Table 1
The primer sequences used.
| For evaluation of LMP-1 expression | For evaluation of LMP-1 expression |
|---|---|
| Primers [Refs.] | Sequences (5’>3’) |
| LMP-1-F [18] | CAG TCA GGC AAG CCT ATG A |
| LMP-1-R [18] | CTG GTT CCG GTG GAG ATG A |
| GAPDH-F [19] | TGC CTC CTG CAC CAC CAA CT |
| GAPDH-R [19] | CGC CTG CTT CAC CAC CTT C |
| For evaluation of E-cadherin methylation | ||||
|---|---|---|---|---|
| Primers [Refs.] | Sequences (5’>3’) | Temperature | Product | |
| Stage 1 | Seq-F [16] Seq-R [16] | TTT TAG GTT AGA GGG TTA T CTA CAA CAA CAA CAA CAA C | 50 °C | 350 bp |
| Stage 2 | E-cad-MF [16] E-cad-MR [16] | GGT GAA TTT TTA GTT AAT TAG CGG TAC CAT AAC TAAC CGA AAA CGC CG | 60 °C | 205 bp |
| Stage 2 | E-cad-UF [16] E-cad-UR [16] | GGT AGG TGA ATT TTT AGT TAA TTA GTG GTA ACC CAT AAC TAA CCA AAA ACA ACA | 60 °C | 211 bp |
Table 2
Characteristics of enrolled clinical nasopharyngeal carcinoma NPC samples.
| n = 93 | n (%) |
|---|---|
| Gender: | |
| males | 68 (73.12) |
| females | 25 (26.88) |
| Age (years: | |
| <20 | 1 (1.08) |
| 20 to 40 | 17 (18.28) |
| 40 to 60 | 43 (46.23) |
| 60 to 80 | 31 (33.33) |
| >80 | 1 (1.08) |
| Histological typea: | |
| type 1 | 4 (4.30) |
| type 2 | 26 (27.96) |
| type 3 | 63 (67.74) |
| Stage: | |
| I | 0 (0.00) |
| II | 33 (35.48) |
| III | 15 (16.13) |
| IV | 45 (48.39) |
Figure 1
The mean Ct value and percentage of GAPDH, LMP-1 and E-cadherin in the case and control groups. Each black dot indicated the Ct value of GAPDH, LMP-1 in each sample. Each bar indicated the positive percentage of GAPDH, LMP-1 and E-cadherin in case and control groups.
Table 3
Association between the expression of LMP-1, methylation of E-cadherin and clinicopathological parameters.
| LMP-1 | E-Cadherin | |||
|---|---|---|---|---|
| Positive, n (%) | Negative, n (%) | Methylated, n (%) | Unmethylated, n (%) | |
| Gender: | ||||
| males | 51 (54.83) | 17 (18.28) | 46 (49.46) | 22 (23.66) |
| females | 20 (21.51) | 5 (5.38) | 22 (23.66) | 3 (3.23) |
| p Value | 0.06 | 0.05 | ||
| Age (years):<20 | 1 (1.08) | 0 (0.00) | 1 (1.08) | 0 (0.00) |
| 20 to 40 | 11 (11.83) | 6 (6.45) | 12 (12.90) | 5 (5.38) |
| 40 to 60 | 36 (38.71) | 7 (7.53) | 32 (34.41) | 11 (11.83) |
| 60 to 80 | 23 (24.73) | 8 (8.60) | 23 (24.73) | 8 (8.60) |
| >80 | 0 (0.00) | 1 (1.08) | 0 (0.00) | 1 (1.08) |
| p Value | 0.19 | 0.52 | ||
| Histological typea: | ||||
| type 1 | 3 (3.23) | 1 (0.00) | 4 (4.30) | 0 (0.00) |
| type 2 | 17 (18.28) | 9 (9.68) | 16 (17.20) | 10 (10.75) |
| type 3 | 51 (54.83) | 12 (12.90) | 48 (51.61) | 15 (16.13) |
| p Value | 0.29 | 0.16 | ||
| Stage: | ||||
| I | 0 (0.00) | 0 (0.00) | 0 (0.00) | 0 (0.00) |
| II | 26 (27.96) | 7 (7.53) | 22 (23.66) | 11 (11.83) |
| III | 8 (8.60) | 7 (7.53) | 9 (9.68) | 6 (6.45) |
| IV | 37 (39.78) | 8 (8.60) | 37 (39.78) | 8 (8.60) |
| p Value | 0.07 | 0.14 | ||
Figure 2
(A) The schematic diagram represented the E-cadherin gene promoter and its CpG island. MF: methylated forward primer; MR: methylated reverse primer; UF: unmethylated forward primer; UR: unmethylated reverse primer; MP: amplified methylated product by methylated primers; UP: amplified unmethylated product by unmethylated primers. Numbers indicated the position of nucleotide. (B) Methylated promoter of the E-cadherin gene was analyzed on some clinical samples by MSP. (The MSP product was 205/211 bps in length. Lanes 1, 2 and 3: NPC biopsy samples; lanes 4, 5 and 6: healthy samples; lane (–): negative control; MW: 100 bps ladder; M: methylated; U: unmethylated.
