Infection of HPV 16 and 18 in cervical tissues with different degrees of lesion
Statistical analysis was performed on HPV16 and HPV18 infection in cervical tissues of 154 women. The results showed that in normal and chronic cervicitis, CIN, and cervical cancer tissues, the HPV16 infection rates were 19.6, 34.3, and 64.7%, respectively, and demonstrated an increasing trend with severity. The infection rates of HPV18 were 2, 8.6, and 13.2%, respectively. The multiple comparison of infection rates of HPV16 in the above groups, and the difference were statistically significant (P < 0.05), while that of HPV18 was not. HPV16 and HPV18 infection rates were statistically significant in the CIN and cervical cancer groups, and the difference was also significant in the normal and cervical cancer groups. (Table 2).
Table 2
The infection status of HPV16/18
groups | HPV16 | HPV18 | HPV16/18 |
infection(%) | χ2 value | P value | infection(%) | χ2 value | P value | infection(%) | χ2 value | P value |
normal | 19.6(10/51) | 45.75 | 0.000* | 2.0(1/51) | 0.826 | 0.363 | 21.6(11/51) | 1.713 | 0.191 |
CIN | 34.3(12/35) | 8.619 | 0.003∆ | 8.6(3/35) | 0.14 | 0.708 | 34.3(12/35) | 9.508 | 0.002∆ |
cancer | 64.7(44/68) | 23.913 | 0.000# | 13.2(9/68) | 3.459 | 0.063 | 66.2(45/68) | 23.277 | 0.000# |
*:P < 0.05, normal group compared with CIN group ;∆ :P < 0.05, CIN group compared with cancer group ; #:P < 0.05, normal group compared with cancer group. |
The ZNF582 gene promoter is highly methylated in cervical cancer tissues.
We tested the methylation level of the ZNF582 gene promoter in the collected samples. ZNF582 methylation occurred in nine of 51 normal cervical tissues (17.6%), 16 of 35 CIN tissues (45.7%), and 50 out of 68 cervical cancer cases (73.5%). The difference in methylation rates of the ZNF582 gene in the three groups was statistically significant (P < 0.05). With the development of the disease, the probability of methylation showed an increasing trend. (Table 3)
Table 3
The frequency of the ZNF582 promoter methylation
Groups | Total | Methylated(%) | Unmethylated(%) | χ2 value | P value |
normal | 51 | 17.6( 9/51) | 82.4(42/51) | 7.930 | 0.005* |
CIN | 35 | 45.7(16/35) | 54.3(19/35) | 7.767 | 0.005∆ |
cancer | 68 | 73.5(50/68) | 26.5(18/68) | 36.406 | 0.000# |
*: Normal group compared with the CIN group ;∆ : CIN group compared with the cancer group ;#: normal group compared with the cancer group |
DNA methylation was modified in the ZNF582 genomic promoter region and we designed methylation and non-methylation specific primers. The non-methylated PCR product was 142 bp. A band corresponding to the methylated promoter was present in the sample with methylation, and absent in the unmethylated sample. (Fig. 1)
Relationship between methylation of ZNF582 gene and HPV infection in cervical cancer tissues
Among 93 high grade cervical lesions and cervical cancer tissue samples, 57 HPV16/18 positive and 36 negative samples were included. In the positive group, 42 cases (73.7%) were methylated at the ZNF582 promoter. The negative group had 23 cases (63.9%) of methylation. There was no statistically significant difference between the two groups (χ2 = 1.006, P > 0.05) (Table 4).
Table 4
The distribution of ZNF582 promoter methylation and HPV16/18 infection of in CINII, III and cervical cancer tissues
Group | Total | Methylated | Unmethylated | Methylation rate(%) |
HPV16/18 positive | 57 | 42 | 15 | 73.7 |
HPV16/18 negative | 36 | 23 | 13 | 63.9 |
χ2 = 1.006 P = 0.316 |
The mRNA expression level of ZNF582 promoter methylation was significantly reduced.
