原发性骨性关节炎膝关节软骨细胞差异表达circRNA、miRNA筛选和生物学功能及其相互作用分析
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摘要
目的筛选原发性骨性关节炎(OA)膝关节软骨细胞中差异表达的环状RNA(circRNA)及miRNA,并分析其生物学功能、相关代谢通路及相互作用。方法首先用Gene Spring software V13. 0软件筛选出5例OA及5例正常对照的膝关节软骨细胞差异表达的CircRNA,然后对差异表达的CircRNA进行基因本体论(GO)及代谢通路分析(采用KEGG通路数据库);将OA和正常对照膝软骨细胞的miRNA原始数据(下载自Array Express,获取号为:EMTAB-3514)进行对数转换,然后使用Quantile方法对数据进行标准化处理,以P <0. 05,OA和正常对照膝软骨细胞的miRNA差异倍数≥2或≤0. 5为筛选条件,用R包limma中的经验贝叶斯模型(e Bayes)筛选差异表达的miRNA;用miRror工具进行差异miRNA的靶基因预测,使用KOBAS软件对找到的靶基因进行GO及代谢通路注释富集分析。用miRanda软件对0A膝软骨细胞差异表达的miRNA进行miRNA-circRNA相互作用预测分析。结果与正常软骨细胞比较,OA膝关节软骨细胞中共筛选出1 380个表达差异的circRNA,其中215个差异circRNA表达上调,1 165个表达下调。分子功能层面,差异表达的circRNA主要影响GTP酶激活剂活性、腺苷核糖核苷酸结合、钙通道调节剂活性;细胞组成层面,差异表达的circRNA主要影响细胞外基质成分、带状胶原纤维、纤维状胶原蛋白三聚体;生物学过程层面,差异表达的circRNA主要影响细胞成分形态发生、软骨细胞发育和肢体发育。差异表达的circRNA相关代谢通路与糖原生物合成、补体和凝血级联、PDGF信号途径、胶原纤维组装和其他多聚体结构有关。与正常软骨细胞比较,OA膝关节软骨细胞中共筛选出差异表达miRNA 24个,其中上调表达及下调表达的miRNA各12个。生物学过程层面,差异表达的miRNA主要参与含核碱基的化合物生物合成过程、细胞质翻译终止、神经生长和细胞发育。细胞组成层面,主要影响细胞内部分、翻译释放因子复合体、膜结合的细胞器、核转录抑制因子复合物;在细胞组成层面,差异表达的miRNA主要影响有机环状化合物结合、核酸结合和杂环化合物结合;差异表达的miRNA主要与蛋白质泛素化、白细胞跨内皮迁移、泛素蛋白酶体途径、基因表达和蛋白质代谢有关。与has-miR-762相关的circRNA有71个,与has-miR-18a-3p相关的circRNA有25个,与has-miR-136-5p相关的circRNA有5个,与has-miR-3131相关的circRNA有17个,与has-miR-3189-3p相关的circRNA有29个。结论 OA膝关节软骨细胞中差异表达的circRNA有1 380个,其中215个上调表达,1 165个下调表达,差异表达的circRNA主要影响GTP酶激活剂活性、腺苷核糖核苷酸结合等分子功能,细胞外基质成分、带状胶原纤维等细胞成分,糖原生物合成、补体和凝血级联等相关代谢通路。OA膝关节软骨细胞差异表达miRNA 24个,其中上、下调表达的miRNA各12个,差异表达的miRNA主要参与含核碱基的化合物生物合成过程、细胞质翻译终止等分子功能,细胞内部分、翻译释放因子复合体等细胞成分,蛋白质泛素化、白细胞跨内皮迁移等相关代谢通路。circRNA和miRNA共同通路有Cell adhesion molecules pathway、Circadian entrainment pathway; circRNA-miRNA相互作用网络中,has-miR-762、has-miR-18a-3p、has-miR-136-5p与circRNA关系最为密切。
Objective To screen out the differentially expressed circRNA and miRNA in knee chondrocytes of primary osteoarthritis( OA),and to analyze their biological functions,metabolic pathways,and interactions. Methods Firstly,Gene Spring software V13. 0 was used to screen out the circRNA differential gene expression profiles in knee cartilage of 5 patients with OA and 5 normal controls,and we performed GO( Gene Ontology) and KEGG( Kyoto Encyclopedia of Genes and Genomes) pathway analysis on differentially expressed circRNA. The miRNA raw data of OA cartilage and normal control cartilage was subjected to logarithmic transformation and normalized by using the Quantile method( original data was downloaded from Array Express,accession number: E-MTAB-3514). The empirical Bayes model( e Bayes) in R-packed limma was used to analyze the differences of microRNAs between OA and normal control cartilage under the screening conditions of P < 0. 05,fold change ≥2 or ≤0. 