Systematic Analysis of Transcriptomic Profile of Chondrocytes in Osteoarthritic Knee Using Next-Generation Sequencing and Bioinformatics
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. RNA Sequencing
2.3. Ingenuity Pathway Analysis (IPA)
2.4. Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources
2.5. Gene Expression Omnibus (GEO) Database
2.6. miRmap Database
2.7. Statistical Analysis
3. Results
3.1. The Sequencing Quality and Mapped Reads for RNA and Small RNA Sequencing of Chondrocytes
3.2. The Differentially Expressed Genes in Osteoarthritic Knee Chondrocytes were Associated with Osteoarthritis Pathway, Cell Adhesion and Extracellular Matrix Organization
3.3. Identification of Dysregulated Genes Related to Joint Structural Damage in OA
3.4. SMAD3 and WNT5A were Involved in Growth of Blood Vessel and Cell Aggregation
3.5. Identification of Differentially Expressed miRNAs and Potential miRNA–mRNA Interactions between Normal and OA Knee Chondrocytes
3.6. Analysis of Candidate Genes with Potential miRNA-mRNA Interactions in Gene Expression Omnibus (GEO) Database and Identification of Potential Molecular Signatures in OA Knee Joint Microenvironment
3.7. Identification of Potential miRNA-mRNA Interactions of LRRC15, MARCKS, and EREG in OA Knee Chondrocytes
3.8. MARCKS and EREG were Potentially Involved in the Pathogenesis of Arthritic Knee Joint Pain
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Category | No. of Genes | p-Value |
---|---|---|
Top canonical pathways | ||
Osteoarthritis Pathway | 23 | 4.72 × 10−10 |
Hepatic Fibrosis/Hepatic Stellate Cell Activation | 20 | 8.99 × 10−9 |
Axonal Guidance Signaling | 31 | 4.32 × 10−8 |
Atherosclerosis Signaling | 15 | 1.77 × 10−7 |
GP6 Signaling Pathway | 13 | 1.06 × 10−5 |
Top predicted upstream regulators | ||
TNF | 2.20 × 10−22 | |
TGFB1 | 1.01 × 10−21 | |
Dexamethasone | 1.57 × 10−16 | |
IFNG | 6.75 × 10−16 | |
CTNNB1 | 1.50 × 10−15 |
Biological Process | Count | p-Value | Up-Regulated Genes | Down-Regulated Genes | Fold Enrichment |
---|---|---|---|---|---|
Positive regulation of bone mineralization | 7 | 2.66 × 10−4 | BMP4, GPM6B, P2RX7, SMAD3, TMEM119 | BMP2, BMP6 | 7.62 |
Positive regulation of chondrocyte differentiation | 5 | 1.32 × 10−3 | ACVRL1, GDF6,SMAD3 | BMP6, SOX9 | 10.02 |
Positive regulation of cartilage development | 4 | 7.76 × 10−3 | BMP4, WNT5A | BMP2, SOX9 | 9.52 |
Chondrocyte differentiation | 5 | 1.86 × 10−2 | BMP4 | BMP2, COL11A2, CYTL1, FGFR3 | 4.88 |
Endochondral ossification | 4 | 2.97 × 10−2 | ALPL, BMP4 | BMP6, FGFR3 | 5.86 |
Gene Symbol | Gene Name | HC-OA FPKM | HC FPKM | Fold-Change (HC-OA/HC) |
---|---|---|---|---|
AKAP12 | A-kinase anchoring protein 12 | 94.69 | 32.02 | 2.96 |
BASP1 | brain abundant, membrane attached signal protein 1 | 80.88 | 38.09 | 2.12 |
BDNF | brain-derived neurotrophic factor | 8.28 | 1.76 | 4.70 |
FGF7 | fibroblast growth factor 7 | 37.21 | 8.52 | 4.37 |
GREM2 | gremlin 2, DAN family BMP antagonist | 9.66 | 3.54 | 2.73 |
LMOD1 | leiomodin 1 | 26.03 | 1.78 | 14.65 |