The mRNA expression in 10 cases of cervical cancer with methylation was 0.46 ± 0.33, while in 10 cases of normal cervical tissue with no methylation the RNA expression was 1.05 ± 0.16. The expression level of mRNA, in the methylation positive group, was significantly lower than the negative group (P < 0.01), the differences were statistically significant. (Table 5, Fig. 2)
Table 5
ZNF582 gene expression analysis
Groups | mRNA expression (`x ± s) |
Methylation negative | 1.0493 ± 0.16315 |
Methylation positive | 0.4554 ± 0.32580 |
P | < 0.000 |
Correlation between promoter methylation of ZNF582 gene and clinical pathological factors
The results showed that there was no correlation between age, FIGO stage or ZNF582 gene promoter methylation (P > 0.05) in 68 cases of cervical cancer (Table 6).
Table 6
ZNF582 promoter methylation with clinical information of cervical cancer patients
Clinical factor | Total | Methylated | Unmethylated | Methylation rate(%) | χ2 value | P value |
Age/year | | | | | | |
< 50 | 38 | 26 | 12 | 68.4(26/38) | 1.155 | 0.283 |
≥ 50 | 30 | 24 | 6 | 80.0(24/30) |
FIGO stage | | | | | | |
I | 42 | 30 | 12 | 71.4(30/42) | 0.753 | 0.785 |
II | 18 | 13 | 5 | 72.2(13/18) |
III | 8 | 7 | 1 | 87.5(7/8) |
Diagnostic efficacy of ZNF582 promoter methylation and HPV16 /18 infection
We compared the sensitivity, specificity, and positive and negative predictive value of ZNF582 promoter methylation and HPV16/18 infection detection in normal cervical tissue, low-grade cervical intraepithelial neoplasia (CINI), high-grade cervical intraepithelial neoplasia (CINII and CINIII) and cervical cancer tissue. The results showed that with the sensitivity and specificity of cervical cancer diagnosis, the ZNF582 promoter methylation was 69.9% and 83.6%, respectively. The sensitivity and specificity of methylation of ZNF582 gene promoter was higher than that of HPV16/18 infection, and its sensitivity and specificity are 61.3% and 82.0%, respectively (Table 7).
Table 7
Sensitivity and positive predictive value detection HSIL and cervical cancer tissues, negative predictive value and specific analysis of the normal tissues and LSIL
Groups | Sensitivity(%) | Specificity(%) | PPV(%) | NPV(%) |
HSIL/cancer | Normal | Normal/LSIL | HSIL/Cancer | Normal | Normal /LSIL |
HPV16/18 | 61.3(57/93) | 78.4(40/51) | 82.0(50/61) | 83.8(57/68) | 52.6(40/76) | 65.8(50/76) |
ZNF582 | 69.9(65/93) | 84.3(42/51) | 83.6(51/61) | 86.7(65/75) | 53.2(42/79) | 64.6(51/79) |
HSIL: High grade cervical squamous cell injury,including CINII and CINIII. LSIL: Low grade cervical squamous cell injury, including CINI. PPV: positive predictive value. NPV: positive predictive value |
ZNF582 protein expression is increased expression in cervical cancer tissues.
We analyzed ZNF582 protein levels in 88 tissues of cervical cancer tissue and normal cervical tissue by immunohistochemistry. Compared with normal tissues, ZNF582 expression was abnormally upregulated; 86 of 87 cancer patient samples were positive, suggesting ZNF582 protein expression levels can be used to diagnosis cervical cancer (Table 8, Fig. 3).
Table 8
Statistical analysis of ZNF582 expression in cervical cancer tissues and cervicitis tissues
| Negative | Weak | Moderate | Strong | Total | Z value | P value |
Cervicitis tissues | 10 | 28 | 26 | 4 | 68 | 47.57 | < 0.0001 |
Cervical cancer tissues | 1 | 2 | 38 | 46 | 87 | 101.78 |
Experimental results were tested by the Wilcoxon rank sum test. Data show that ZNF582 was highly expressed in cancer tissues, and the difference was significant. |