【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。更多还原

【Abstract】 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.更多还原