5. The target genes of differential miRNAs were predicted by using the miRror tool. For the target gene,GO and KEGG enrichment were performed by using KOBAS software. The predictive analysis of the interaction between microRNAs and circRNAs were performed by using miRanda software. Results There were 1 380 circRNAs differentially expressed in OA cartilages and normal controls,of which 215 were up-regulated and 1 165 were down-regulated. In terms of molecular function,the differentially expressed circRNAs mainly affected GTPase activator activity,adenosine ribonucleotide binding,and calcium channel modulator activity; in terms of cell component,the differentially expressed circRNAs mainly affected the composition of extracellular matrix,ribbon collagen fibers and fibrous collagen trimer; in terms of biological processes,the differentially expressed circRNAs mainly affected cell component morphogenesis,chondrocyte development and limb development. The differentially expressed circRNA-related metabolic pathways were involved in glycogen biosynthesis,complement and coagulation cascades,PDGF signaling pathways,collagen fiber assembly and other multimeric structures. Compared with the normal chondrocytes,24 differentially expressed miRNAs were screened out in OA cartilages,of which 12 were up-regulated and 12 down-regulated. In terms of biological process,the differentially expressed miRNAs were mainly involved in the biosynthesis of compounds containing nuclear bases,the termination of cytoplasmic translation,nerve growth and cell development; in terms of cell component,it mainly affected intracellular parts,translational release factor complexes,membrane-bound organelles,and nuclear transcription inhibitory factor complexes; in molecular function,the differentially expressed miRNAs mainly affected the binding of organic cyclic compounds,nucleic acids and heterocyclic compounds. The differentially expressed miRNAs were mainly involved in protein ubiquitination,leukocyte transendothelial migration,ubiquitin proteasome pathway,gene expression and protein metabolism. Seventy-one circRNAs were related to has-miRNA-762,25 circRNAs were related to has-miRNA-18 a-3 p,5 circRNAs were related to has-miRNA-136-5 p,17 circRNAs were related to has-miRNA-3131,and 29 circRNAs were related to has-miRNA-3189-3 p. Conclusions There are 1 380 differentially expressed circRNAs in OA cartilages,of which 215 are up-regulated and 1 165 are down-regulated. Differentially expressed circRNAs mainly affect GTPase activator activity,adenosine ribonucleotide binding,extracellular matrix component,ribbon collagen fiber,glycogen biosynthesis,complement and coagulation cascades and so on-related metabolic pathways. Twenty-four differentially expressed miRNAs are screened out in OA knee chondrocytes,12 of which are up-regulated and 12 are down-regulated. The differentially expressed miRNAs are mainly involved in the biosynthesis of compounds containing nucleotides,termination of cytoplasmic translation,translation release factor complexes,ubiquitination of proteins,transendothelial migration of leukocytes. The common pathways enriched by the two are the cell adhesion molecules pathway and the circadian entrainment pathway,among the circRNA-miRNA interaction networks,has-miR-762,has-miR-18 a-3 p,and has-miR-136-5 p are most closely related to circRNA.
