Significance of Hypermethylation of Tumor-Suppressor Genes PTGER4 and ZNF43 at CpG Sites in the Prognosis of Colorectal Cancer
Abstract
:1. Introduction
2. Results
2.1. Clinical Features and Follow-Up Information of Patients
2.2. Correlation between PTGER4 and ZNF43 Methylation Status and CRC
2.3. Methylation Level at Certain CpG Sites in Candidate Genes and Its Influence
3. Discussion
4. Methods and Materials
4.1. Study Design
4.2. Study Cohort and Specimens
4.3. Gene Selection
4.4. DNA Extraction and Bisulfite Modification
4.5. Qualitative and Quantitative Analysis of DNA Methylation
4.6. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Variables | Total | Methylation Status | |||
---|---|---|---|---|---|
PTGER4 | ZNF43 | ||||
Normal | Tumor | Normal | Tumor | ||
Sex | |||||
Male | 103 (49.5) | 29 (42.0) | 41 (59.4) | 22 (31.9) | 49 (71.0) |
Female | 105 (50.5) | 36 (48.0) | 47 (62.7) | 20 (26.7) | 57 (76.0) |
χ2 (p value) | 0.30 (0.581) | 0.05 (0.820) | 0.26 (0.614) | 0.24 (0.625) | |
Age at surgery | |||||
<50 | 35 (16.8) | 12 (54.5) | 12 (54.5) | 5 (22.7) | 15 (68.2) |
≥50 | 173 (83.2) | 53 (43.4) | 76 (62.3) | 37 (30.3) | 91 (74.6) |
χ2 (p value) | 0.53 (0.465) | 0.20 (0.654) | 0.22 (0.640) | 0.13 (0.715) | |
Stage | |||||
I + II | 106 (51.0) | 33 (45.8) | 42 (58.3) | 29 (40.3) | 53 (73.6) |
III + IV | 102 (49.0) | 32 (44.4) | 46 (63.9) | 13 (18.1) | 53 (73.6) |
χ2 (p value) | <0.01 (1.000) | 0.26 (0.608) | 7.56 (0.006) a | <0.01 (1.000) | |
Tumor location 1 | |||||
Colon | 37 (20.1) | 11 (40.7) | 14 (51.9) | 30 (28.3) | 75 (70.8) |
Rectum | 147 (79.9) | 50 (47.2) | 66 (62.3) | 7 (25.9) | 23 (85.2) |
χ2 (p value) | 0.15 (0.702) | 0.59 (0.443) | <0.01 (0.996) | 1.63 (0.202) | |
Tumor size 1 | |||||
≤5 cm | 114 (63.0) | 40 (48.8) | 46 (56.1) | 18 (22.0) | 57 (69.5) |
>5 cm | 67 (37.0) | 20 (40.0) | 33 (66.0) | 19 (38.0) | 40 (80.0) |
χ2 (p value) | 0.64 (0.422) | 0.89 (0.346) | 3.21(0.073) | 1.26 (0.262) | |
Lymph node counts 1 | |||||
0–11 | 34 (18.4) | 7 (35.0) | 11 (55.0) | 6 (30.0) | 15 (75.0) |
≥12 | 151 (81.6) | 54 (47.4) | 70 (61.4) | 31 (27.2) | 84 (73.7) |
χ2 (p value) | 0.61 (0.435) | 0.09 (0.770) | <0.01 (1.000) | <0.01 (1.000) | |
Histological grade 1 | |||||
Well or Moderate | 156 (89.7) | 49 (44.5) | 66 (60.0) | 31 (28.2) | 83 (75.5) |
Poor or undifferentiated | 18 (10.3) | 8 (53.3) | 8 (53.3) | 5 (33.3) | 9 (60.0) |
χ2 (p value) | 0.13 (0.715) | 0.05 (0.831) | 0.01 (0.913) | 0.93 (0.336) | |
Adjuvant chemotherapy 1 | |||||
