Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts
Abstract
:1. Introduction
2. Results
2.1. Morphological Changes and Myoblast Fusion Index
2.2. Gene Expression
2.2.1. Time-Dependent Expression of Individual Genes Relative to 18S Ribosomal RNA Using Real Time qPCR Analysis
Gpr30, Erα and Erβ
Mapk11 and Akt1
MyoD1, Myog, Myh1 and Myomaker (Tmem8c)
2.3. Next Generation Sequencing (NGS)
2.3.1. Pathway Enrichment Analyses, Gene Ontology and Gene Functional Category in Cultures at 48 h Treatment with Con-Ve, E2 and EPA
2.3.2. Gene Expression Profile in the Myogenic and the Striated Muscle Contraction Pathways at 48 h Treatment with Con-Ve, E2 and EPA
2.3.3. Genes Unique to E2 and EPA Treated Cells
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. E2 and EPA Treatments
4.3. Immunofluorescence, Mytube Formation and Fusion Parameters
4.4. Transcriptome Analyses
4.4.1. RNA Extraction
4.4.2. Real Time Quantitative PCR
4.4.3. Next Generation Sequencing (NGS) Using Illumina HiSeq 2500 RNA-seq
4.5. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviation
Akt1 | serine/threonine kinase 1 |
BSA | bovine serum albumin |
DAPI | 4′,6-diamidino-2-phenylindole |
DMEM | Dulbecco’s modified eagle medium |
DEG | digital expression of genes |
E2 | 17β-estradiol |
EPA | eicosapentaenoic acid |
Erα | E2 receptor alpha |
Erβ | E2 receptor beta |
ERT | estrogen-based hormone replacement therapy |
FBS | foetal bovine serum |
FC | fold change |
FDR | false discovery |
GO | gene ontology |
GFC | gene functional category |
Gpr30 | G protein-coupled estrogen receptor-30 |
h | hour |
HRT | hormone replacement therapy |
HS | horse serum |
Mapk11 | mitogen-activated protein kinase 11 |
Mrfs | myogenic regulatory factors |
MyoD | myoblast determination protein |
Myh1 | myosin heavy chain 1 |
Myog | myogenin |
μL | microliter |
μM | micromolar |
nM | nanomolar |
n-3PUFA | 3n polyunsaturated fatty acids |
NGS | next generation sequencing |
PE | pathway enrichment |
Pen/Strep | penicillin/streptomycin |
qPCR | quantitative polymerase chain reaction |
RNA | ribosomal nucleic acid |
SCs | satellite cells |
Tmem8c | transmembrane protein 8C |
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Gene Name | E2 vs. Con-Ve | EPA vs. Cont-Ve | EPA vs. E2 | |||||||
---|---|---|---|---|---|---|---|---|---|---|
logFC | p Value | FDR | logFC | p Value | FDR | logFC | p Value | FDR | ||
Acta1 | actin, alpha 1, skeletal muscle | 0.56 | 0.048 | 0.606 | −2.11 | 1 × 105 | 0.0004 | −2.67 | 1 × 106 | 0.0001 |