LRRC15 | leucine rich repeat containing 15 | 6.47 | 2.56 | 2.53 |
LRRC32 | leucine rich repeat containing 32 | 9.64 | 2.30 | 4.19 |
MARCKS | myristoylated alanine-rich protein kinase C substrate | 278.54 | 133.99 | 2.08 |
PCSK9 | proprotein convertase subtilisin/kexin type 9 | 1.51 | 0.53 | 2.86 |
PDE3A | phosphodiesterase 3A | 19.30 | 1.88 | 10.25 |
PDE7B | phosphodiesterase 7B | 4.98 | 1.53 | 3.27 |
PPM1L | protein phosphatase, Mg2+/Mn2+ dependent 1L | 4.44 | 0.97 | 4.59 |
RALGPS2 | Ral GEF with PH domain and SH3 binding motif 2 | 48.94 | 18.20 | 2.69 |
RGS5 | regulator of G-protein signaling 5 | 90.50 | 6.24 | 14.50 |
RNF152 | ring finger protein 152 | 15.71 | 3.83 | 4.10 |
RPP25 | ribonuclease P/MRP 25kDa subunit | 4.69 | 2.02 | 2.32 |
SESN3 | sestrin 3 | 14.57 | 6.95 | 2.10 |
SLIT3 | slit guidance ligand 3 | 29.08 | 2.46 | 11.80 |
SMAD3 | SMAD family member 3 | 408.29 | 166.01 | 2.46 |
TFPI | tissue factor pathway inhibitor | 56.10 | 8.72 | 6.43 |
THSD4 | thrombospondin type 1 domain containing 4 | 3.44 | 0.65 | 5.31 |
KIAA1644 | KIAA1644 | 22.55 | 61.04 | 0.37 |
SEMA3A | semaphorin 3A | 12.52 | 39.96 | 0.31 |
EREG | epiregulin | 1.50 | 4.65 | 0.32 |
SDK2 | sidekick cell adhesion molecule 2 | 2.04 | 15.99 | 0.13 |
GEO Accession Number | GSE114007 | GSE51588 | GSE55235 | GSE55457 | |
---|---|---|---|---|---|
Specimen | Cartilage | Subchondral bone | Synovial tissue | ||
Normal/OA | Normal/OA | Normal/OA | Normal/OA | ||
Medial | Lateral | ||||
Numbers | 18/20 | 5/20 | 5/20 | 10/10 | 10/10 |
Up-Regulated mRNA * | |||||
AKAP12 | DOWN | n.s. † | n.s. | n.s. | n.s. |
BASP1 | UP | n.s. | DOWN | n.s. | n.s. |
BDNF | UP | n.s. | n.s. | n.s. | n.s. |
FGF7 | UP | n.s. | n.s.† | n.s. | n.s. |
GREM2 | n.s. | n.s. | n.s. | n.s. | n.s. |
LMOD1 | n.s. | n.s. | n.s. | DOWN | n.s. |
LRRC15 | UP | UP | n.s. | UP | UP |
LRRC32 | n.s. | n.s. | n.s. | n.s. | n.s. |
MARCKS | UP | UP | UP | UP | n.s. † |
PCSK9 | UP | UP | n.s. | -- | -- |
PDE3A | UP | UP | UP | n.s. | n.s. |
PDE7B | n.s. | n.s. | n.s. | DOWN | n.s. |
PPM1L | n.s. | n.s. | n.s. | -- | -- |
RALGPS2 | n.s. | n.s. | n.s. | n.s. | n.s. |
RGS5 | n.s. | n.s. | n.s. | n.s. | n.s. |
RNF152 | n.s. | n.s. | n.s. | -- | -- |
RPP25 | n.s. | UP | n.s. | n.s. | n.s. |
SESN3 | n.s. | n.s. | n.s. | -- | -- |
SLIT3 | n.s. | UP | n.s. † | n.s. † | n.s. † |
SMAD3 | DOWN | UP | UP | n.s. † | n.s. † |
TFPI | n.s. | n.s. † | n.s. | DOWN | n.s. |
THSD4 | DOWN | UP | UP | n.s. | n.s. |
Down-Regulated mRNA * | |||||
KIAA1644 | UP | n.s. † | n.s. | UP | n.s. |
SEMA3A | n.s. | n.s. | n.s. | UP | n.s. |
EREG | n.s. | DOWN | DOWN | DOWN | n.s. |
SDK2 | n.s. | UP | UP | n.s. | n.s. |
Down-Regulated miRNA | Fold-Change | Predicted Target Up-Regulated mRNA | miRmap Score | TargetScan | miRDB |
hsa-miR-140-5p | −3.11 | LRRC15 | 99.03 | − | − |
hsa-miR-140-3p | −2.87 | MARCKS | 99.27 | + | + |
hsa-miR-495-3p | −4.80 | PDE3A | 99.93 | − | + |
Up-Regulated miRNA | Fold-Change | Predicted Target Down-Regulated mRNA | miRmap Score | TargetScan | miRDB |