No | 54 (29.3) | 21 (52.5) | 26 (65.0) | 22 (23.7) | 32 (80.0) |
Yes | 130 (70.7) | 40 (43.0) | 54 (58.1) | 15 (37.5) | 66 (71.0) |
χ2 (p value) | 0.67 (0.414) | 0.31 (0.614) | 2.03 (0.155) | 0.76 (0.384) | |
5-year recurrence | |||||
No | 149 (71.6) | 48 (45.3) | 22 (57.9) | 32 (31.1) | 74 (71.8) |
Yes | 59 (28.4) | 17 (44.7) | 66 (62.3) | 10 (24.4) | 32 (78.0) |
χ2 (p value) | <0.01 (1.000) | 0.08 (0.779) | 0.35 (0.554) | 0.31 (0.580) | |
5-year progression | |||||
No | 116 (55.8) | 36 (46.2) | 45 (57.7) | 28 (29.5) | 68 (71.6) |
Yes | 92 (44.2) | 29 (43.9) | 43 (65.2) | 14 (28.6) | 38 (77.6) |
χ2 (p value) | <0.01 (0.922) | 0.55 (0.457) | <0.01 (1.000) | 0.33 (0.568) | |
5-year all-cause death | |||||
No | 168 (80.8) | 59 (49.6) | 69 (58.0) | 33 (27.7) | 88 (73.9) |
Yes | 40 (19.2) | 6 (24.0) | 19 (76.0) | 9 (36.0) | 18 (72.0) |
χ2 (p value) | 4.48 (0.034) a | 2.12 (0.146) | 0.34 (0.559) | <0.01 (1.000) |
Methylation Status | |||
---|---|---|---|
PTGER4 a | ZNF43 a | ||
Normal | Tumor | Normal | Tumor |
65 (45.1) | 88 (61.1) | 42 (29.2) | 106 (73.6) |
Normal | Tumor | p Value 3 | |||||
---|---|---|---|---|---|---|---|
n 1 | Median (Q1–Q3) (%) | Mean ± SD 2 (%) | n 1 | Median (Q1–Q3) (%) | Mean ± SD 2 (%) | ||
PTGER4 | |||||||
CpG_1.2 | 143 | 6 (3–8) | 6.78 ± 2.4 | 144 | 6 (4.3–8) | 7.37 ± 7.0 | 0.322 |
CpG_3 | 144 | 4 (3–6) | 4.74 ± 2.7 | 140 | 3 (2–4) | 4.64 ± 8.3 | <0.001 |
CpG_4.5 | 141 | 11 (8–14) | 12.06 ± 6.5 | 141 | 10 (7–13) | 11.24 ± 7.5 | 0.044 |
CpG_6.7 | 142 | 6 (5–8) | 6.65 ± 3.2 | 144 | 5 (3.3–7) | 6.38 ± 7.4 | <0.001 |
CpG_9.10 | 143 | 9 (8–12) | 10.14 ± 4.4 | 143 | 7 (5–10) | 8.71 ± 8.4 | <0.001 |
CpG_11 | 144 | 10.5 (9–13) | 11.56 ± 5.0 | 144 | 8 (6–11.8) | 10.10 ± 8.5 | <0.001 |
CpG_13 | 144 | 16 (13–18) | 16.42 ± 5.8 | 144 | 11.5 (9–15) | 13.07 ± 7.6 | <0.001 |
CpG_15 | 142 | 16 (12–21) | 18.05 ± 10.8 | 139 | 11 (6–16) | 13.24 ± 11.1 | <0.001 |
CpG_16 | 141 | 29 (26–33) | 29.27 ± 5.8 | 143 | 23 (18–30) | 25.36 ± 10.0 | <0.001 |
CpG_17 | 84 | 18 (14–22) | 18.21 ± 6.9 | 85 | 14 (10–18) | 15.05 ± 8.2 | 0.052 |
CpG_18 | 143 | 27 (23–32) | 28.31 ± 8.5 | 144 | 20 (16–29) | 24.10 ± 13.8 | <0.001 |
ZNF43 | |||||||
CpG_2 | 143 | 4 (3.0–6.0) | 5.17 ± 0.25 | 143 | 9 (4.0–29.0) | 18.10 ± 1.56 | <0.001 |
CpG_3 | 144 | 3 (2.0–3.0) | 3.15 ± 0.26 | 144 | 9 (3.0–36.0) | 20.39 ± 1.84 | <0.001 |
CpG_4 | 131 | 1 (1.0–3.0) | 1.98 ± 0.24 | 140 | 11 (2.0–36.0) | 20.31 ± 1.87 | <0.001 |
CpG_5 | 144 | 6 (4.0–9.0) | 8.99 ± 0.77 | 143 | 16 (6.0–38.0) | 23.08 ± 1.77 | <0.001 |