Figure 3
Sequencing profile of (A) methylated and (B) unmethylated of the E-cadherin gene promoter. (1) DNA sequence was without bisulfite modification; (2) DNA sequence was bisulfite-modified; (3) amplified sequencing DNA by forward primer; (4) amplified sequencing DNA by reversed primer; square symbols: methylated cytosine; triangle symbols: unmethylated cytosine.
Table 4
Statistical analysis of association between LMP-1 expression and methylation of E-cadherin in nasopharyngeal carcinoma samples.
| LMP-1 Expression | |||
|---|---|---|---|
| Positive, n (%) | Negative, n (%) | p Value | |
| E-cadherin methylation: | |||
| positive, n (%) | 61 (65.59) | 7 (7.53) | <0.0001 |
| negative, n (%) | 10 (10.75) | 15 (16.13) | |
Figure 4
Summary of LMP-1 expression and methylation of the E-cadherin gene in 45 stage IV NCP samples. Filled box: positive, open box: negative.
Table 5
Association between the positive index and clinicopathological parameters.
| LMP-1, E-Cadherin | |||
|---|---|---|---|
| PI = 0, n (%) | PI = 0.5, n (%) | PI = 1.0, n (%) | |
| Gender: | |||
| males | 14 (15.06) | 11 (11.83) | 43 (46.24) |
| females | 1 (1.08) | 6 (6.45) | 18 (19.35) |
| p Value | 0.14 | ||
| Age (years): | |||
| <20 | 0 (0.00) | 0 (0.00) | 1 (1.08) |
| 20 to 40 | 3 (3.23) | 5 (5.38) | 9 (9.68) |
| 40 to 60 | 6 (6.45) | 6 (6.45) | 31 (33.33) |
| 60 to 80 | 5 (5.38) | 6 (6.45) | 20 (21.51) |
| >80 | 1 (1.08) | 0 (0.00) | 0 (0.00) |
| p Value | 0.42 | ||
| Histological typea: | |||
| type 1 | 0 (0.00) | 1 (1.08) | 3 (3.23) |
| type 2 | 7 (7.53) | 5 (5.38) | 14 (15.05) |
| type 3 | 8 (8.60) | 11 (11.83) | 44 (47.31) |
| p Value | 0.42 | ||
| Stage: | |||
| I | 0 (0.00) | 0 (0.00) | 0 (0.00) |
| II | 4 (4.30) | 10 (10.75) | 19 (20.43) |
| III | 5 (5.38) | 3 (3.23) | 7 (7.53) |
| IV | 6 (6.45) | 4 (4.30) | 35 (37.63) |
| p Value | 0.04 | ||
[i] PI: positive index; PI = 0: both LMP-1 and methylation of E-cadherin were negative; PI = 0.5: either LMP-1 or E-cadherin was positive;
PI = 1.0: both LMP-1 and methylation of E-cadherin were positive.
a Type 1: keratinizing squamous cell carcinoma; type 2: nonkeratinizing squamous cell carcinoma; type 3: undifferentiated carcinoma.