Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines
Abstract
:1. Introduction
2. Results
2.1. CSPG mRNA Expression in WERI-RB1 and WERI-ETOR
2.2. Expression of ECM Glycoproteins in WERI-RB1 and WERI-ETOR
2.3. Expression of MMPs and TIMPs in WERI-RB1 and WERI-ETOR
2.4. Expression of Integrin Receptor Subunits in WERI-RB1 and WERI-ETOR
2.5. Expression of CSPGs, ECM Glycoproteins, MMPs, TIMPs, and Integrin Receptor Subunits in Etoposide Sensitive and Resistant Y79 RB cells
2.6. Expression of RPTPβ/ζ Isoforms in WERI-RB1 and WERI-ETOR
2.7. Impact of the ECM on WERI-RB1 and WERI-ETOR Cell Survival
2.8. Influence of the ECM on WERI-RB1 and WERI-ETOR Cell Proliferation
2.9. Cluster Formation Capacity of WERI-RB1 and WERI-ETOR Cells on Different ECM Substrates
3. Discussion
3.1. Expression of CSPGs in RB Cells
3.2. Expression of Glycoproteins and Collagen IV in RB Cells
3.3. Expression of Tenascin-C and Tenascin-R in RB Cells
3.4. Expression of MMPs and TIMPs in RB Cells
3.5. Integrin Expression in RB Cells
3.6. Expression of RPTPβ/ζ in RB Cells
3.7. ECM Influence on the Apoptotic Rate of RB Cells
3.8. ECM Influence on the Proliferation Rate of RB Cells
3.9. ECM Influence on Cluster Formation of RB Cells
3.10. Limitations and Future Perspectives of the Study
4. Materials and Methods
4.1. Cultivation and Authentication Verification of Human RB Cell Lines
4.2. Cultivation of the Human Glioblastoma Cell Line U-251-MG and Purification of Human Tenascin-C
4.3. Cell Proliferation and Cluster Formation Analyses Via Time-Lapse Video Microscopy
4.4. RNA Isolation and cDNA Synthesis
4.5. Quantitative Real-Time PCR Analyses (RT-qPCR)
4.6. Semi-Quantitative Reverse-Transcription PCR Analyses (RT-PCR)
4.7. Immunocytochemistry
4.8. Protein Lysis and Western Blot
4.9. Statistical Analyses
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A
References
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Primer Name | Gene | Primer Sequence | Accession Number | Product Size (bp) |
---|---|---|---|---|
Aggrecan for | ACAN | tcaccagtgaggacctcgt | NM_001135.3 | 84 |
Aggrecan rev | ggcggtagtggaagacgac | NM_013227.3 | ||
β-Actin for | ACTB | ccaaccgcgagaagatga | NM_001101.3 | 97 |
β-Actin rev | ccagaggcgtacagggatag | |||
Brevican for | BCAN | tggaagctccactccagaa | NM_021948.4 | 76 |
Brevican rev | cggtaccatggattgtgtttc | |||
Collagen IV for | COL4A1 | gggagaaaagggtgaagca | NM_001845.5 | 72 |
Collagen IV rev | ccaaaggtcctgtgcctataa | |||
Fibronectin for | FN1 | cttccacaggaggcctacac | NM_212482.1 | 114 |
NM_002026.2 | ||||
NM_212478.1 | ||||
Fibronectin rev | cgcaaaatatgctggaacttt | NM_212476.1 | ||
NM_212474.1 | ||||
GAPDH for | GAPDH | tccactggcgtcttcacc | NM_001289746.1 | 114 |
NM_001289745.1 | ||||
GAPDH rev | ggcagagatgatgaccctttt | NM_001256799.2 | ||
NM_002046.5 | ||||
α4-Integrin for | ITGA4 | gatgaaaatgagcctgaaacg | NM_000885.4 | 80 |
α4-Integrin rev | gccatactattgccagtgttga | |||
