Identification of Key Candidate Genes Related to Inflammatory Osteolysis Associated with Vitamin E-Blended UHMWPE Debris of Orthopedic Implants by Integrated Bioinformatics Analysis and Experimental Confirmation

Figures & data

Figure 1 The distribution of expression of all samples before and after normalization.

Figure 2 (A) The distribution of expression of samples involving principal component analysis (PCA) for confirming biological variability between different samples; (B) 112 DEGs are shown in the volcano plot, containing 44 upregulated genes in red and 68 downregulated genes in green; (C) the heatmap shows the 112 most significant DEGs. Red indicates relatively high expression, and blue indicates relatively low expression. DEGs were identified by the criteria of|log₂fold change (FC)|> 2 and adj. p. val < 0.05.

Abbreviation: DEGs, differentially expressed genes.

Table 1 Top 20 Biological Processes of DEGs

Term	Description	Genes	Gene Counts	P-value
GO:0005515	Protein binding	PTGS2, RRAD, QARS, ANKRD1, SDC4, SLC1A5, SEMA7A, TGFBI, INSIG1, SLC2A1, USP12, FAM129A, DPP4, PTPRJ, ICAM1, NRIP3, EGR2, BST2, SOCS3, NPC1, SERPINF1, RPS14, PFDN5, TUBA4A, PEBP1, G0S2, TMSB4X, PPP1R15B, PPP1R15A, ARL4C, EMP1, CCL3, FGR, CXCL9, IFI30, RSAD2, FKBP1A, HSPA1A, HSPA1B, CCL5, CCL4, CCL7, TSC22D1, CCL20, METTL1, TAP1, NUMB, THBS1, GPNMB, PHLDA1, MAFB, IL1RN, DPYSL2, SPARC, STAT1, SLAMF1, PSMB8, SH3BP5, RASSF3, DUSP5, ATF4, NUP62, STAB1, ITGA5, UCP2, HOPX, CKS2, RAP1B, MERTK	69	0.003967971
GO:0005886	Plasma membrane	C3AR1, FGR, IFI30, RRAD, SDC4, GPC4, RGS10, SLC1A5, CLEC4E, SEMA7A, NUMB, SLC2A1, TGFBI, FAM129A, GPNMB, HCAR2, DPP4, ATP8B4, PTPRJ, ICAM1, C5AR1, IL2RA, BST2, SLCO4A1, IL1RN, SLAMF7, SPARC, ANKRD13A, RASSF3, ITGA5, STAB1, SERPINB2, RAP1B, MERTK, ARL4C, IL3RA, EMP1, GRASP	38	0.004446979
GO:0070062	Extracellular exosome	FGR, FKBP1A, SDC4, GPC4, SLC1A5, TGFBI, SLC2A1, IL1B, FAM129A, THBS1, DPP4, PTPRJ, ICAM1, BST2, IL1RN, MYO1D, DPYSL2, SLAMF1, CPVL, FUCA1, PSMB8, QPCT, NPC1, SERPINF1, RPS14, SLC37A2, PEBP1, TUBA4A, FABP4, RAP1B, CP, VSIG4, CTSF	33	1.62E-04
GO:0005615	Extracellular space	CCL1, CXCL1, ICAM1, CCL3, CXCL5, ARSG, CRTAP, IL1RN, CXCL9, SPARC, CCL5, CCL4, CCL7, GPC4, CCL22, SERPINF1, CCL20, SEMA7A, TGFBI, SERPINB2, IL1B, CP, MERTK, THBS1, CTSF	25	1.06E-06
GO:0005576	Extracellular region	CCL1, CXCL1, CCL3, CXCL5, OLFML2B, F13A1, CXCL9, IFI30, IL4I1, SPARC, CCL5, CCL4, CCL7, NPC1, CCL22, SERPINF1, CCL20, TGFBI, SERPINB2, TUBA4A, IL1B, TMSB4X, CP, THBS1	24	6.67E-05
GO:0006954	Inflammatory response	CCL1, CXCL1, C3AR1, CCL3, IL2RA, C5AR1, CXCL5, PTGS2, CXCL9, CCL5, CCL4, CD180, CCL7, CCL22, CCL20, STAB1, SEMA7A, IL1B, THBS1	19	2.87E-11