Acta2 | actin, alpha 2, smooth muscle, aorta | 0.35 | 0.034 | 0.566 | −0.328 | 0.045 | 0.1138 | −0.68 | 0.0008 | 0.006 |
Actc1 | actin, alpha, cardiac muscle 1 | 0.66 | 0.024 | 0.551 | −2.53 | 2 × 106 | 0.0001 | −3.19 | 4 × 107 | 4 × 105 |
Actg1 | actin, gamma, cytoplasmic 1 | −0.02 | 0.831 | 0.985 | −0.49 | 0.0005 | 0.005 | −0.47 | 0.0006 | 0.005 |
Actn2 | actinin alpha 2 | 0.92 | 0.001 | 0.364 | −2.29 | 4 × 106 | 0.0002 | −3.22 | 1 × 107 | 2 × 105 |
Actn3 | actinin alpha 3 | 0.24 | 0.029 | 0.557 | −1.10 | 6 × 106 | 9 × 105 | −1.35 | 1 × 107 | 2 × 105 |
Actn4 | actinin alpha 4 | 0.05 | 0.352 | 0.886 | −0.36 | 0.0001 | 0.002 | −0.42 | 3 × 105 | 0.0007 |
Casq2 | calsequestrin 2 | 0.53 | 0.034 | 0.566 | −2.43 | 1 × 106 | 0.0001 | −2.97 | 2 × 107 | 3 × 105 |
Des | Desmin | 0.03 | 0.705 | 0.977 | −0.52 | 5 × 105 | 0.001 | −0.55 | 10 × 103 | 0.0007 |
Jsrp1 | junctional sarcoplasmic reticulum protein 1 | 0.17 | 0.325 | 0.877 | −0.69 | 0.001 | 0.014 | −0.86 | 0.0004 | 0.003 |
Mybpc1 | myosin binding protein C, slow-type | 1.16 | 0.0002 | 0.226 | −1.05 | 0.001 | 0.013 | −2.21 | 3 × 106 | 0.0001 |
Mybpc2 | myosin binding protein C, fast-type | 0.67 | 0.128 | 0.740 | −2.23 | 0.002 | 0.017 | −2.91 | 0.0003 | 0.003 |
Myh1 | myosin, heavy polypeptide 1, skeletal muscle, adult | 0.95 | 0.0006 | 0.300 | −2.00 | 2 × 102 | 0.0001 | −2.96 | 9 × 109 | 2 × 105 |
Myh3 | myosin, heavy polypeptide 3, skeletal muscle, embryonic | 0.60 | 0.016 | 0.529 | −3.21 | 7 × 108 | 3 × 105 | −3.828 | 1 × 108 | 1 × 105 |
Myh4 | myosin, heavy polypeptide 4, skeletal muscle | 0.99 | 6 × 105 | 0.183 | −0.55 | 0.005 | 0.029 | −1.55 | 1 × 106 | 0.0001 |
Myh6 | myosin, heavy polypeptide 6, cardiac muscle, alpha | 1.10 | 0.0001 | 0.183 | −1.69 | 2 × 105 | 0.0008 | −2.79 | 2 × 107 | 3 × 105 |
Myh7 | myosin, heavy polypeptide 7, cardiac muscle, beta | 0.93 | 0.0004 | 0.262 | −1.34 | 0.0005 | 0.006 | −2.15 | 8 × 106 | 0.0002 |
Myh8 | myosin, heavy polypeptide 8, skeletal muscle, perinatal | 1.18 | 0.0006 | 0.295 | −2.21 | 1 × 105 | 0.0004 | −3.39 | 2 × 107 | 3 × 105 |
Myl1 | myosin, light polypeptide 1 | 0.50 | 0.035 | 0.566 | −0.83 | 0.002 | 0.016 | −1.34 | 8 × 105 | 0.001 |
Myl4 | myosin, light polypeptide 4 | 0.61 | 0.047 | 0.604 | −1.74 | 0.0001 | 0.002 | −2.36 | 1 × 105 | 0.0003 |
Myl9 | myosin, light polypeptide 9, regulatory | 0.09 | 0.661 | 0.971 | −0.90 | 0.002 | 0.016 | −1.00 | 0.001 | 0.008 |
Myom1 | myomesin 1 | 0.52 | 0.021 | 0.539 | −2.00 | 2 × 106 | 0.0001 | −2.52 | 3 × 107 | 4 × 105 |
Myom2 | myomesin 2 | 0.17 | 0.138 | 0.754 | −0.86 | 1 × 105 | 0.0005 | −1.03 | 2 × 106 | 0.0001 |