Objective To screen out the differentially expressed circRNA and miRNA in knee chondrocytes of primary osteoarthritis( OA),and to analyze their biological functions,metabolic pathways,and interactions. Methods Firstly,Gene Spring software V13. 0 was used to screen out the circRNA differential gene expression profiles in knee cartilage of 5 patients with OA and 5 normal controls,and we performed GO( Gene Ontology) and KEGG( Kyoto Encyclopedia of Genes and Genomes) pathway analysis on differentially expressed circRNA. The miRNA raw data of OA cartilage and normal control cartilage was subjected to logarithmic transformation and normalized by using the Quantile method( original data was downloaded from Array Express,accession number: E-MTAB-3514). The empirical Bayes model( e Bayes) in R-packed limma was used to analyze the differences of microRNAs between OA and normal control cartilage under the screening conditions of P < 0. 05,fold change ≥2 or ≤0. 5. The target genes of differential miRNAs were predicted by using the miRror tool. For the target gene,GO and KEGG enrichment were performed by using KOBAS software. The predictive analysis of the interaction between microRNAs and circRNAs were performed by using miRanda software. Results There were 1 380 circRNAs differentially expressed in OA cartilages and normal controls,of which 215 were up-regulated and 1 165 were down-regulated. In terms of molecular function,the differentially expressed circRNAs mainly affected GTPase activator activity,adenosine ribonucleotide binding,and calcium channel modulator activity; in terms of cell component,the differentially expressed circRNAs mainly affected the composition of extracellular matrix,ribbon collagen fibers and fibrous collagen trimer; in terms of biological processes,the differentially expressed circRNAs mainly affected cell component morphogenesis,chondrocyte development and limb development. The differentially expressed circRNA-related metabolic pathways were involved in glycogen biosynthesis,complement and coagulation cascades,PDGF signaling pathways,collagen fiber assembly and other multimeric structures. Compared with the normal chondrocytes,24 differentially expressed miRNAs were screened out in OA cartilages,of which 12 were up-regulated and 12 down-regulated. In terms of biological process,the differentially expressed miRNAs were mainly involved in the biosynthesis of compounds containing nuclear bases,the termination of cytoplasmic translation,nerve growth and cell development; in terms of cell component,it mainly affected intracellular parts,translational release factor complexes,membrane-bound organelles,and nuclear transcription inhibitory factor complexes; in molecular function,the differentially expressed miRNAs mainly affected the binding of organic cyclic compounds,nucleic acids and heterocyclic compounds. The differentially expressed miRNAs were mainly involved in protein ubiquitination,leukocyte transendothelial migration,ubiquitin proteasome pathway,gene expression and protein metabolism. Seventy-one circRNAs were related to has-miRNA-762,25 circRNAs were related to has-miRNA-18 a-3 p,5 circRNAs were related to has-miRNA-136-5 p,17 circRNAs were related to has-miRNA-3131,and 29 circRNAs were related to has-miRNA-3189-3 p. Conclusions There are 1 380 differentially expressed circRNAs in OA cartilages,of which 215 are up-regulated and 1 165 are down-regulated. Differentially expressed circRNAs mainly affect GTPase activator activity,adenosine ribonucleotide binding,extracellular matrix component,ribbon collagen fiber,glycogen biosynthesis,complement and coagulation cascades and so on-related metabolic pathways. Twenty-four differentially expressed miRNAs are screened out in OA knee chondrocytes,12 of which are up-regulated and 12 are down-regulated. The differentially expressed miRNAs are mainly involved in the biosynthesis of compounds containing nucleotides,termination of cytoplasmic translation,translation release factor complexes,ubiquitination of proteins,transendothelial migration of leukocytes. The common pathways enriched by the two are the cell adhesion molecules pathway and the circadian entrainment pathway,among the circRNA-miRNA interaction networks,has-miR-762,has-miR-18 a-3 p,and has-miR-136-5 p are most closely related to circRNA.
引文
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[12]Miyaki S,Sato T,Inoue A,et al. MicroRNA-140 plays dual roles in both cartilage development and homeostasis[J]. Genes Dev,2010,24(11):1173-1185.
