Transcriptome Analysis Reveals Modulation of Human Stem Cells from the Apical Papilla by Species Associated with Dental Root Canal Infection
Abstract
:1. Introduction
2. Results
2.1. Mapping and Quantifying SCAP Transcriptomes by RNA-Seq
2.2. SCAP Transcriptome Elicits Distinct Profiles Based on Bacterial Stimulation
2.3. E. faecalis Supernatant Has the Strongest Influence on SCAPs Whereas Supernatant of F. nucleatum Has a Mild Effect
2.4. F. nucleatum Upregulates Immune and Inflammatory Response whereas E. faecalis Downregulates Cell Division and Proliferation in SCAP
2.5. Osteogenic/Odontogenic Genes in SCAPs Are Strongly Influenced by F. nucleatum and E. faecalis Associated with Endodontic Infections
3. Discussion
4. Materials and Methods
4.1. Cell Isolation and Culture
4.2. Bacterial Strains and Culture Conditions
4.3. SCAP Infection by Bacterial Strains: Co-culture Experiments
4.4. RNA Extraction and Quality Evaluation
4.5. Transcriptomic Analysis, Data Preprocessing and Bioinformatics
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Sample | Type of Treatment | Raw Data (Read Count) | Clean Data (Read Count) | Total Mapped Reads | Total Mapping Rate (%) | Percent of Genome Regions (%) | ||
---|---|---|---|---|---|---|---|---|
Exon | Intron | Intergenic | ||||||
1 | DI NB | 43,633,856 | 43,606,890 | 43,210,852 | 99.1 | 93.0 | 2.5 | 4.5 |
2 | DI F.n_B | 54,476,736 | 54,444,824 | 53,964,109 | 99.1 | 93.3 | 2.4 | 4.3 |
3 | DI F.n._S | 42,956,122 | 42,933,526 | 42,576,137 | 99.2 | 93.7 | 2.0 | 4.3 |
4 | DI E.f_B | 55,629,374 | 55,600,608 | 54,824,199 | 98.6 | 88.9 | 5.9 | 5.2 |
5 | DI E.f_S | 55,430,062 | 55,401,924 | 54,885,257 | 99.1 | 92.3 | 3.2 | 4.5 |
6 | DII NB | 47,027,536 | 47,002,190 | 46,541,473 | 99.0 | 93.5 | 2.2 | 4.3 |
7 | DII F.n_B | 48,806,760 | 48,784,160 | 48,246,591 | 98.9 | 91.6 | 2.9 | 5.5 |
8 | DII F.n._S | 46,427,206 | 46,406,140 | 45,986,861 | 99.1 | 92.1 | 2.7 | 5.2 |
9 | DII E.f_B | 39,262,262 | 39,242,992 | 38,806,871 | 98.9 | 90.9 | 4.2 | 4.9 |
10 | DII E.f_S | 49,064,034 | 49,042,722 | 48,616,751 | 99.1 | 91.9 | 3.3 | 4.8 |
11 | DIII NB | 48,797,068 | 48,773,676 | 48,361,861 | 99.1 | 93.7 | 2.1 | 4.2 |
12 | DIII F.n_B | 52,910,564 | 52,883,784 | 52,349,588 | 98.9 | 93.2 | 2.5 | 4.3 |
13 | DIII F.n._S | 52,127,896 | 52,105,306 | 51,686,329 | 99.2 | 91.9 | 2.7 | 5.4 |
14 | DIII E.f_B | 46,191,244 | 46,171,130 | 45,806,664 | 99.2 | 90.8 | 4.2 | 5.0 |
15 | DIII E.f_S | 49,193,684 | 49,173,978 | 48,823,399 | 99.3 | 93.4 | 2.3 | 4.3 |
Gene | Function | Reference | |
---|---|---|---|
Dentinogenic genes | Vascular endothelial growth factor A (VEGFA) | Inducing proliferation and differentiation of hDPSCs into odontoblasts | Matsushita et al., 2000 [32] |
Fibroblast growth factor 2 (FGF2) | Potent regulator of mineralization | Roberts-Clark and Smith, 2000; Madan and Kramer, 2005; Cooper et al., 2010; Miraoui and Marie, 2010; Marie et al., 2012; Smith et al., 2012 [33,34,35,36,37,38] | |
Platelet-derived growth factor C (PDGFC) | Enhancement of DPSCs proliferation, odontoblast differentiation, and regeneration of dentin–pulp complex | Tsutsui, 2020 [39] | |
Transforming growth factor β-1 (TGF-β1) | Importance in regulating reparative dentinogenesis | Toyono et al., 1997; Piatelli et al., 2004; Unterbrink et al., 2002 [40,41,42] | |
Osteogenic cell surface markers | Gamma-aminobutyric acid B receptor 1 (GABABR1) | Negative regulation of osteoblastogenesis | Takahata et al., 2011 [43] |
Gamma-aminobutyric acid B receptor 2 (GABABR2) | |||
Parathyroid hormone 1 receptor (PTH1R) | Committing MSCs to the osteoblast lineage and promoting bone formation | Yu et al., 2012 [44] | |
Receptor activator of nuclear factor-κB (RANK) or TNFRSF11A | Suppression of osteoblast differentiation | Chen et al., 2018 [45] | |
Integrin alpha-V (ITGAV) | Osteoblast differentiation promotion | Cheng et al., 2001 [46] | |
Osteoclast-associated receptor (OSCAR) | Regulator of osteoclast differentiation | Barrow et al., 2011 [47] | |
Activated leukocyte cell adhesion molecule (ALCAM/CD166) | Immature osteoblast marker, promotes osteoblast differentiation | Hooker et al., 2015 [48] | |
Osteogenic intracellular markers | Nuclear factor interleukin-3-regulated (NFIL3) | Transcriptional repressor in osteoblasts | Hariri et al., 2020 [49] |
Runt-related transcription factor 2 (RUNX2) | Essential for initial commitment of MSCs to the osteoblastic lineage | Camilleri et al., 2006 [50] | |
