Identification of DEGs
There were 10 normal tissues, 10 OA tissues and 10 RA tissues in our present study. From GEO2R online tools, we extracted 321 and 538 DEGs from OA and RA, respectively (Fig. 1). A total of 162 DEGs in common were detected, including 73 down-regulated genes (logFC <0) and 89 up-regulated genes (logFC >0) (Table. 1, Fig. 2)
Figure.1 Volcanic map of all genes.
Note: Red dots indicate up-regulated genes, blue dots indicate down-regulated genes and gray dots indicate genes that are not regulated. . a. OA. b. RA.
Table.1 Differentially expressed genes.
DEGs | Genes Name |
Up-regulated | CCDC102B///IGKV1OR2-108///IGHV3-23///IGHV4-31///IGHM///IGHG3///IGHG1///IGHD///IGHA1///IGH///CRIP1///ADAM12///IGHV4-31///IGHM///IGHG4///IGHG3///IGHG1///IGHD///IGHA2///IGHA1///IGHD///CCL5/// IGHM///RTN1///LILRB2///CECR///GPR65 ///HLA-DPA1///IGLJ3///TRAF3IP3///IGLJ3///IGLV1-44///CKAP2///IGLV@///IGLC1///FAP///SORL1///ABCC3///HLA-DMB///NREP///OSBPL3/// IGHV3-23///IGHV4-31///IGHM/// IGHG4///IGHG3///IGHG1///IGHD///IGHA2///IGHA1///IGH FAM198B ARHGAP25 RAC2 COL1A1 WNT5A CSF2RA///CALML4///IGKC///CLEC4A///TNFSF11///IGK///IKZF1/// ATM///IGLJ3///IGLV@/// IGLC1///NCF1C///NCF1B///NCF1/// SDC1///TLR7///TRBC1///LST1///LRRC15///POU2AF1/// TPPP3///BTN3A1///LOC100509457///HLA-DQA2///HLA-DQA1///IGLJ3///CKAP2///MXRA5///DOCK10///GAP43///IGHV3-23///IGHV4-31///IGHM///IGHG1///IGHA1/// XIST///CX3CR1///LOC101060835///HLA-DQB1///LOC102725526///IGHV4-31///IGHM///IGHG3///IGHG2///IGHG1///IGHA2///IGHA1///IGH///RRM2///TRIM14///NDC80/// IGLC1///CD52///TOP2A///IGHM///IGHG1///IGHA2///IGHA1///IGH///IGHV4-31///IGHM/// IGHG3///IGHG2///IGHG1///IGHD//////IGHA2///IGHA1///IGH///CYAT1///IGLV1-44///IGLC1///EPHB2///AIF1/// IGK///IGKC///COL1A2///IGHG1///APLNR///IGLL3P/// CIITA/// MMP1///MIR8071-2///MIR8071-1///IGHV4-31///IGHM///IGHG2///IGHG1///GUSBP11///PLXNC1///TNFRSF25///JCHAIN///KCNQ1///IGLV1-44///HK3///UCP2///LOC100506718///FLRT2///PTPRC///NKG7 |
Down-regulated | FKBP5///EIF1///ELL2///NPAS2///RRAD///ARC///MIR6732///ZC3H12A///LAMA2///FOSL2///NR1D1///THRA///PTX3///TIMP4///CEBPD///PPP1R15A/// JUND///FNDC4///ZFP36///BCL6///MIR1908/// FADS1///VEGFA///APOD///APOLD1///C10orf10///ANGPTL7///LOC100129518//////MYOC///MTSS1/// ADH1B///MYC///CDKN1A///LDLR///JUN///SOCS3///NR4A2///DUOX2///ZBTB16///SLC6A8///KLF9///ACADL/// CCNL1///CHST7///PMAIP1///LAMA3///CYR61/// NFIL3///MAFF///ADM///ADCY2///GADD45A///MT1M///FAM107A///AASS///CDH19///ATF3///PTGS2///DUSP1/// HSPA1L///HSPA1B///HSPA1A///PIGA///CYP4B1///CXCL8///EGR1//////FOSB///SLC19A2///GADD45B/// FOXO3B///FOXO3///MAOA///HSPA1B///HSPA1A///SAA2-SAA4///SAA2///SAA1///MIR6883///PER1///NR4A1///KLF4///TFPI |
Note: All 162 commonly differentially expressed genes (DEGs) were detected from profile datasets, includ ing 89 up-regulated genes and 73 down-regulated genes.
Fig.2. The common DEGs between OA and normal tissues, RA and normal tissues.
Note: Different color meant different datasets. The common DEGs between OA and normal tissues, RA and normal tissues in GSE55235 were verified through Venn diagrams software(available online: http://bioinformatics. psb.ugent. be/webtools /Venn/ ). a 89 DEGs were up-regulated (logFC> 0). b 73 DEGs were down-regulated (logFC < 0).