GO:0007165	Signal transduction	CCL1, CXCL1, C5AR1, CXCL5, CXCL9, RRAD, SPARC, DPYSL2, SDC4, CCL4, SLAMF1, CCL7, SH3BP5, RASSF3, NPC1, CCL22, CCL20, IL1B, GRASP	19	3.70E-04
GO:0005887	Integral component of plasma membrane	PTPRJ, ICAM1, C3AR1, C5AR1, BST2, SLCO4A1, SDC4, CD180, GPC4, SLC1A5, NPC1, STAB1, SLC2A1, TAP1, TM4SF1, MERTK, GPNMB	17	0.0098955
GO:0006955	Immune response	CXCL1, CCL3, IL2RA, C5AR1, CXCL5, IL1RN, CXCL9, CCL5, CCL4, CCL22, CCL20, SEMA7A, IL1B, THBS1	14	2.75E-06
GO:0005739	Mitochondrion	PDK4, RSAD2, QARS, HSPA1A, HSPA1B, SPARC, DPYSL2, SH3BP5, CYP27B1, SDS, RPS14, TAP1, G0S2, PPP1R15A	14	0.05425545
GO:0070374	Positive regulation of ERK1 and ERK2 cascade	CCL1, ICAM1, CCL22, CCL3, C5AR1, CCL20, SEMA7A, RAP1B, CCL5, GPNMB, SLAMF1, CCL4, CCL7	13	1.18E-09
GO:0007267	Cell-cell signaling	CCL22, CCL3, CXCL5, BST2, CCL20, STAB1, CXCL9, IL1B, CCL5, MERTK, CCL4, CCL7	12	6.58E-07
GO:0007186	G-protein coupled receptor signaling pathway	CXCL1, CCL1, C3AR1, CCL22, CCL3, CXCL5, CCL20, CXCL9, CCL5, HCAR2, CCL4, CCL7	12	0.028053381
GO:0071356	Cellular response to tumor necrosis factor	CCL1, ICAM1, CCL22, CCL3, CCL20, FABP4, ANKRD1, THBS1, CCL5, CCL4, CCL7	11	1.99E-09
GO:0006935	Chemotaxis	CXCL1, CCL1, C3AR1, CCL22, CCL3, C5AR1, CXCL5, CCL20, CXCL9, CCL5, CCL7	11	5.48E-09
GO:0043547	Positive regulation of GTPase activity	CCL1, ICAM1, RGS10, CCL22, CCL3, IL2RA, CCL20, CCL5, CCL4, CCL7, IL3RA	11	0.00324408
GO:0070098	Chemokine-mediated signaling pathway	CXCL1, CCL1, CCL22, CCL3, CXCL5, CCL20, CXCL9, CCL5, CCL4, CCL7	10	6.60E-10
GO:0008283	Cell proliferation	CXCL1, GPC4, IL2RA, BST2, SERPINF1, TGFBI, INSIG1, CKS2, RAP1B, EMP1	10	5.52E-04
GO:0009986	Cell surface	PTPRJ, ICAM1, C5AR1, BST2, ITGA5, SPARC, THBS1, SDC4, SLAMF1, DPP4	10	0.005226755
GO:0008009	Chemokine activity	CXCL1, CCL1, CCL22, CCL3, CXCL5, CCL20, CXCL9, CCL5, CCL4, CCL7	10	1.50E-11

Abbreviation: DEGs, differentially expressed genes.

Figure 3 GO including biological process (A), cellular components (B) and molecular function (C) analysis, and KEGG (D) enrichment analysis of the DEGs between the VE-UHMWPE group and the UHMWPE group.

Abbreviations: DEGs, differentially expressed genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4 Protein–protein interaction (PPI) network analysis. (A) The PPI network of DEGs visualized in Cytoscape. Red indicates upregulated genes, and green indicates the downregulated genes. (B) Four significant modules in the PPI network.

Figure 5 The biological process analysis of the top 20 DEGs was constructed using BiNGO. The color depth of nodes refers to the corrected p-value of ontologies. The size of nodes refers to the numbers of genes that are involved in the ontologies. P < 0.01 was considered statistically significant.

