Epigenetic DNA Modifications Upregulate SPRY2 in Human Colorectal Cancers
Abstract
:1. Introduction
2. Materials and Methods
2.1. Human Tissue Collection
2.2. Isolation of Coloncytes from Colonic Mucosa
2.3. Cell Culture
2.4. Western Blotting
2.5. Combined Bisulfite Restriction Analysis
- Region #1: Promoter: putative CTCF binding site [2 CpGs] HpyCH4IV
- Forward primer: (5′→3′)TTAAAGTTTGGGTGTGTAAGGTTAG;
- Reverse primer: (5′→3′)AAAACCAACAAAACACAAATATCTC;
- Region #2: Promoter: upstream of the TSS [1 CpG] HpyCH4IV
- Forward primer: (5′→3′)TTAGAGATGGTAAGGGAAGTGGTAT;
- Reverse primer: (5′→3′)AACCAAAAAAACCCAACTATTTTAC;
- Region #3: Promoter: closest to the TSS [3 CpGs] BstUI
- Forward primer: (5′→3′)GTAGGAGGTATTTAAAAGGGTAAT;
- Reverse primer: (5′→3′)AAAAAAAATTCCCTAAATCAACTC;
- Region #4: Intragenic CpG island: putative CTCF binding site [4 CpGs] HpyCH4IV
- Forward primer: (5′→3′)TTGTTAGGTTTTTTGTAAAGTTTTT;
- Reverse primer: (5′→3′)CAAATTACAAACAATACCCCCTTC;
2.6. 5hmC-Modified Locations in the Gene Body
2.7. Bioinformatics Analysis
3. Results
3.1. SPRY2 Is Upregulated in Adenomas and Colorectal Cancers
3.2. CTCF Binding Sites within Transcriptional Regulatory Regions of SPRY2 Are Differentially Methylated in CRC
3.3. Increased SPRY2 5hmC Deposition Occurs in CRC
3.4. SPRY2 Is Differentially Methylated in CRC Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Caco2 Cells | RKO Cells | HCT116 Cells | |
---|---|---|---|
SPRY2 protein expression | High | Medium | Low |
5mC presence region #1 | No | Yes | Yes |
5mC presence region #2 | No | No | Yes |
5mC presence region #3 | No | No | Yes |
5mC presence region #4 | No | No | Yes |
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Stuckel, A.J.; Zeng, S.; Lyu, Z.; Zhang, W.; Zhang, X.; Dougherty, U.; Mustafi, R.; Zhang, Q.; Joshi, T.; Bissonnette, M.; et al. Epigenetic DNA Modifications Upregulate SPRY2 in Human Colorectal Cancers. Cells 2021, 10, 2632. https://doi.org/10.3390/cells10102632
Stuckel AJ, Zeng S, Lyu Z, Zhang W, Zhang X, Dougherty U, Mustafi R, Zhang Q, Joshi T, Bissonnette M, et al. Epigenetic DNA Modifications Upregulate SPRY2 in Human Colorectal Cancers. Cells. 2021; 10(10):2632. https://doi.org/10.3390/cells10102632
Chicago/Turabian StyleStuckel, Alexei J., Shuai Zeng, Zhen Lyu, Wei Zhang, Xu Zhang, Urszula Dougherty, Reba Mustafi, Qiong Zhang, Trupti Joshi, Marc Bissonnette, and et al. 2021. "Epigenetic DNA Modifications Upregulate SPRY2 in Human Colorectal Cancers" Cells 10, no. 10: 2632. https://doi.org/10.3390/cells10102632