Table 1
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 |
[i] DKK2, gene for Dickkopf 2; F, forward primer; GAPDH, gene for glyceraldehyde-3-phosphate dehydrogenase; M, methylated-specific primers; PCR, polymerase chain reaction; R, reverse primer; U, unmethylated-specific primers.
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
Table 2
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 |
[i] DKK2, Dickkopf 2; HSIL, high-grade squamous intraepithelial lesions; LSIL, low-grade squamous intraepithelial lesions.
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.
Table 3
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%) | ||
[i] DDK2, Dickkopf 2.