摘要
为探究miR-881-3p对白细胞介素9受体(IL9R)的靶向性及其对小鼠脑微血管内皮细胞(MBMEC)增殖和迁移的调控作用,本研究将MBMEC分为模拟物组(转染miR-881-3p模拟物)、模拟物对照组(转染miR-881-3p模拟物对照物)、抑制剂组(转染miR-881-3p抑制剂)、抑制剂对照组(转染miR-881-3p抑制剂对照物),通过双荧光素酶试验检测miR-881-3p对IL9R的靶向性;采用RT-qPCR检测各组细胞中miR-881-3p、IL9R、增殖细胞核抗原(PCNA)、小型染色体维持蛋白复合体3(MCM3)、紧密连接蛋白-5(Claudin-5)的相对转录水平;采用CCK-8法检测各组细胞增殖水平;采用划痕试验检测各组细胞的迁移情况;通过western blot检测IL9R、PCNA蛋白、MCM3、claudin-5的表达水平。双荧光素酶试验结果显示,miR-881-3p对IL9R具有直接靶向作用。RTqPCR结果显示,转染48 h后,与对照组相比,模拟物组中miR-881-3p的相对转录水平极显著升高(P<0.01),而IL9R的相对转录水平极显著降低(P<0.01),与抑制剂对照组相比,抑制剂组中miR-881-3p的相对转录水平极显著降低(P<0.01),而IL9R的相对转录水平极显著升高(P<0.01),与抑制剂组相比,模拟物组中PCNA、MCM3、claudin-5的相对转录水平极显著降低(P<0.001)。CCK-8法检测结果显示,miR-881-3p通过负调控IL9R进而抑制MBMEC的增殖。划痕试验结果显示,miR-881-3p通过负调控IL9R进而抑制MBMEC的迁移。Western blot检测结果显示,过表达miR-881-3p后MBMEC中的IL9R、MCM3、claudin-5蛋白表达量极显著降低(P<0.001),PCNA蛋白表达量显著降低(P<0.05)。上述结果首次证实miR-881-3p通过负调控IL9R抑制MBMEC的增殖和迁移,本研究为探究细菌性脑膜炎损伤机制提供了可靠依据。
To explore the miR-881-3p-targeting to interleukin 9 receptor(IL9R)and its regulatory effects on the proliferation and migration of brain microvascular endothelial cells in mouse(MBMEC),in this study,MBMEC was divided into several groups:a mimic group(transfected with miR-881-3p mimic),a mimic control group(transfected with miR-881-3p mimic control),an inhibitor group(transfected with miR-881-3p inhibitor),and an inhibitor control group(transfected with miR-881-3p inhibitor control).A dualluciferase assay was conducted to test the targeting relationship between miR-881-3p and IL9R.The RT-qPCR was used to detect the transcription levels of miR-881-3p,IL9R,PCNA,MCM3,and claudin-5 in each group of cells.The CCK-8 method was employed to assess cell proliferation levels,and a scratch assay was used to evaluate cell migration.The western blot(WB)was used to detect the protein expression levels of IL9R,proliferating cell nuclear antigen(PCNA),minichromosome maintenance complex component 3(MCM3),and tight junction protein 5(claudin-5).The dual-luciferase assay results indicated that miR-881-3p has a direct targeting effect on IL9R.The RT-qPCR results showed that after 48 hours transfection,compared to the control group,the transcription level of miR-881-3p significantly increased(P<0.01)in the mimic group,while the transcription level of IL9R significantly decreased(P<0.01).Compared to the inhibitor control group,the transcription level of miR-881-3p significantly decreased(P<0.01)in the inhibitor group,and the transcription level of IL9R noticeably increased(P<0.01).Compared to the inhibitor group,the transcription levels of PCNA,MCM3,and claudin-5 significantly decreased(P<0.001)in the mimic group.The CCK-8 assay results revealed that miR-881-3p inhibits the proliferation of MBMEC by negatively regulating IL9R.The scratch assay results demonstrated that miR-881-3p inhibits the migration of MBMEC by negatively regulating IL9R.The WB detection results showed that overexpression of miR-881-3p significantly reduced the protein expression levels of IL9R,MCM3,and claudin-5(P<0.001)and notably decreased the protein expression level of PCNA(P<0.05)in MBMEC.All the results above,for the first time,demonstrated that the miR-881-3p inhibits the proliferation and migration of MBMEC by negatively regulating IL9R,providing a reliable basis for studying the injury mechanisms of bacterial meningitis.
作者
曹芳
魏勇
吴忧
任静静
齐亚银
CAO Fang;WEI Yong;WU You;REN Jing-jing;QI Ya-yin(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;Xinjiang Tianrun Technology Co.,Ltd.,Urumqi 830063,China)
出处
《中国预防兽医学报》
CSCD
北大核心
2024年第11期1107-1116,共10页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(32460898、31760737)
天山英才“三农骨干人才”项目(2024-97)
石河子大学科技成果转化项目(CGZH202104)
奶牛信息智能化管理技术研究与示范(NYHXGG、2023AA310)
石河子大学高层次人才科研启动项目(RCZK202042)。
作者简介
共同第一作者:曹芳(1996-),女,山东邹平人,硕士研究生,主要从事动物传染病与细菌学研究;共同第一作者:魏勇(1980-),男,四川资中人,硕士研究生,高级兽医师,主要从事奶牛健康养殖方面研究;共同第一作者:吴忧(1999-),女,河南平顶山人,硕士研究生,主要从事动物传染病与细菌学研究;通信作者:任静静,E-mail:qiyayin@163.com;通信作者:齐亚银,E-mail:renjingjing1990@sina.com。
