miR-30b-5p抑制Notch信号通路活化对实验性自身免疫性葡萄膜炎的治疗作用

Therapeutic effect of miR-30b-5p on experimental autoimmune uveitis by inhibiting the activation of Notch signaling pathway

目的探讨miR-30b-5p对实验性自身免疫性葡萄膜炎(EAU)大鼠Notch信号通路活化的调控机制及治疗作用。方法通过Target Scan(http//www.targetscan.org/)对Notch1和Dll4基因与miR-30b的关系进行生物信息学预测。将雌性Lewis大鼠随机分为对照组、EAU组、miR-30b-5p组和miR-30b-5p空载体(miR-30b-5p-N)组。EAU组、miR-30b-5p组和miR-30b-5p-N组大鼠首先诱导EAU模型,miR-30b-5p组和miR-30b-5p-N组大鼠分别采用携带miR-30b-5p和miR-30b-5p-N的慢病毒于EAU大鼠脾脏进行注射干预处理,EAU组大鼠于同一部位注射相同体积的生理盐水。处理后12 d,实时荧光定量PCR检测4组大鼠脾脏、淋巴结和眼组织中Notch1和Dll4 mRNA的表达;免疫组织化学检测4组大鼠脾脏、淋巴结及眼组织中Notch1和Dll4蛋白的表达,分析miR-30b-5p对葡萄膜炎大鼠Notch信号通路的调控作用。结果生物信息学预测分析结果表明,Notch信号通路上Notch1和Dll4基因均为miR-30b调控的靶基因。免疫后12 d,相比于对照组,EAU组、miR-30b-5p组和miR-30b-5p-N组大鼠脾脏、淋巴结和眼组织中Notch1和Dll4 mRNA水平均升高(均为P<0.05);经miR-30b-5p干预治疗后,miR-30b-5p组脾脏、淋巴结和眼组织中Notch1和Dll4 mRNA表达均显著降低(均为P<0.05)。免疫组织化学检测结果显示,免疫后12 d,EAU组、miR-30b-5p组和miR-30b-5p-N组大鼠脾脏、淋巴结和眼组织中Notch1和Dll4蛋白表达均上调(均为P<0.05);miR-30b-5p干预治疗后,脾脏、淋巴结和眼组织中Notch1和Dll4蛋白表达均显著下调(均为P<0.05)。结论miR-30b-5p可明显下调EAU大鼠各组织中Notch1和Dll4等Notch信号通路相关分子的表达进而抑制Notch信号通路活化,从而达到治疗葡萄膜炎的作用。

Objective To investigate the regulatory role of miR-30b-5p on the activation of the Notch signaling pathway and its therapeutic effect on experimental autoimmune uveitis(EAU)in rats.Methods The relationship of Notch1 and Dll4 genes with miR-30b was predicted based on bioinformatics analysis using Target Scan(http//www.targetscan.org/).Female Lewis rats were randomly divided into the control group,EAU group,miR-30b-5p group,and miR-30b-5p empty vector(miR-30b-5p-N)group.EAU model was first induced in rats in the EAU,miR-30b-5p,and miR-30b-5p-N groups.Then lentiviruses carrying either miR-30b-5p or miR-30b-5p-N were respectively injected into the spleen of rats in the miR-30b-5p and miR-30b-5p-N groups for intervention treatment,while rats in the EAU group were injected with the same volume of normal saline at the same position.Twelve days after the treatment,the expression levels of Notch1 and Dll4 mRNA in the spleen,lymph nodes,and eye tissues were assessed by quantitative real-time polymerase chain reaction,and the expression levels of Notch1 and Dll4 proteins in the spleen,lymph nodes,and eye tissues were assessed by immunohistochemistry(IHC),so as to analyze the regulation of miR-30b-5p on the Notch signaling pathway in uveitis rats.Results Bioinformatics analysis showed that both Notch1 and Dll4 in the Notch signaling pathway were target genes of miR-30b.Compared with the control group,the mRNA levels of Notch1 and Dll4 in the spleen,lymph nodes,and eye tissues of rats in the EAU,miR-30b-5p,and miR-30b-5p-N groups increased significantly on day 12 after immunization(all P<0.05);whereas after miR-30b-5p intervention,the mRNA levels of Notch1 and Dll4 in the spleen,lymph nodes,and eye tissues of rats in the miR-30b-5p group decreased significantly(all P<0.05).IHC results indicated that,on day 12 after immunization,the expression levels of Notch1 and Dll4 proteins in the spleen,lymph nodes,and eye tissues were up-regulated in the EAU,miR-30b-5p,and miR-30b-5p-N groups(all P<0.05);whereas after miR-30b-5p intervention,the expression levels of Notch1 and Dll4 proteins in the spleen,lymph nodes,and eye tissues were significantly down-regulated(all P<0.05).Conclusion miR-30b-5p can significantly down-regulate the expression of molecules related to the Notch signaling pathway,including Notch1 and Dll4,in EAU rats,thereby inhibiting the activation of the Notch signaling pathway to achieve therapeutic effects in treating uveitis.