Methylation Patterns of DKK1, DKK3 and GSK3β Are Accompanied with Different Expression Levels in Human Astrocytoma
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Tissue Samples
2.2. DNA Extraction
2.3. Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP), Loss of Heterozygosity (LOH)
2.3.1. Polymerase Chain Reaction
2.3.2. Restriction Fragment Length Polymorphism/Loss of Heterozygosity
2.3.3. Methylation-Specific PCR (MSP)
2.4. Immunohistochemistry (IHC)
2.5. Microscopic Analysis
2.6. Statistical Analysis
3. Results
3.1. Methylation Status of Promoter Regions of GSK3β, DKK,1 and DKK3
3.2. pGSK3β-S9 and pGSK3β-Y216 Expression Levels
3.3. Total β-Catenin and Unphosphorylated β-Catenin Expression Levels in Glioblastoma
3.4. APC Exon 11 Genetic Changes in Glioblastoma
3.5. The Correlations of Molecular Findings and Clinical Parameters
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primer | Sequence | Product Size | |
---|---|---|---|
DKK1 | MR-F | 5′-CGTTCGTTGGTAGTTTTTATTTCGA-3′ | 175 bp |
MR-R | 5′-GCGACTACCTTTATACCGCGAA-3′ | ||
UMR-F | 5′-TGTTTGTTGGTAGTTTTTATTTTGA-3′ | 173 bp | |
UMR-R | 5′-ACCACAACTACCTTTATACCACAAA-3′ | ||
DKK3 | MR-F | 5′-CGGTTTTTTTTCGTTTTCGGG-3′ | 154 bp |
MR-R | 5′-CAAACCGCTACATCTCCGCT-3′ | ||
UMR-F | 5′-TTTTGGTTTTTTTTTGTTTTTGGG-3′ | 155 bp | |
UMR-R | 5′-CCAA ACCACTACATCTCCACT-3′ | ||
GSK3β | MR-F | 5′ CGTCGTTATCGTTATCGTTC 3′ | 135 bp |
MR-R | 5′ AATAACTCGAAAATACGACG 3′ | ||
UMR-F | 5′ GAGGAGTTGTTGTTATTGTTATTGTTT 3′ | 136 bp | |
UMR-R | 5′ AAAAAAATAACTCAAAAATACAACA 3′ |
Gene | Initial Denaturation | Cycle Conditions | Final Elongation | |||
---|---|---|---|---|---|---|
DKK1 | MR | 95 °C 5 min | 95 °C 30 s | 61 °C 30 s | 72 °C 30 s | 72 °C 7 min |
UMR | 95 °C 5 min | 95 °C 30 s | 61 °C 30 s | 72 °C 30 s | 72 °C 7 min | |
DKK3 | MR | 95 °C 5 min | 95 °C 30 s | 61 °C 30 s | 72 °C 30 s | 72 °C 7 min |
UMR | 95 °C 5 min | 95 °C 30 s | 61 °C 30 s | 72 °C 30 s | 72 °C 7 min | |
GSK3β | MR | 95 °C 5 min | 95 °C 30 s | 55 °C 30 s | 72 °C 30 s | 72 °C 7 min |
UMR | 95 °C 5 min | 95 °C 30 s | 56,5 °C 30 s | 72 °C 30 s | 72 °C 7 min |
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Kafka, A.; Bukovac, A.; Brglez, E.; Jarmek, A.-M.; Poljak, K.; Brlek, P.; Žarković, K.; Njirić, N.; Pećina-Šlaus, N. Methylation Patterns of DKK1, DKK3 and GSK3β Are Accompanied with Different Expression Levels in Human Astrocytoma. Cancers 2021, 13, 2530. https://doi.org/10.3390/cancers13112530
Kafka A, Bukovac A, Brglez E, Jarmek A-M, Poljak K, Brlek P, Žarković K, Njirić N, Pećina-Šlaus N. Methylation Patterns of DKK1, DKK3 and GSK3β Are Accompanied with Different Expression Levels in Human Astrocytoma. Cancers. 2021; 13(11):2530. https://doi.org/10.3390/cancers13112530
Chicago/Turabian StyleKafka, Anja, Anja Bukovac, Emilija Brglez, Ana-Marija Jarmek, Karolina Poljak, Petar Brlek, Kamelija Žarković, Niko Njirić, and Nives Pećina-Šlaus. 2021. "Methylation Patterns of DKK1, DKK3 and GSK3β Are Accompanied with Different Expression Levels in Human Astrocytoma" Cancers 13, no. 11: 2530. https://doi.org/10.3390/cancers13112530
APA StyleKafka, A., Bukovac, A., Brglez, E., Jarmek, A.-M., Poljak, K., Brlek, P., Žarković, K., Njirić, N., & Pećina-Šlaus, N. (2021). Methylation Patterns of DKK1, DKK3 and GSK3β Are Accompanied with Different Expression Levels in Human Astrocytoma. Cancers, 13(11), 2530. https://doi.org/10.3390/cancers13112530