[1]孙园园,郭大东,陈美清,等.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[J].眼科新进展,2017,37(4):330-334.[doi:10.13389/j.cnki.rao.2017.0083]
　SUN Yuan-Yuan,GUO Da-Dong,CHEN Mei-Qing,et al.Regulatory roles of rno-miR-30b-5p in expressions of IL-10 and TLR4 in rats with experimental autoimmune uveitis[J].Recent Advances in Ophthalmology,2017,37(4):330-334.[doi:10.13389/j.cnki.rao.2017.0083]
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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/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
37卷
期数:
2017年4期
页码:
330-334
栏目:
实验研究
出版日期:
2017-04-05

文章信息/Info

Title:
Regulatory roles of rno-miR-30b-5p in expressions of IL-10 and TLR4 in rats with experimental autoimmune uveitis
作者:
孙园园; 郭大东; 陈美清; 李少玉; 刘滨; 唐凯; 毕宏生
250014　山东省济南市，山东中医药大学［孙园园、陈美清、李少玉（2015级硕士研究生），刘滨（2016级硕士研究生）］；250002　山东省济南市，山东中医药大学眼科研究所（郭大东，唐凯，毕宏生）
Author(s):
SUN Yuan-Yuan; GUO Da-Dong; CHEN Mei-Qing; LI Shao-Yu; LIU Bin; TANG Kai; BI Hong-Sheng
Shandong University of Traditional Chinese Medicine（SUN Yuan-Yuan，CHEN Mei-Qing，LI Shao-Yu，LIU Bin），Jinan 250014，Shandong Province，China；Eye Institute of Shandong University of Traditional Chinese Medicine（GUO Da-Dong，TANG Kai，BI Hong-Sheng），Jinan 250002,Shandong Province,China
关键词:
rno-miR-30b-5p; 荧光素酶质粒载体; 白细胞介素-10; Toll样受体4; 葡萄膜炎
Keywords:
rno-miR-30b-5p; luciferase plasmid vector; IL-10; TLR4; uveitis
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2017.0083
文献标志码:
A
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rno-miR-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P<0.01）。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P<0.01），而IL-10 mRNA、TLR4 mRNA表达明显升高（均为P<0.01）；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P<0.05）。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 regulatory role of rno-miR-30b-5p in the expressions of interleukin-10（IL-10） and toll-like receptor 4（TLR4） in uveitis.Methods　Both IL-10 and TLR4 gene 3’UTR luciferase vectors and relevant binding site mutant vectors were constructed.Further,both rno-miR-30b-5p mimics and reporter gene vector were co-transferred into 293 T cells to validate the fluorescent alterations of the reporter gene expression to detect the interactions between rno-miR-30b-5p and the related target genes.Moreover,an experimental autoimmune uveitis (EAU) model was induced with IRBP peptide emulsion in rats,and both lymph node and spleen were isolated on day 12 after EAU induction.In order to measure rno-miR-30b-5p levels and IL-10,TLR4 expressions in spleen and lymph node,quantitative PCR and ELISA techniques were applied.Results　The results of double luciferase reporter gene expression analysis showed rno-miR-30b-5p mimic apparently down-regulated the fluorescence intensity of both IL-10 and TLR4 in wild type cells.After the mutation of the target site,the fluorescence intensity of the mutant vector was significantly reduced,accompanied by a significantly statistical difference (all P<0.01).Moreover,animal results revealed the expressions of rno-miR-30b-5p were apparently decreased,whereas IL-10 and TLR4 were markedly increased in both lymph node and spleen (all P<0.05).Conclusion　Target identification shows that rno-miR-30b-5p can obviously regulate the expressions of 3’UTR gene with either IL-10 or TLR4 gene fragment,though its regulation might not be through the predicted site.The down-regulated expression of rno-miR-30b-5p in both spleen and lymph node in EAU rats result in the up-regulated expressions of both IL-10 and TLR4,further influence the development of uveitis.This study paves a way for the modulation of microRNA on the occurrence and development of uveitis,and will provide a new insight on treating uveitis.

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备注/Memo

备注/Memo:
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）
更新日期/Last Update: 2017-04-26