin的mRNA和蛋白水平以及HRP泄漏率呈现相反趋势。结论 在缺氧损伤条件下，HO - 1Cδ23可能通过上调miR - 125a - 5p的表达促进参与连接和黏附相关基因的转录和翻译，降低BSCB的通透性。
