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Promoter methylation analysis of DKK2 may be a potential biomarker for early detection of cervical cancer

Xian Zhang
Xian Zhang
, 
Aihua Li
Aihua Li
, 
Jie Wu
Jie Wu
, 
Yu Wu
Yu Wu
, 
Xiaoping Ma
Xiaoping Ma
, 
Yanjun Liu
Yanjun Liu
, 
Qingfa Chen
Qingfa Chen
 and 
Yan Zhang
Yan Zhang
   | Aug 31, 2022
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Article Category: Original article
Published Online: Aug 31, 2022
Page range: 181 - 189
DOI: https://doi.org/10.2478/abm-2022-0022
Keywords
© 2022 Xian Zhang et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

DKK2 mRNA expression in cervical cells and tissues. (A–E) RT-PCR analysis of DKK2 mRNA expression in cervical normal and cancer tissues. **P < 0.01 compared with normal tissues. (F) RT-PCR analysis of DKK2 mRNA expression in normal cervical epithelial cell lines and in 9 human cervical cancer cell lines. *P < 0.05 compared with normal cervical epithelial cell lines. (G) RT-PCR analysis of DKK2 mRNA relative expression in normal cervical epithelial cell lines and 4 human cervical cancer cell lines (Hela, Caski, HT-3, and C33A cell lines). **P < 0.01 compared with normal cervical epithelial cell lines. (H) DKK2 mRNA expression in LSIL, HSIL, tumor, and normal cervical tissues. The PCR products were separated in 2% agarose gels using a DL1000 marker set (catalog No. 3591A; Takara) marker set; the band of interest is between markers 100 bp and 200 bp at 169 bp. DKK2, Dickkopf 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H, HSIL; L, LSIL; N, normal; RT-PCR, reverse transcription-polymerase chain reaction; T, tumor; TCGA, The Cancer Genome Atlas; bp, base pairs
DKK2 mRNA expression in cervical cells and tissues. (A–E) RT-PCR analysis of DKK2 mRNA expression in cervical normal and cancer tissues. **P < 0.01 compared with normal tissues. (F) RT-PCR analysis of DKK2 mRNA expression in normal cervical epithelial cell lines and in 9 human cervical cancer cell lines. *P < 0.05 compared with normal cervical epithelial cell lines. (G) RT-PCR analysis of DKK2 mRNA relative expression in normal cervical epithelial cell lines and 4 human cervical cancer cell lines (Hela, Caski, HT-3, and C33A cell lines). **P < 0.01 compared with normal cervical epithelial cell lines. (H) DKK2 mRNA expression in LSIL, HSIL, tumor, and normal cervical tissues. The PCR products were separated in 2% agarose gels using a DL1000 marker set (catalog No. 3591A; Takara) marker set; the band of interest is between markers 100 bp and 200 bp at 169 bp. DKK2, Dickkopf 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H, HSIL; L, LSIL; N, normal; RT-PCR, reverse transcription-polymerase chain reaction; T, tumor; TCGA, The Cancer Genome Atlas; bp, base pairs

Figure 2

DKK2 methylation level in cervical cancer cell lines and tissues. (A) MSP analysis of DKK2 hypermethylation in Hela, Caski, HT-3, and C33A cervical cancer cell lines. (B) DKK2 expression after treatment with 5-Aza-dC for 4 d in cervical cancer cell lines. GAPDH was used as a housekeeping control gene. (C) DKK2 methylation levels in TCGA samples analyzed by data mining. **P < 0.01 compared with normal tissues. (D) MSP analysis of DKK2 CGIs methylation frequency in primary cervical cancer tissues. CGIs, CpG islands; DKK2, Dickkopf 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M or MSP, methylation-specific polymerase chain reaction (PCR); TCGA, The Cancer Genome Atlas; U, unmethylated-specific PCR.
DKK2 methylation level in cervical cancer cell lines and tissues. (A) MSP analysis of DKK2 hypermethylation in Hela, Caski, HT-3, and C33A cervical cancer cell lines. (B) DKK2 expression after treatment with 5-Aza-dC for 4 d in cervical cancer cell lines. GAPDH was used as a housekeeping control gene. (C) DKK2 methylation levels in TCGA samples analyzed by data mining. **P < 0.01 compared with normal tissues. (D) MSP analysis of DKK2 CGIs methylation frequency in primary cervical cancer tissues. CGIs, CpG islands; DKK2, Dickkopf 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M or MSP, methylation-specific polymerase chain reaction (PCR); TCGA, The Cancer Genome Atlas; U, unmethylated-specific PCR.

DKK2 mRNA expression and methylation in cervical neoplasms

Diagnosis n mRNA expression (%) P Methylation (%) P
Normal 29 29/29 (100) 0/29 (0)
LSIL 25 25/25 (100) 0.00003 1/25 (4.0) 0.005
HSIL 38 21/38 (55.2) 0.001 9/38 (23.7) 0.037
Cervical cancer 79 25/79 (31.6) 0.014 41/79 (51.9) 0.004

Relationship between HR-HPV infection type and DKK2 methylation rate

Group HR-HPV (+)n DKK2 methylation P
n %
Cervical cancer 0.015
HPV16/18(+) 48 32 66.7
HPV16/18(−) 25 9 36
Precancerous lesions 0.48
HPV16/18(+) 29 7 24.1
HPV16/18(−) 23 3 15

DKK2 mRNA expression, DKK2 promoter methylation and clinicopathological characteristics of cervical cancer

Clinical status n DKK2 mRNA expression DKK2 methylation
+ P + P
Clinical stage
Stage IA2 28 13 (46.4%) 15 (53.6%) 0.099 12 (42.9%) 16 (57.1%) 0.32
Stage IB1+IB2 41 9 (22.0%) 32 (78.0%) 22 (53.7%) 19 (46.3%)
Stage IIA 10 3 (30.0%) 7 (70.0%) 7 (70.0%) 3 (30.0%)
Histology
Squamous cell carcinoma 70 21 (30.0%) 49 (70.0%) 0.38 36 (51.4%) 34 (48.6%) 0.55
Adenocarcinoma 9 4 (44.4%) 5 (55.6%) 5 (55.6%) 4 (44.4%)
Tumor grade
Grade 1 15 5 (33.3%) 10 (66.7%) 0.81 6 (40.0%) 9 (60.0%) 0.17
Grade 2 42 12 (28.6%) 30 (71.4%) 20 (47.6%) 22 (52.4%)
Grade 3 22 8 (36.4%) 14 (63.6%) 15 (68.2%) 7 (31.8%)
Lymph node metastasis
Negative 62 23 (37.1%) 39 (62.9%) 0.047 28 (45.2%) 34 (54.8%) 0.022
Positive 17 2 (11.8%) 15 (88.2%) 13 (76.5%) 4 (23.5%)

Primer pairs used for mRNA expression and the size of PCR products

Gene Primer information (5′-3′) Product size
DKK2 5′-GTACCAAGGACTGGCATTCG-3′ (F)5′-ATCTCGGTGGCAGCGCTTCT-3′ (R) 169 bp
GAPDH 5′-CCAGCAAGAGCACAAGAGGAA-3′ (F)5′-CAAGGGGTCTACATGGCAACT-3′ (R) 114 bp
DKK2 (M) 5′-AGAGTTAAATCGTCGAGATTTC-3′ (F)5′-CTAAAAACAATCAAATACGAAACG-3′ (R) 146 bp
DKK2 (U) 5′-GGAGAGTTAAATTGTTGAGATTTT-3′ (F)5′-ACTAAAAACAATCAAATACAAAACA-3′ (R) 149 bp
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