Establishment of a Novel In Vitro Model of Endometriosis with Oncogenic KRAS and PIK3CA Mutations for Understanding the Underlying Biology and Molecular Pathogenesis
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Development of Immortalized Epithelial Cell Lines from Ovarian Endometriotic Tissue
2.2. Western Blot Analysis of pan-Cytokeratin, pan-AKT, pan-MAPK, p-MAPK, and p-AKT Expression in Immortalized Cells
2.3. Short Tandem Repeat (STR) Analysis
2.4. Migration, Invasion, Proliferation, and Anchorage-Independent Assays of Mutant Cells
2.5. Constitutive Effect of Tumorigenicity in HMOsisEC10 KRAS and HMOsisEC10 PIK3CA Mutant Cells
2.6. Lack of Other Genetic Mutations in Immortalized Epithelial Cells
2.7. Identification and Analysis of Genes Involved in Cell Migration, Invasion, and Proliferation
2.8. Validation of Microarray Data by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
2.9. Signaling Pathways Targeting KRAS and PIK3CA Mutations in Endometriosis Due to the Upregulation of Lysyl Oxidase (LOX) and Pentraxin3 (PTX3)
2.10. LOX and PTX3 mRNA Expression Levels Are Upregulated in Immortalized Endometriotic Mutant Epithelial Cells
2.11. Constitutive Knockdown of LOX and PTX3 Leads to Decreased Migration, Invasion, and Proliferation in KRAS and PIK3CA Mutant Cell Lines
3. Discussion
4. Materials and Methods
4.1. Ethical Approval
4.2. Isolation and Purification of the Tissue Samples
4.3. Vector Construction and Cell Transfection
4.4. Cell Lines and Cell Culture
4.5. Western Blot Analysis
4.6. Immunocytochemistry
4.7. Short Tandem Repeat (STR) Analysis
4.8. Simulated Wound Healing Assay to Assess Cell Motility
4.9. Matrigel Invasion Assay
4.10. Cell Proliferation Assay
4.11. Anchorage Independent Assay
4.12. Nude Mice Xenograft
4.13. Whole-Exome Sequencing
4.14. Microarray Analysis
4.15. siRNA Transfection
4.16. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
KRAS: | Kristen Rat Sarcoma |
PIK3CA | Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha |
LOX | lysyl oxidase |
PTX3 | Pentraxin3 |
RT-PCR | Reverse Transcriptase Polymerase Chain Reaction |
RhoA | Ras homolog family member A, |
TNF-α | Tumor Necrotic Factor- α |
SRF | Serum Response Factor |
hTERT | humans Telomerase Reverse Transcriptase |
CDK4 | Cyclin Dependent Kinase 4 |
STR | Short Tandem Repeat |
siRNA | small interfering RNA |
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Name of Primer | Type | Sequence (5′-3′) |
---|---|---|
LOX | Forward | TGCCAGTGGATTGATATTAC |
Reverse | TACGGTGAAATTGTGCAGCC | |
PTX3 | Forward | GCATCTCCTTGCGATTCTGTT |
Reverse | CATTCCGAGTGCTCCTGACC | |
GAPDH | Forward | ACGGGAAGCTTGTCATCAAT |
Reverse | TGGACTCCACGACGTACTCA |
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Hossain, M.M.; Nakayama, K.; Shanta, K.; Razia, S.; Ishikawa, M.; Ishibashi, T.; Yamashita, H.; Sato, S.; Iida, K.; Kanno, K.; et al. Establishment of a Novel In Vitro Model of Endometriosis with Oncogenic KRAS and PIK3CA Mutations for Understanding the Underlying Biology and Molecular Pathogenesis. Cancers 2021, 13, 3174. https://doi.org/10.3390/cancers13133174
Hossain MM, Nakayama K, Shanta K, Razia S, Ishikawa M, Ishibashi T, Yamashita H, Sato S, Iida K, Kanno K, et al. Establishment of a Novel In Vitro Model of Endometriosis with Oncogenic KRAS and PIK3CA Mutations for Understanding the Underlying Biology and Molecular Pathogenesis. Cancers. 2021; 13(13):3174. https://doi.org/10.3390/cancers13133174
Chicago/Turabian StyleHossain, Mohammad Mahmud, Kentaro Nakayama, Kamrunnahar Shanta, Sultana Razia, Masako Ishikawa, Tomoka Ishibashi, Hitomi Yamashita, Seiya Sato, Kouji Iida, Kosuke Kanno, and et al. 2021. "Establishment of a Novel In Vitro Model of Endometriosis with Oncogenic KRAS and PIK3CA Mutations for Understanding the Underlying Biology and Molecular Pathogenesis" Cancers 13, no. 13: 3174. https://doi.org/10.3390/cancers13133174
APA StyleHossain, M. M., Nakayama, K., Shanta, K., Razia, S., Ishikawa, M., Ishibashi, T., Yamashita, H., Sato, S., Iida, K., Kanno, K., Ishikawa, N., Kiyono, T., & Kyo, S. (2021). Establishment of a Novel In Vitro Model of Endometriosis with Oncogenic KRAS and PIK3CA Mutations for Understanding the Underlying Biology and Molecular Pathogenesis. Cancers, 13(13), 3174. https://doi.org/10.3390/cancers13133174