rno-miR-30b-5p在葡萄膜炎发病过程中对白细胞介素-10和Toll样受体4表达的调控作用

发布时间：2019-01-29 01:36

【摘要】：目的探讨rno-miR-30b-5p在葡萄膜炎中对白细胞介素10(interleukin-10,IL-10)和Toll样受体4(toll-like receptor 4,TLR4)表达的调控作用。方法构建IL-10、TLR4基因的3’端非编码区域(3’UTR)荧光素酶质粒载体以及结合位点突变质粒载体,将rnomiR-30b-5p模拟物(mimic)与构建好的报告基因载体共转染293T细胞,通过报告基因相对荧光值的表达改变检测rno-miR-30b-5p对IL-10、TLR4表达的调控作用;制备实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis,EAU)模型,通过注射视网膜光感受器间维生素A类结合蛋白乳糜液,在造模后12 d分离EAU大鼠的脾脏和淋巴结,实时荧光定量PCR检测rno-miR-30b-5p和IL-10、TLR4基因的表达情况;ELISA检测IL-10和TLR4蛋白的表达情况。结果双荧光素酶报告基因表达结果表明,经rno-miR-30b-5p mimic干预后IL-10、TLR4野生型的报告荧光有明显的下调作用,对其预测靶位点进行突变后,突变型载体中的报告荧光也存在明显的下调作用,与阴性对照相比,差异均有统计学意义(均为P0.01)。实时荧光定量PCR检测结果发现rno-miR-30b-5p在EAU大鼠脾脏和淋巴结中的表达均较对照组明显降低(均为P0.01),而IL-10 mRNA、TLR4 mRNA表达明显升高(均为P0.01);ELISA检测发现IL-10、TLR4蛋白表达升高,与对照组相比,差异均有统计学意义(均为P0.05)。结论rno-miR-30b-5p对带有IL-10、TLR4基因片段3’UTR基因表达的调控作用可能不是通过预测到的位点起作用而是通过其他的调控结合位点发挥作用;rno-miR-30b-5p在EAU大鼠脾脏和淋巴结的下调表达导致IL-10和TLR4的表达水平升高,进而调控葡萄膜炎的发展。本研究为进一步探索microRNA在葡萄膜炎发生发展进程中的调控作用、治疗葡萄膜炎奠定了基础。
[Abstract]:Objective to investigate the effect of rno-miR-30b-5p on the expression of interleukin 10 (interleukin-10,IL-10) and Toll like receptor 4 (toll-like receptor 4) in uveitis. Methods the 3 '-terminal non-coding region (3'UTR) luciferase plasmid vector of IL-10,TLR4 gene and the plasmid vector with binding site mutation were constructed. The rnomiR-30b-5p mimic (mimic) was co-transfected into 293T cells with the constructed reporter gene vector. The regulation of rno-miR-30b-5p on the expression of IL-10,TLR4 was detected by the change of relative fluorescence value of the reporter gene. The experimental autoimmune uveitis (experimental autoimmune uveitis,EAU) model was established. The spleen and lymph nodes of EAU rats were isolated 12 days after injection of vitamin A binding protein chyme between retinal photoreceptors. The expression of rno-miR-30b-5p and IL-10,TLR4 genes was detected by real-time fluorescence quantitative PCR. The expression of IL-10 and TLR4 protein was detected by ELISA. Results the results of double luciferase reporter gene expression showed that IL-10,TLR4 wild-type reporter fluorescence was significantly down-regulated after rno-miR-30b-5p mimic intervention, and the predicted target sites were mutated. There was also a significant down-regulation of the reported fluorescence in the mutant vector, compared with the negative control, the difference was statistically significant (all P0.01). The results of real-time fluorescence quantitative PCR showed that the expression of rno-miR-30b-5p in spleen and lymph nodes of EAU rats was significantly lower than that in control group (P0.01), while the expression of IL-10 mRNA,TLR4 mRNA was significantly increased (P0.01). ELISA detection showed that the expression of IL-10,TLR4 protein increased, compared with the control group, the difference was statistically significant (P0.05). Conclusion the regulatory effect of rno-miR-30b-5p on the expression of 3'UTR gene with IL-10,TLR4 gene fragment may play a role not by the predicted sites but by other regulatory binding sites. The down-regulation of rno-miR-30b-5p expression in spleen and lymph nodes of EAU rats leads to the increase of IL-10 and TLR4 expression, which regulates the development of uveitis. This study laid a foundation for further exploring the regulatory role of microRNA in the development of uveitis and for the treatment of uveitis.
【作者单位】：山东中医药大学;山东中医药大学眼科研究所;
【基金】：高等学校博士学科点专项科研基金(编号:20133731110004) 山东中医药科技发展计划(编号:2015-145) 山东省自然科学基金(编号:ZR2016HP27)~~
【分类号】：R773.9

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