α5-Integrin for | ITGA5 | cccattgaatttgacagcaa | NM_002205.2 | 92 |
α5-Integrin rev | tgcaaggacttgtactccaca | |||
β1-Integrin for | ITGB1 | cgatgccatcatgcaagt | NM_002211 | 71 |
β1-Integrin rev | acaccagcagccgtgtaac | NM_133376 | ||
α1-Laminin for | LAMA1 | aggatgacctccattctgactt | NM_005559.3 | 60 |
α1-Laminin rev | ccttacatgggcactgacct | |||
MMP-2 for | MMP2 | ctacgaccgcgacaagaagt | NM_001302510.1 | 114 |
NM_001302509.1 | ||||
NM_001302508.1 | ||||
MMP-2 rev | agttcccaccaacagtggac | NM_001127891.2 | ||
NM_004530.5 | ||||
MMP-7 for | MMP7 | gctgacatcatgattggcttt | NM_002423.3 | 113 |
MMP-7 rev | tctcctccgagacctgtcc | |||
MMP-9 for | MMP9 | gtaccacggccaactacgac | NM_004994.2 | 74 |
MMP-9 rev | gccgtcctgggtgtagagt | |||
Neurocan for | NCAN | aggggttcggagctatgg | NM_004386.2 | 95 |
Neurocan rev | ggcccacgtagaagacctc | |||
S18 for | RPS18 | cttccacaggaggcctacac | NM_022551.2 | 82 |
S18 rev | cgcaaaatatgctggaacttt | |||
TIMP-1 for | TIMP1 | agtggcactcattgcttgtg | NM_003254.2 | 104 |
TIMP-1 rev | ggactggaagcccttttca | |||
TIMP-2 for | TIMP2 | gaagagcctgaaccacaggt | NM_003255.4 | 85 |
TIMP-2 rev | cggggaggagatgtagcac | |||
Tenascin-C for | TNC | gtctccagcctgccacag | NM_002160.3 | 82 |
Tenascin-C rev | cgcctttccagccaggttc | |||
Tenascin-R for | TNR | agtgacctctgagcccattg | NM_003285.2 | 61 |
Tenascin-R rev | gatgtcttttgggggatcaa | |||
Versican for | VCAN | tcccaggaaacttacgatg | NM_004385.4 | 70 |
Versican rev | ggggacagtgaggtgggaa |
Primer Name | Gene | Primer Sequence | Accession Number | Amplicon Size (bp) |
---|---|---|---|---|
β-Actin for | ACTB | tgacggggtcacccacactgtgcccatcta | NM_001101.3 | 661 |
β-Actin rev | ctagaagcatttgcggtggacgatggaggg | |||
CP RPTPβ/ζ for | PTPRZ1 | agtgtgcaagtgcttgcctat | XM_005250519.1 | 555 |
CP RPTPβ/ζ rev | tgcagaatagtcactctgctg | XM_017012477.1 | ||
EC RPTPβ/ζ for | tgcctacttcccaactgag | NM_002851.2 | 509 | |
EC RPTPβ/ζ rev | cagcatgagtagtggac | |||
TEC RPTPβ/ζ for | tgcctacttcccaactgag | NM_001206839.1 | 369 | |
TEC RPTPβ/ζ rev | attgctccgacatcatctg | 2949 |
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Reinhard, J.; Wagner, N.; Krämer, M.M.; Jarocki, M.; Joachim, S.C.; Dick, H.B.; Faissner, A.; Kakkassery, V. Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines. Int. J. Mol. Sci. 2020, 21, 4322. https://doi.org/10.3390/ijms21124322
Reinhard J, Wagner N, Krämer MM, Jarocki M, Joachim SC, Dick HB, Faissner A, Kakkassery V. Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines. International Journal of Molecular Sciences. 2020; 21(12):4322. https://doi.org/10.3390/ijms21124322
Chicago/Turabian StyleReinhard, Jacqueline, Natalie Wagner, Miriam M. Krämer, Marvin Jarocki, Stephanie C. Joachim, H. Burkhard Dick, Andreas Faissner, and Vinodh Kakkassery. 2020. "Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines" International Journal of Molecular Sciences 21, no. 12: 4322. https://doi.org/10.3390/ijms21124322