[13]Dudek M,Meng QJ. Running on time:the role of circadian clocks in the musculoskeletal system[J]. Biochem J,2014,463(1):1-8.
[14]Schett G,Kiechl S,Bonora E,et al. Vascular cell adhesion molecule 1 as a predictor of severe osteoarthritis of the hip and knee joints[J]. Arthritis Rheum,2009,60(8):2381-2389.
[15] Kalichman L,Pantsulaia I,Kobyliansky E. Association between vascular cell adhesion molecule 1 and radiographic hand osteoarthritis[J]. Clin Exp Rheumatol,2011,29(3):544-546.
[16]Karatay S,Kiziltunc A,Yildirim K,et al. Effects of different hyaluronic acid products on synovial fluid levels of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in knee osteoarthritis[J]. Ann Clin Lab Sci,2004,34(3):330-335.
[17]张申华,戴婷,宋成文. miR-762对卵巢癌细胞增殖的影响[J].华南国防医学杂志,2017,31(9):582-585.
[2]Li HZ,Lin Z,Xu XH,et al. The potential roles of circRNAs in osteoarthritis:a coming journey to find a treasure[J]. Biosci Rep,2018,38(5):pii:BSR20180542.
[3]Lasda E,R Parker. Circular RNAs:diversity of form and function[J]. Rna,2014,20(12):1829-1842.
[4]Hansen TB,Jensen T,Clausen BH,et al. Natural RNA circles function as efficient microRNA sponges[J]. Nature,2013,495(7441):384-388.
[5]Zhou Z,Du D,Chen A,et al. Circular RNA expression profile of articular chondrocytes in an IL-1beta-induced mouse model of osteoarthritis[J]. Gene,2018,644:20-26.
[6]Yi R,Fuchs E. MicroRNAs and their roles in mammalian stem cells[J]. J Cell Sci,2011,124(Pt11):1775-1783.
[7]Yu C,Chen WP,Wang XH. MicroRNA in osteoarthritis[J]. J Int Med Res,2011,39(1):1-9.
[8]Wu C,Tian B,Qu X,et al. MicroRNAs play a role in chondrogenesis and osteoarthritis(review)[J]. Int J Mol Med,2014,34(1):13-23.
[9]Sondag GR,Haqqi TM. The role of micrornas and their targets in osteoarthritis[J]. Curr Rheumatol Rep,2016,18(8):56.
[10]Cong L,Zhu Y,Tu G. A bioinformatic analysis of microRNAs role in osteoarthritis[J]. Osteoarthritis Cartilage,2017,25(8):1362-1371.
[11]Jones SW,Watkins G,Le Good N,et al. The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13[J]. Osteoarthritis Cartilage,2009,17(4):464-472.
[12]Miyaki S,Sato T,Inoue A,et al. MicroRNA-140 plays dual roles in both cartilage development and homeostasis[J]. Genes Dev,2010,24(11):1173-1185.
[13]Dudek M,Meng QJ. Running on time:the role of circadian clocks in the musculoskeletal system[J]. Biochem J,2014,463(1):1-8.
[14]Schett G,Kiechl S,Bonora E,et al. Vascular cell adhesion molecule 1 as a predictor of severe osteoarthritis of the hip and knee joints[J]. Arthritis Rheum,2009,60(8):2381-2389.
[15] Kalichman L,Pantsulaia I,Kobyliansky E. Association between vascular cell adhesion molecule 1 and radiographic hand osteoarthritis[J]. Clin Exp Rheumatol,2011,29(3):544-546.
[16]Karatay S,Kiziltunc A,Yildirim K,et al. Effects of different hyaluronic acid products on synovial fluid levels of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in knee osteoarthritis[J]. Ann Clin Lab Sci,2004,34(3):330-335.
[17]张申华,戴婷,宋成文. miR-762对卵巢癌细胞增殖的影响[J].华南国防医学杂志,2017,31(9):582-585.