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Simultaneously both expression of LMP-1 and methylation of E-cadherin: Molecular biomarker in stage IV of nasopharyngeal carcinoma patients

TD Lao
TD Lao
, 
PK Truong
PK Truong
, 
HH Thieu
HH Thieu
, 
DH Nguyen
DH Nguyen
, 
MT Nguyen
MT Nguyen
 et 
TAH Le
TAH Le
   | 27 juil. 2021
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Article Category: Original Article
Publié en ligne: 27 juil. 2021
Pages: 57 - 66
DOI: https://doi.org/10.2478/bjmg-2021-0005
Mots clés
© 2021 Lao TD, Truong PK, Thieu HH, Nguyen DH, Nguyen MT, Le TAH, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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.
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.

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.
(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.
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.

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.
Summary of LMP-1 expression and methylation of the E-cadherin gene in 45 stage IV NCP samples. Filled box: positive, open box: negative.

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

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

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

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)

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)
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