Illumina高通量测序技术研究肌少-骨质疏松症与骨质疏松症患者骨组织microRNAs差异表达谱及差异分析

The differential expression profile and difference analysis of microRNAs in bone tissue of patients with sarco-osteoporosis and osteoporosis based on Illumina high-throughput sequencing technology

目的采用高通量测序技术筛选“肌少-骨质疏松症”(sarco-osteoporosis,SO)与骨质疏松症(osteoporosis,OP)患者骨组织标本中miRNA差异表达谱,并对差异显著的miRNA进行生物信息分析。方法运用Illumina Hiseq 2500测序技术筛查2017年至2019年期间福建省老年医院SO组与OP组患者的6对骨组织样品中miRNA表达谱;差异显著表达的miRNA运用层次聚类图和火山图分析,并结合基因本体论(GO)富集分析、京都基因与基因组百科全书(KEGG)信号通路分析探讨miRNA参与成肌细胞、成骨细胞增殖和分化的生物过程。结果SO组与OP组进行组间对比,12份骨组织中共鉴定到3064个(P<0.05;log2 Fold Change>0.0)差异表达的miRNAs,其中上调的1432个,下调的1632个,筛选出其中差异表达显著的22个miRNAs︱log2 Fold Change︱≥2且P<0.05包括15个在SO组骨组织中表达上调的miRNAs和7个表达下调的miRNAs;层次聚类图和火山图分析提示,SO和OP骨组织中的miRNA差异表达谱具有统计学意义;GO分析结果显示,miRNA的靶基因富集于成肌细胞与成骨细胞的生物过程,还参与细胞的氧化代谢、增殖和分化以及凋亡等生物过程。KEGG通路分析显示,hsa-miR-382-3p、hsa-miR-27a-5p、hsa-miR-1226-5p、hsa-miR-3934-5p和hsa-miR-451a的靶基因集合显著富集于NF-κB、Wnt、MAPK和P53等信号通路中。结论Illumina高通量测序技术能有效筛查出肌少-骨质疏松症骨组织中差异表达的miRNA;通过分析5个差异显著基因的下游靶基因,并结合分子生物学相关数据库与查阅大量文献发现这些差异miRNA可能参与肌少-骨质疏松症疾病状态下成骨细胞和成肌细胞多种生物活性过程。这一研究结果为后续深入研究miRNA及其靶基因调控机体骨髓间充质干细胞增殖与成骨分化的机制提供了新思路和理论依据。

Objective high-throughput sequencing technology was used to screen the differential expression profiles of miRNAs in bone tissue specimens from sarco-osteoporosis(SO)and osteoporosis(OP)patients,and to perform biological analysis on the significantly different miRNAs.Methods The Illumina Hiseq 2500 sequencing technology was used to screen the miRNA expression profiles of 6 pairs of bone tissue samples of patients in the SO group and OP group in Fujian Provincial Geriatric Hospital from 2017 to 2019.miRNAs with significantly different expression were analyzed with hierarchical clustering diagram and volcano Map analysis,combined with gene ontology(GO)enrichment analysis,Kyoto Encyclopedia of Genes,and Genomes(KEGG)signaling pathway analysis to explore the biological process of miRNA involved in the proliferation and differentiation of myoblasts and osteoblasts.Results The SO group was compared with the OP group.A total of 3064(P<0.05;log2 Fold Change>0.0)differentially expressed miRNAs were identified in 12 bone tissues.Among them,1432 were up-regulated and 1632 were down-regulated.The 22 miRNAs with significant differential expression(︱log2(fold change)︱≥2 and P<0.05)included 15 miRNAs with up-regulated expression in the bone tissue of SO group and 7 miRNAs with down-regulated expression.Hierarchical cluster diagram and volcano diagram analysis suggested that the differential expression profile of miRNAs in SO and OP bone tissues was statistically significant.GO analysis result showed that miRNA target genes were enriched in the biological processes of myoblasts and osteoblasts,and were also involved in biological processes such as cell oxidative metabolism,proliferation and differentiation,and apoptosis.KEGG pathway analysis showed that the target genes hsa-miR-382-3p,hsa-miR-27a-5p,hsa-miR-1226-5p,hsa-miR-3934-5p,and hsa-miR-451a were significantly enriched in NF-κB,Wnt,MAPK,and P53 signaling pathways.Conclusion Illumina high-throughput sequencing technology effectively screens the differentially expressed miRNAs in bone tissues of SO.By analyzing the downstream target genes of 5 significantly different genes,combining molecular biology related databases,and consulting a large number of literature,it is found that these differential miRNAs may be involved in various biological activities of osteoblasts and myoblasts in the condition of SO.This research result provides a new idea and theoretical basis for the follow-up in-depth study of the mechanism of the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells regulated by miRNA and its target genes.