DEGs gene ontology and KEGG pathway analysis
All DEGs of OA and RA were analyzed by DAVID software, and the GO analysis results were as follows 1) for BP, the DEGs of OA and RA were enriched in immune response and inflammatory response, OA DEGs were enriched in cellular response to fibroblast growth factor stimulus, while RA DEGs wereenriched in response to cAMP. 2) for GO CC, DEGs of OA and RA were enriched in extracellular region and integral component of plasma membrane, OA DEGs were enriched in extracellular space, and RA DEGs were enriched in external side of plasma membrane. 3) for MF, they were both enriched in immunoglobulin receptor binding, OA DEGs were enriched in antigen binding, transcription factor activity, and RNA polymerase II core promoter proximal region sequence-specific binding, while RA DEGs were enriched in chemokine activity and immunoglobulin receptor binding (Fig. 3).
KEGG analysis results demonstrated that OA and RA DEGs both enriched in Rheumatoid arthritis, Osteoclast differentiation. OA DEGs were particularly enriched in TNF signaling pathwaywhile RA DEGs were particularly enriched in Leishmaniasis (P <0.05) (Fig. 4).
Fig. 3 . KEGG pathway analysis of DEGs in osteoarthritis and Rheumatoid arthritis
Note: The top 3 biological processes, cellular component and molecular function analysis in DEGs. a. OA. b. RA.
Fig. 4 KEGG pathway analysis of DEGs in osteoarthritis and Rheumatoid arthritis
Note: The dot size indicates the counts of genes and the redder the color, the richer the gene. a. OA. b. RA.
Protein–protein interaction network (PPI) and modular analysis
The DEGs of OA and the DEGs of RA were respectively imported into the DEG PPI network complex. OA contained 190 nodes and 882 edges, including 141 down-regulated and 180 up-regulated genes . RA contains 319 nodes and 2194 edges, including 199 down-regulated and 339 up-regulated genes (Fig. 5). Then we applied Cytotype MCODE for further analysis, and the results showed that there were 23 central nodes in OA DEGs and 17 central nodes in RA DEGs (figure. 6).
Fig. 5 Common DEGs PPI network
- Note: Common DEGs PPI network constructed by STRING online database and Module analysis. The nodes meant proteins; the edges meant the interaction of proteins; blue circles meant down-regulated DEGs and red circles meant up-regulated DEGs.a. OA. b. RA.
Fig. 6 Module analysis of OA and RA
Note: Degree cutoff=2, node score cutoff=0.2, k-core= 2, and max. Depth= 100
- b. RA.
Re-analysis of selected genes via KEGG pathway enrichment
To understand the possible pathway of these selected DEGs, KEGG pathway enrichment was re-analyzed via DAVID (P <0.05). Results showed that twelve genes (CCL5, CXCL1, CXCL2, CXCL3, IL-1β, IL-6, NFKBIA, SOCS3, TNFAIP3, JUN, MMP9 and PTGS2 ) in osteoarthritis markedly enriched in the TNF signaling pathway ((P =1.24E-11, Table 2) and eleven genes (CCL5, CCR5, CXCL5, CXCL6, CXCL9, CXCL10, CXCL11, CXCL13, ADCY2, CX3CL1, CX3CR1) in rheumatoid arthritis markedly enriched in the Chemokine signaling pathway (P =4.39E-13,Table 3).
Table 2 Re-analysis of 23 genes for osteoarthritis via KEGG pathway enrichment
Pathyway ID | Name | Cout | % | P-value | Genes |
bta04668 | TNF signaling pathway | 10 | 0.278473962684489 | 1.24E-11 | IL6, PTGS2, SOCS3, MMP9, CXCL3, JUN, IL1B, NFKBIA, TNFAIP3, CCL5 |
bta05323 | Rheumatoid arthritis | 9 | 0.250626566 | 1.96E-10 | IL6, TNFSF11, JUN, VEGFA, IL1B, CXCL8, CCL5, CXCL12, MMP1 |
Table 3 Re-analysis of 17 genes for rheumatoid arthritis via KEGG pathway enrichment.
Pathyway ID | Name | Cout | % | P-value | Genes |
hsa04062 | Chemokine signaling pathway | 11 | 0.533462658 | 4.39E-13 | ADCY2, CXCL5, CCR5, CXCL13, CX3CR1, CXCL9, CXCL6, CX3CL1, CCL5, CXCL11, CXCL10 |
hsa04060 | Cytokine-cytokine receptor interaction | 10 | 0.484966052 | 3.08E-10 | CXCL5, CCR5, CXCL13, CX3CR1, CXCL9, CXCL6, CX3CL1, CCL5, CXCL11, CXCL10 |
Verify genes withGSE55457 and evaluated by ROC
The reliability of the above results is further verified by GSE55457. Results showed that three genes markedly enriched(JUN,VEGFA,HGF) in osteoarthritisand five genes (CCL5,CXCL9,CXCL10,CXCL11,CXCL13 ) markedly enriched in rheumatoid arthritis. The results of extracting the intersection of GSE55235 and GSE55457 core genes showed as JUN, CCL5, CXCL9,CXCL10,CXCL11,CXCL13. ROC curves were constructed by R software to calculate the AUCs of JUN (99.0%), CCL5 (88.5%), CXCL9 (97.7%), CXCL10 (95.4%),CXCL11 (96.6%) and CXCL13 (96.5%) (Fig. 7).
Figure 7. Receiver operating characteristic curves.
Note: Receiver operating characteristic curves of the six genes in the core network using data from the GSE55457. Receiver operating characteristic curves of JUN, CCL5, CXCL9, CXCL10, CXCL11, CXCL13 are presented.