Table 2 List of the Top 10 Hub Genes Selected by MCC, MNC and Degree Methods in cytoHubba

MCC	MNC	Degree
IL1B	IL1B	IL1B
CXCL1	CXCL1	CXCL1
CCL20	ICAM1	ICAM1
CCL4	CCL5	CCL5
CXCL5	CCL4	CCL4
CXCL9	STAT1	STAT1
CCL5	CCL20	C3AR1
C3AR1	CXCL9	CCL20
HCAR2	CXCL5	CXCL9
C5AR1	C3AR1	PTGS2

Abbreviations: MCC, maximal clique centrality; MNC, maximum neighborhood component.

Figure 6 Identification of the hub genes. (A) Hub genes were identified by the overlap of the three methods in CytoHubba; (B) the selected hub genes were all included in the most significant module.

Figure 7 The network of TF-DEG was obtained from the ENCODE database.

Abbreviations ENCODE, Encyclopedia of DNA Elements; TF, transcription factor; DEG, differentially expressed gene.

Figure 8 The network of DEG-miRNA was obtained from the tarbase, miRTarBase, and miRecords databases.

Abbreviations: DEG, differentially expressed gene; miRNA, microRNA.

Table 3 Co-DEGs Regulated by TF and miRNAs

TF	DEGS	Gene Counts
RELA	Socs3, Ptgs2, Icam1, Cxcl1, Ccl5, Ccl20	5
NFKB1	Socs3, Ptgs2, Icam1, Cxcl1, Ccl5, Il1rn	5
SP1	Ptgs2, Icam1, Cxcl1, Ccl20	4
REL	Ptgs2, Icam1, Cxcl1, Ccl5	4
JUN	Ptgs2, Icam1, Cxcl1, Ccl5	4
TP53	Ptgs2, Icam1, Thbs1	3
SPI1	Icam1, Ccl5, Il1rn	3
5915	Ptgs2, Icam1, Il1rn	3
NFKB2	Ptgs2, Icam1, Ccl5	3
CTCF	Ptgs2, Thbs1, Ccl20	3
CREB1	Ptgs2, Icam1, Ccl5	3
CEBPB	Ptgs2, Icam1, Ccl5	3
STAT1	Socs3, Icam1	2
USF1	Ptgs2, Thbs1	2
STAT5A	Socs3, Il1rn	2
SRF	Ptgs2, Thbs1	2
MYC	Icam1, Ccl20	2
FOS	Ptgs2, Ccl5	2
EGR1	Ccl5, Thbs1	2
CEBPD	Ptgs2, Ccl5	2
CEBPA	Ptgs2, Ccl5	2
ATF1	Ptgs2, Ccl5	2
STAT3	Socs3	1
TFAP2A	Ptgs2	1
STAT2	Socs3	1
STAT5B	Socs3	1
IRF5	Ccl5	1
miRNA	DEGS	Gene counts
hsa-miR-144	Ptgs2, Thbs1, Ccl20	3
hsa-miR-21	Icam1, Thbs1, Ccl20	3
hsa-miR-221	Socs3, Icam1, Thbs1	3
hsa-let-7b	Ptgs2, Thbs1	2
hsa-miR-17	Icam1, Thbs1	2
hsa-miR-98	Ccl5, Thbs1	2
hsa-let-7a	Thbs1	1
hsa-miR-203	Socs3	1
hsa-miR-30e	Socs3	1
hsa-miR-590-5p	Ccl20	1

Abbreviations: DEGs, differentially expressed genes; miRNAs, microRNAs.

Figure 9 Integrative regulatory network of TF-DEG-miRNA.

Abbreviations: TF, transcription factor; DEG, differentially expressed gene; miRNA, microRNA.

Figure 10 Macrophage intervention, qRT-PCR and ELISA. (A) Macrophage-engulfed VE-UHMWPE particles observed under a transmission electron microscope (3000× magnification); (B) macrophage-engulfed VE-UHMWPE particles observed under transmission electron microscope (15,000× magnification); (C) comparison of the top five hub genes with high connectivity degree by qRT-PCR and (D) comparison of inflammatory factors in supernatant by ELISA. *p-value < 0.05, **p-value < 0.01 and ***p-value < 0.001.