miRNAs参与养血柔肝法治疗骨关节炎的生物信息学分析

Bioinformatics analysis of miRNAs in rat osteoarthritis with Yangxue Ruanjian capsule treatment

关键词:  miRNAs  骨关节炎  养血柔肝法  基因测序

Key words:  miRNA  osteoarthritis  nourishing blood and softening the liver  gene sequencing

摘要:

目的　分析miRNAs在养血柔肝法“养血软坚胶囊”治疗骨关节炎中的生物学信息。方法　将40只SD大鼠进行前交叉切除(ACLT)造模成骨关节炎模型，模型验证造模成功后，随机分为模型组（n=20）和中药组（n=20）。模型组不干预，中药组予大鼠灌胃饲养，药物采用养血柔肝法中成药养血软坚胶囊。4周后取材，提取关节软骨组织的RNA，制备文库，通过数据预处理、测序错误率分布检查、测序数据过滤、差异表达的miRNAs的鉴定、差异表达的miRNA的靶基因的鉴定、差异表达的miRNA的靶基因的功能分析、网络图构建等步骤，对差异表达的miRNAs进行数据分析。并RT-PCR验证所得结果。结果　中药组与模型组并共有35个miRNAs表达有差异。其中7个上调（共调控了1174个靶基因），28个下调（共调控了2216个靶基因）；对差异miRNAs的靶基因进行蛋白互作网络分析（PPI）后，通过连接度最高的几个节点，获得了19个重要的靶基因。关节软骨中，中药组rno-miR-199a-5p、rno-miR-140-3p的表达高于模型组，中药组rno-miR-300-3p、rno-miR-9a-3p的表达低于模型组，差异具有统计学意义（P<0.05）。结论　养血柔肝法中药“养血软坚胶囊”可以调控骨关节炎大鼠模型的miR-199a-5p, miR-140-3p, miR-300-3p和miR-9a-3p等miRNAs。

Abstract:

ObjectiveTo analyze miRNAs changes of rat osteoarthritis with the treatment of nourishing blood and softening the liver-“Yangxue Ruanjian capsule”.Methods Forty SD rats were randomly divided into control group (n=20) and Chinese medicine group (n=20). All rats were build osteoarthritis models with ACLT model method. After the verificaiton of these models, the rats in the Chinese medicine group were fed with “Yangxue Ruanjian capsule” which was based on mechanism of nourishing blood and liver therapy. After 4 weeks, RNA of articular cartilage was extracted. The library was prepared and the FASTX toolkit was used for data preprocessing. Bowtie tool was used to translate the filtered sequencing and sequence into the value matrix of RNA. The differential expression of miRanda’s target gene was identified by miRanda. DAVID tools were used in the GO enrichment analysis and Pathway GO enrichment analysis on differential expressed target gene. The results were verified by fluorescence quantitative PCR assay.ResultsThere were 35 differentially expressed miRNAs totally, of which 7 were up-regulated (1174 hub genes were regulated), 28 were down-regulated (2216 hub genes were regulated). In PPI network, by analyzing the different miRNAs, which regulated hub genes, 19 important hub genes were obtained. The miR-199a-5p, miR-140-3p, miR-300-3p, miR-9a-3p expression in the two groups had significant difference. The difference of miR-142-5p and miR-485-5p was not statistically different.Conclusion The nourishing blood and liver therapy, the traditional Chinese medicine “ Yangxue Ruanjian capsule” could play a role in the regulation of miR-199a-5p, miR-140-3p, miR-300-3p and miR-9a-3p.