CpG_6 | 84 | 3 (2.0–4.8) | 4.07 ± 0.37 | 115 | 16 (4.0–39.0) | 22.65 ± 1.82 | <0.001 |
CpG_7.8 | 144 | 4 (4.0–5.0) | 4.60 ± 0.22 | 144 | 9 (4.0–35.0) | 21.24 ± 1.76 | <0.001 |
RFS | PFS | OS | ||||
---|---|---|---|---|---|---|
cHR (95% CI) | aHR (95% CI) 1 | cHR (95% CI) | aHR (95% CI) 1 | cHR (95% CI) | aHR (95% CI) 2 | |
PTGER4 in tumor tissue | ||||||
all CpG sites | ||||||
hypomethylation | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) |
hypermethylation | 1.22 (0.64–2.30) | 1.03 (0.51–2.08) | 1.47 (0.91–2.40) | 1.33 (0.76–2.31) | 2.18 (0.94–5.04) | 1.77 (0.74–4.24) |
p value | 0.550 | 0.945 | 0.121 | 0.263 | 0.070 | 0.201 |
CpG_4.5 a | ||||||
hypomethylation | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) |
hypermethylation | 1.36 (0.71–2.58) | 1.04 (0.51–2.12) | 1.60 (0.97–2.63) | 1.33 (0.76–2.30) | 1.98 (0.85–4.58) | 1.72 (0.72–4.09) |
p value | 0.354 | 0.917 | 0.064 | 0.317 | 0.112 | 0.222 |
CpG_15 a | ||||||
hypomethylation | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) |
hypermethylation | 1.86 (0.94–3.66) | 1.06 (0.48–2.33) | 1.39 (0.85–2.29) | 1.07 (0.60–1.90) | 2.11 (0.91–4.88) | 1.55 (0.63–3.79) |
p value | 0.075 | 0.895 | 0.195 | 0.829 | 0.082 | 0.339 |
CpG_17 a | ||||||
hypomethylation | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) |
hypermethylation | 1.87 (0.79–4.41) | 2.08 (0.79–5.48) | 1.60 (0.84–3.04) | 1.85 (0.89–3.84) | 2.60 (0.83–8.17) | 2.55 (0.80–8.05) |
p value | 0.155 | 0.137 | 0.156 | 0.099 | 0.103 | 0.112 |
ZNF43 in normal tissue | ||||||
CpG_5 | ||||||
hypomethylation | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) |
hypermethylation | 1.81 (0.94–3.49) | 2.33 (1.07–5.08) b | 1.78 (0.98–3.23) | 2.42 (1.19–4.91) b | 1.16 (0.52–2.58) | 1.13 (0.48–2.67) |
p value | 0.077 | 0.014 | 0.059 | 0.014 | 0.716 | 0.775 |
n 1 | RFS | PFS | OS | ||||
---|---|---|---|---|---|---|---|
cHR (95% CI) | aHR (95% CI) 2 | cHR (95% CI) | aHR (95% CI) 3 | cHR (95% CI) | aHR (95% CI) 4 | ||
CpG_4.5 + CpG_15 hypermethylation | |||||||
No | 90 | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) |
Yes | 49 | 2.14 (1.12–4.07) a | 1.54 (0.74–3.18) | 1.73 (1.06–2.84) a | 1.29 (0.73–2.28) | 2.63 (1.19–5.80) a | 2.19 (0.95–5.05) |
p value | 0.021 | 0.248 | 0.030 | 0.385 | 0.016 | 0.066 | |
CpG_4.5 + CpG_17 hypermethylation | |||||||
No | 79 | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) |