T cell immune regulator 1 (TCIRG1) | Osteoclastogenesis regulation | Zhang et al., 2020 [51] | |
WD repeat domain 5 protein (WDR5) | Critical for MSCs osteogenic differentiation | Zhu et al., 2016 [52] | |
T-box 2 (TBX2) | Positive regulation of osteogenic differentiation | Govoni et al., 2009; Abrahams et al., 2010 [53,54] | |
T-box 3 (TBX3) | |||
Distal-less homeobox 5 (DLX-5) | Transcriptional regulation of osteoblast differentiation | Hassan et al., 2004 [55] | |
Early B cell factor 2 (EBF2) | Inhibition of osteoblast differentiation | Kieslinger et al., 2005 [56] | |
Osteogenic secreted markers | Signal peptide, CUB, and EGF-like domain-containing protein 3 (SCUBE3) | Controlling growth, morphogenesis, and bone and teeth development | Lin et al., 2021 [57] |
Type 1 collagen A (COL1A1) | Early marker of osteoblast | Kannan et al., 2020 [58] | |
Insulin-like growth factor binding protein-3 (IGFBP-3) | Osteoblasts differentiation suppression | Li et al., 2013 [59] | |
Insulin-like growth factor binding protein-4 (IGFBP-4) | Osteoclastogenesis regulation | Maridas et al., 2017 [60] | |
Secreted protein acidic and rich in cysteine (SPARC) | Regulation of bone remodeling and bone mass maintenance | Rosset et al., 2016 [61] | |
SPARC-related modular calcium-binding protein 1 (SMOC1) | Increases the expression of osteoblast differentiation-related genes in BMSCs | Choi et al., 2010 [62] | |
Acid phosphatase 5 (ACP5) | Promotion of odontoblast differentiation and mineralization during tooth development | Choi et al., 2016 [63] | |
Biorientation of chromosomes in cell division1-like 1(BOD1L1) | Positive regulation of osteoblasts differentiation | Okamura et al., 2017 [64]; NCBI [65] | |
Aggrecan (ACAN) | Bone tissue formation | Viti et al., 2016 [66] | |
Tissue-nonspecific alkaline phosphatase (ALPL) | Essential for bone mineralization; osteoblast marker | Nakamura et al., 2020 [67] | |
Biglycan (BGN) | Modulation of osteoblast differentiation | Parisuthiman et al., 2005 [68] | |
Decorin (DCN) | Key marker of odontoblasts | Matsuura et al., 2001 [69] | |
Fibronectin 1 (FN1) | Essential for osteoblast differentiation and mineralization | Globus et al., 1998 [70] | |
Bone morphogenetic protein (BMP2) | Important in osteoblast differentiation | Yang et al., 2012 [71] |
Sample | SCAP Source | Treatment | Concentration, pg/μL | Absorbance Ratio 260/280 | RNA Integrity Number |
---|---|---|---|---|---|
1 | Donor I | No Bacteria | 4405 | 2.086 | 9.9 |
2 | Donor I | F. nucleatum B * | 3737 | 2.113 | 9.4 |
3 | Donor I | F. nucleatum S ** | 3016 | 2.088 | 9.1 |
4 | Donor I | E. faecalis B | 3977 | 2.089 | 6.4 |
5 | Donor I | E. faecalis S | 3078 | 2.102 | 9.8 |
6 | Donor II | No Bacteria | 4791 | 2.097 | 9.8 |
7 | Donor II | F. nucleatum B | 6566 | 2.123 | 9.2 |
8 | Donor II | F. nucleatum S | 5288 | 2.096 | 10.0 |
9 | Donor II | E. faecalis B | 5208 | 2.087 | 8.0 |
10 | Donor II | E. faecalis S | 4465 | 2.080 | 9.5 |
11 | Donor III | No Bacteria | 4193 | 2.068 | 10.0 |
12 | Donor III | F. nucleatum B | 4929 | 2.058 | 8.7 |
13 | Donor III | F. nucleatum S | 4673 | 2.064 | 9.5 |
14 | Donor III | E. faecalis B | 4871 | 2.068 | 7.6 |
15 | Donor III | E. faecalis S | 4089 | 2.069 | 10.0 |
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Razghonova, Y.; Zymovets, V.; Wadelius, P.; Rakhimova, O.; Manoharan, L.; Brundin, M.; Kelk, P.; Romani Vestman, N. Transcriptome Analysis Reveals Modulation of Human Stem Cells from the Apical Papilla by Species Associated with Dental Root Canal Infection. Int. J. Mol. Sci. 2022, 23, 14420. https://doi.org/10.3390/ijms232214420
Razghonova Y, Zymovets V, Wadelius P, Rakhimova O, Manoharan L, Brundin M, Kelk P, Romani Vestman N. Transcriptome Analysis Reveals Modulation of Human Stem Cells from the Apical Papilla by Species Associated with Dental Root Canal Infection. International Journal of Molecular Sciences. 2022; 23(22):14420. https://doi.org/10.3390/ijms232214420
Chicago/Turabian StyleRazghonova, Yelyzaveta, Valeriia Zymovets, Philip Wadelius, Olena Rakhimova, Lokeshwaran Manoharan, Malin Brundin, Peyman Kelk, and Nelly Romani Vestman. 2022. "Transcriptome Analysis Reveals Modulation of Human Stem Cells from the Apical Papilla by Species Associated with Dental Root Canal Infection" International Journal of Molecular Sciences 23, no. 22: 14420. https://doi.org/10.3390/ijms232214420