hsa-miR-301a-3p | 2.45 | EREG | 99.06 | + | + |
Biological Process | p-Value | Related Genes | Fold Enrichment |
---|---|---|---|
Negative regulation of TORC1 signaling | 0.013 | RNF152, SESN3 | 149.26 |
Cytokine-mediated signaling pathway | 0.015 | EREG, LRRC15, GREM2 | 15.38 |
Axon extension involved in axon guidance | 0.017 | SEMA3A, SLIT3 | 111.95 |
cAMP catabolic process | 0.021 | PDE7B, PDE3A | 89.56 |
Axon guidance | 0.021 | BDNF, SEMA3A, SLIT3 | 12.67 |
Oocyte maturation | 0.025 | EREG, PDE3A | 74.63 |
Positive regulation of GTPase activity | 0.045 | RALGPS2, FGF7, EREG, RGS5 | 4.76 |
Negative chemotaxis | 0.048 | SEMA3A, SLIT3 | 39.51 |
MAPK cascade | 0.053 | FGF7, EREG, PPM1L | 7.69 |
Positive regulation of cell division | 0.065 | FGF7, EREG | 28.58 |
Synapse assembly | 0.084 | BDNF, SDK2 | 22.02 |
Top Diseases and Functions | Score | Focus Molecules | Molecules in Network | |
---|---|---|---|---|
1 | Cellular Movement, Cardiovascular System Development and Function, Organismal Development | 32 | 13 | ↑AKAP12, Akt, Ap1, ↑BDNF, Calmodulin, Creb, ↓EREG, ERK,ERK1/2, estrogen receptor, F Actin, ↑FGF7, Fibrinogen, FSH, Gsk3, Histone h3, LDL, Lh, ↑LRRC32, Mapk, ↑MARCKS, ↑PCSK9, PDGF BB, PI3K (complex), Pka, Pkc(s), Proinsulin, Ras, ↑RGS5, ↓SEMA3A, ↑SESN3, ↑SLIT3, ↑SMAD3, ↑TFPI, Vegf |
2 | Cardiovascular Disease, Organismal Injury and Abnormalities, Cardiovascular System Development and Function | 15 | 7 | 26s Proteasome, BMP2, CLU, F10, G protein alphai, GAS6, ↑GREM2, HNF4A, Jnk, LPA, LRPAP1, ↑LRRC15, MAGI3, MAP2K5, MAP3K, MAPK9, mir-25, mir-181, MMP9, MMP12, MZB1, Neurotrophin, NFkB (complex), NRG (family), P38 MAPK, PP1/PP2A, Pp2c, ↑PPM1L, PTPN13, ↑RALGPS2, ↑RNF152, SAA, ↓SDK2, ↑THSD4, tyrosine kinase |
3 | Gastrointestinal Disease, Hepatic System Disease, Organismal Injury and Abnormalities | 12 | 6 | ABCC4, AR, ↑BASP1, C2CD5, CENPE, CENPH, CEPT1, EGR3, ELAVL1, GADD45GIP1, INPP5K, ↓KIAA1644, KIF11, KIFC3, LMNB1, ↑LMOD1, miR-149-3p, miR-185-5p, MRRF, NONO, Pde, ↑PDE3A, PDE4A, PDE5A, ↑PDE7B, PFKFB3, PITX3, PLBD2, PRMT1, PSMF1, Rb, RNF14, ↑RPP25, TAF12, ZNF281 |
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Chen, Y.-J.; Chang, W.-A.; Wu, L.-Y.; Hsu, Y.-L.; Chen, C.-H.; Kuo, P.-L. Systematic Analysis of Transcriptomic Profile of Chondrocytes in Osteoarthritic Knee Using Next-Generation Sequencing and Bioinformatics. J. Clin. Med. 2018, 7, 535. https://doi.org/10.3390/jcm7120535
Chen Y-J, Chang W-A, Wu L-Y, Hsu Y-L, Chen C-H, Kuo P-L. Systematic Analysis of Transcriptomic Profile of Chondrocytes in Osteoarthritic Knee Using Next-Generation Sequencing and Bioinformatics. Journal of Clinical Medicine. 2018; 7(12):535. https://doi.org/10.3390/jcm7120535
Chicago/Turabian StyleChen, Yi-Jen, Wei-An Chang, Ling-Yu Wu, Ya-Ling Hsu, Chia-Hsin Chen, and Po-Lin Kuo. 2018. "Systematic Analysis of Transcriptomic Profile of Chondrocytes in Osteoarthritic Knee Using Next-Generation Sequencing and Bioinformatics" Journal of Clinical Medicine 7, no. 12: 535. https://doi.org/10.3390/jcm7120535