Han Ning, Li Zengchun
Department of Emergency Trauma Surgery, Shanghai East Hospital of Tongji University, Shanghai, China.
Orthop Surg. 2021 May;13(3):1067-1076. doi: 10.1111/os.12834. Epub 2021 Mar 21.
To investigate the regulatory network of long non-coding RNA (lncRNA) as competing endogenous RNAs (ceRNAs) in osteonecrosis of the femoral head (ONFH).
The gene expression profile GSE74089 of ONFH and microRNA (miRNA) expression profile of GSE89587 were obtained from the Gene Expression Omnibus (GEO) database. The GSE74089 contained four ONFH samples and four controls. The GSE89587 included 10 ONFH samples and 10 control samples. The differentially expressed lncRNAs (DE-lncRNAs) and DE-mRNAs between ONFH group and control group were identified from GSE74089 using the limma package based on criteria of adjusted P value <0.05 and |log fold change (FC)| ≥2. The DEmiRNAs between ONFH group and control group were screened from GSE89587 on the basis of adjusted P value <0.05. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway for DE-mRNAs were analyzed using DAVID 6.7 and GSEA 3.0, respectively. Coexpressed lncRNA-mRNA pairs were identified by corr.test method in R based on the criteria of adjusted P value <0.01 and |r| ≥ 0.9. A ceRNA network was constructed and visualized using cytoscape 3.7.0 by integrating the DE-lncRNA, DE-miRNA, and DEmRNA data. The key mRNAs and lncRNAs in the ceRNA network were further validated in an independent dataset of GSE123568.
Based on our analysis, a total of 28 DE-lncRNAs, 1403 DE-mRNAs, and 134 DE-miRNAs were identified, respectively. The DE-mRNAs were significantly enriched in the function of "skeletal system development," "collagen fibril organization," "blood vessel development," and "regulation of nervous system development." Besides, 72 KEGG pathways, including eight active pathways and 64 suppressed pathways were identified, including which immune pathway was the most significantly activated one and which ribosome-related function was the most suppressed. A co-expression network including 161 DE-mRNAs and 16 DE-lncRNAs was built. Highly connected nodes were identified among lncRNAs such as H19, C20orf203, LINC00355, SFTA3, CRNDE, CASC2, LINC00494, C9orf163, C10orf91, and LINC00301. The ceRNA network indicated that lncRNA H19 functioned as a ceRNA of hsa-miR-519b-3p and hsa-miR-296-5p in ANKH and ECHDC1 regulation; lncRNA C9orf163 functioned as a ceRNA of hsa-miR-424-5p in CCNT1 regulation. The expression trends of ANKH, CCNT1, and C9orf163 were successfully validated in independent dataset of GSE123568.
The ceRNAs of lncRNA H19- hsa-miR-519b-3p/hsa-miR-296-5p-ANKH and lncRNA c9orf163- hsa-miR-424-5p-CCNT1 might play important roles in ONFH development. Our research provided an understanding of the important role of lncRNA-related ceRNAs in ONFH.
研究长链非编码RNA（lncRNA）作为竞争性内源性RNA（ceRNA）在股骨头坏死（ONFH）中的调控网络。
从基因表达综合数据库（GEO）获取ONFH的基因表达谱GSE74089和微小RNA（miRNA）表达谱GSE89587。GSE74089包含4个ONFH样本和4个对照样本。GSE89587包括10个ONFH样本和10个对照样本。使用limma软件包基于调整后P值<0.05和|log倍数变化（FC）|≥2的标准，从GSE74089中鉴定ONFH组和对照组之间差异表达的lncRNA（DE-lncRNA）和DE-mRNA。基于调整后P值<0.05，从GSE89587中筛选ONFH组和对照组之间的DEmiRNA。分别使用DAVID 6.7和GSEA 3.0对DE-mRNA进行基因本体（GO）和京都基因与基因组百科全书（KEGG）通路分析。基于调整后P值<0.01和|r|≥0.9的标准，通过R语言中的corr.test方法鉴定共表达的lncRNA-mRNA对。通过整合DE-lncRNA、DE-miRNA和DEmRNA数据，使用Cytoscape 3.7.0构建并可视化ceRNA网络。在独立数据集GSE123568中进一步验证ceRNA网络中的关键mRNA和lncRNA。
基于我们的分析，分别鉴定出28个DE-lncRNA、1403个DE-mRNA和134个DE-miRNA。DE-mRNA在“骨骼系统发育”“胶原纤维组织”“血管发育”和“神经系统发育调控”功能中显著富集。此外，鉴定出72条KEGG通路，包括8条激活通路和64条抑制通路，其中免疫通路激活最为显著，核糖体相关功能抑制最为明显。构建了一个包含161个DE-mRNA和16个DE-lncRNA的共表达网络。在lncRNA如H19、C20orf203、LINC00355、SFTA3、CRNDE、CASC2、LINC00494、C9orf163、C10orf91和LINC00301中鉴定出高度连接的节点。ceRNA网络表明lncRNA H19在ANKH和ECHDC1调控中作为hsa-miR-519b-3p和hsa-miR-296-5p的ceRNA发挥作用；lncRNA C9orf163在CCNT1调控中作为hsa-miR-424-5p的ceRNA发挥作用。ANKH、CCNT1和C9orf163的表达趋势在独立数据集GSE123568中得到成功验证。
lncRNA H19-hsa-miR-519b-3p/hsa-miR-296-5p-ANKH和lncRNA c9orf163-hsa-miR-424-5p-CCNT1的ceRNA可能在ONFH发展中起重要作用。我们的研究提供了对lncRNA相关ceRNA在ONFH中重要作用的认识。
