rno-miR-30b-5p调控Atg5、Atg12和Becn1自噬基因表达在实验性自身免疫性葡萄膜炎中的作用

Regulatory roles of rno-miR-30b-5p in the expression of Atg5,Atg12 and Becn1 autophagy genes in rats with experimental autoimmune uveitis

目的探讨rno-miR-30b-5p对Atg5、Atg12和Becn1自噬基因表达的调控作用及其在实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis,EAU)中的表达变化。方法 双荧光素酶检测mo-miR-30b-5p对Atg5、Atg12和Becn1自噬基因表达的调控作用。Lewis大鼠随机分为正常对照组和EAU组,每组各6只,EAU组诱导EAU模型,两组大鼠每天用Genesis-A眼底相机观察眼底情况。免疫后12 d,取眼球观察睫状体、视网膜的病理学表现;分离大鼠的脾脏和淋巴结,实时荧光定量PCR(Q-PCR)检测rno-miR-30b-5p、Atg5、Atg12和Becn1 mRNA的表达情况;ELISA方法检测Atg5、Atg12和Becn1蛋白的表达水平。结果双荧光素酶检测结果证实Atg5、Atg12和Becn1为rno-miR-30b-5p调控的靶基因。免疫后12 d,EAU组大鼠眼底血管严重肿胀,病理学检测可见睫状体、视网膜内大量炎性细胞浸润。Q-PCR检测结果示,与正常对照组相比,免疫后12 d EAU组大鼠脾脏和淋巴结中rno-miR-30b-5p mRNA水平分别为0.46±0.01和0.29±0.17,均呈下调表达(均为P<0.01);Atg5、Atg12和Becn1 mRNA水平均呈上调表达,差异均有统计学意义(均为P<0.05)。ELISA检测结果示,EAU组大鼠脾脏和淋巴结中Atg5、Atg12和Becn1蛋白表达水平均明显高于正常对照组大鼠(均为P<0.05)。结论 rno-miR-30b-5p可调控Atg5、Atg12和Becn1的表达。在EAU大鼠脾脏和淋巴结中,rno-miR-30b-5p的下调表达使Atg5、Atg12和Becn1基因和蛋白的表达水平均明显升高,从而影响葡萄膜炎的发展进程。

Objective To investigate the regulatory roles of rno-miR-30b-5p in the expression of Atg5,Atg12 and Becn1 autophagy genes and their expressions in rats with experimental autoimmune uveitis(EAU).Methods Application of dual luciferase report system was conducted to detect the regulatory roles of rno-miR-30b-5p in Atg5,Atg12 and Becn1 gene expression.Lewis rats were randomly divided into control group and EAU group,with 6 rats in each group.Next,rats in EAU group were immunized to establish EAU model.After treatments,Genesis-D fundus camera was used to observe the fundus inflammation every day.After immunization for 12 days,the pathological features of rat ciliary body and retina were detected,and meanwhile,the spleen and lymph nodes in both groups were isolated to detect the expressions of rno-miR-30b-5p,Atg5,Atg12 and Becn1 genes by quantitative PCR(Q-PCR);the levels of autophagy related proteins were determined by ELISA.Results Dual luciferase report gene expression assay confirmed that Atg5,Atg12 and Becn1 were target genes of rno-miR-30b-5p.Twelve days after immunization,compared with the control group,rats in the EAU group had severe iris adhesions and severe blood vessel swelling,and pathological examination revealed massive infiltration of inflammatory cells in the ciliary body and retina.Furthermore,rno-miR-30b-5p mRNA was 0.46±0.01,0.29±0.17in the spleen and lymph nodes in EAU group,respectively,which was down-regulated when compared with the control group(P<0.01);whereas the expressions of Atg5,Atg12 and Becn1 were significantly up-regulated,and the differences were statistically significant(all P<0.05).ELISA results showed that Atg5,Atg 12 and Becn 1 protein expression levels in the spleen and lymph nodes in EAU rats were significantly higher than those in the control group(all P<0.05).Conclusion rno-miR-30b-5p can regulate the expressions of Atg5,Atg12 and Becn1 autophagy-related genes.The down-regulation of rno-miR-30b-5p expression in the spleen and lymph nodes in EAU rats can significantly up-regulate the expressions of Atg5,Atg12 and Becn1 genes,thereby regulating the pathogenesis of uveitis.