Yes | 26 | 2.92 (1.37–6.22) a | 3.44 (1.53–7.72) a,b | 2.74 (1.53–4.92) a,b | 2.45 (1.26–4.76) a | 4.11 (1.62–10.4) a,b | 3.79 (1.46–9.88) a |
p value | 0.005 | 0.003 | 0.001 | 0.008 | 0.003 | 0.006 | |
CpG_15 + CpG_17 hypermethylation | |||||||
No | 79 | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) |
Yes | 34 | 1.91 (0.89–4.11) | 1.71 (0.73–4.01) | 1.56 (0.87–2.78) | 1.42 (0.74–2.74) | 2.89 (1.17–7.11) a | 2.36 (0.93–5.97) |
p value | 0.099 | 0.214 | 0.133 | 0.295 | 0.021 | 0.071 | |
CpG_4.5 + CpG_15 + CpG_17 hypermethylation | |||||||
No | 96 | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) | 1.00 (Reference) |
Yes | 21 | 3.18 (1.45–6.97) a,b | 3.26 (1.38–7.73) a | 2.77 (1.52–5.05) a,b | 2.35 (1.17–4.71) a | 4.79 (2.00–11.4) a,b | 4.32 (1.8–10.5) a,b |
p value | 0.004 | 0.007 | 0.001 | 0.016 | <0.001 | 0.001 |
Genes | Forward Primer (5′→3′) | Annealing Temperature (°C) | Product Size (bp) | |
---|---|---|---|---|
PTGER4 | M | F: GTTTTATTTCGTTCGTGGTGA | 58.6 | 247 |
R: AAAAAAAAAACCCAAACTTCC | ||||
U | F: GGGTTGGGGTTTTATTTGGTT | 64.3 | 315 | |
R: CAACAAACTCCCCTCCACATC | ||||
Q | F: ATTTTTTTGGTGGTGTTTATTTGTT a | 57.9 | 440 | |
R: TCAAATTTTACAATTCACAATTCACA a | ||||
ZNF43 | M | F: GGAGGAAGTTTTGTTTGAAAAGGC | 61.3 | 323 |
R: TTCTAAACTTCCGAAAAATCCTAAC | ||||
U | F: GAAGTTTTGTTTGAAAAGGTGG | 59.5 | 328 | |
R: ACCATTTCTAAACTTCCAAAA | ||||
Q | F: AAGGTTAAAGGTAAATATTTTTTGGG a | 60.8 | 267 | |
R: TCCAACTACAACCAAAAACAAAAAC a |
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Chen, C.-Y.; Wu, J.-J.; Lin, Y.-J.; Hsu, C.-H.; Hu, J.-M.; Chang, P.-K.; Sun, C.-A.; Yang, T.; Su, J.-Q.; Chou, Y.-C. Significance of Hypermethylation of Tumor-Suppressor Genes PTGER4 and ZNF43 at CpG Sites in the Prognosis of Colorectal Cancer. Int. J. Mol. Sci. 2022, 23, 10225. https://doi.org/10.3390/ijms231810225
Chen C-Y, Wu J-J, Lin Y-J, Hsu C-H, Hu J-M, Chang P-K, Sun C-A, Yang T, Su J-Q, Chou Y-C. Significance of Hypermethylation of Tumor-Suppressor Genes PTGER4 and ZNF43 at CpG Sites in the Prognosis of Colorectal Cancer. International Journal of Molecular Sciences. 2022; 23(18):10225. https://doi.org/10.3390/ijms231810225
Chicago/Turabian StyleChen, Chao-Yang, Jia-Jheng Wu, Yu-Jyun Lin, Chih-Hsiung Hsu, Je-Ming Hu, Pi-Kai Chang, Chien-An Sun, Tsan Yang, Jing-Quan Su, and Yu-Ching Chou. 2022. "Significance of Hypermethylation of Tumor-Suppressor Genes PTGER4 and ZNF43 at CpG Sites in the Prognosis of Colorectal Cancer" International Journal of Molecular Sciences 23, no. 18: 10225. https://doi.org/10.3390/ijms231810225