Neb | Nebulin | 0.40 | 0.036 | 0.566 | −2.02 | 4 × 107 | 8 × 105 | −2.42 | 9 × 108 | 2 × 105 |
Smpx | small muscle protein, X-linked | 0.49 | 0.047 | 0.604 | −1.67 | 4 × 105 | 0.001 | −2.16 | 4 × 106 | 0.0002 |
Tcap | titin-cap | 0.87 | 0.031 | 0.561 | −0.59 | 0.169 | 0.285 | −1.47 | 0.003 | 0.015 |
Tmod1 | tropomodulin 1 | 0.54 | 0.008 | 0.482 | −0.84 | 0.0005 | 0.005 | −1.38 | 1 × 105 | 0.0003 |
Tnnc1 | troponin C, cardiac/slow skeletal | 0.51 | 0.060 | 0.624 | −1.43 | 0.0002 | 0.003 | −1.94 | 1 × 105 | 0.0004 |
Tnnc2 | troponin C2, fast | 0.51 | 0.072 | 0.646 | −1.72 | 7 × 105 | 0.001 | −2.23 | 9 × 106 | 0.0003 |
Tnni1 | troponin I, skeletal, slow 1 | 0.49 | 0.068 | 0.639 | −1.90 | 2 × 105 | 0.0007 | −2.40 | 3 × 106 | 0.0001 |
Tnni2 | troponin I, skeletal, fast 2 | 0.39 | 0.187 | 0.800 | −1.65 | 0.0001 | 0.003 | −2.05 | 3 × 105 | 0.0007 |
Tnni3 | troponin I, cardiac 3 | 0.27 | 0.457 | 0.918 | −0.90 | 0.05 | 0.129 | −1.18 | 0.015 | 0.050 |
Tnnt1 | troponin T1, skeletal, slow | 0.32 | 0.146 | 0.764 | −1.31 | 0.0001 | 0.002 | −1.64 | 1 × 105 | 0.0004 |
Tnnt2 | troponin T2, cardiac | 0.29 | 0.268 | 0.855 | −1.97 | 2 × 105 | 0.0007 | −2.27 | 7 × 106 | 0.0002 |
Tnnt3 | troponin T3, skeletal, fast | 0.39 | 0.152 | 0.769 | −1.63 | 0.0001 | 0.001 | −2.025 | 1 × 105 | 0.0004 |
Tpm1 | tropomyosin 1, alpha | 0.13 | 0.139 | 0.756 | −0.36 | 0.001 | 0.009 | −0.49 | 0.0001 | 0.001 |
Tpm2 | tropomyosin 2, beta | 0.33 | 0.061 | 0.629 | −0.68 | 0.001 | 0.012 | −1.02 | 8 × 105 | 0.001 |
Tpm3 | tropomyosin 3, gamma | −0.09 | 0.342 | 0.885 | −0.42 | 0.001 | 0.009 | −0.33 | 0.005 | 0.023 |
Tpm4 | tropomyosin 4 | −0.08 | 0.299 | 0.868 | −0.14 | 0.072 | 0.157 | −0.06 | 0.38 | 0.506 |
Ttn | titin | 0.19 | 0.152 | 0.769 | −1.17 | 4 × 106 | 0.0002 | −1.37 | 1 × 106 | 7E-05 |
Vim | vimetin | 0.09 | 0.419 | 0.904 | −0.03 | 0.728 | 0.810 | −0.13 | 0.258 | 0.381 |
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Lacham-Kaplan, O.; Camera, D.M.; Hawley, J.A. Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts. Int. J. Mol. Sci. 2020, 21, 745. https://doi.org/10.3390/ijms21030745
Lacham-Kaplan O, Camera DM, Hawley JA. Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts. International Journal of Molecular Sciences. 2020; 21(3):745. https://doi.org/10.3390/ijms21030745
Chicago/Turabian StyleLacham-Kaplan, Orly, Donny M. Camera, and John A. Hawley. 2020. "Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts" International Journal of Molecular Sciences 21, no. 3: 745. https://doi.org/10.3390/ijms21030745