Abstract
Objective: To analyze the distribution of HLA-DRB1 and HLA-DQB1 alleles and its correlation with IFN-γ, IL-2, IL-6, IL-10 in HPV16 infected women with advanced cervical carcinoma. Methods: We collected 137 blood samples of cervical carcinoma patients diagnosed by pathology as cervical cancer in stage IIb-IVb before the treatment, and we gathered 175 blood samples of healthy women living in the local. We determined the genetic subtypes of HLA-DRB1 and HLA-DQB1, and we measured the concentration of IFN-γ, IL-2, IL-6 and IL-10. We compared the difference of cytokines in patients with different clinical stages and the healthy in the control group. According to genetic subtypes of HLA-DRB1 and HLA-DQB1, we also compared the concentration of cytokine (CK) in different genetic subtypes. Results: Eight HLA-DRB1 alleles and four HLA-DQB1 alleles were found. There were not significant differences between each allele in the concentration of IFN-γ, IL-2, IL-6, and IL-10. Conclusion: HLA-DRB1*07, HLA-DQB1*02 and HLA-DQB1*03 were the differentially expressed gene in HPV16 infected patients with advanced cervical cancer. There may be correlations between the occurrence, development of cervical cancer and IFN-γ, IL-2, IL-6, IL-10.
Keywords: HLA-DRB1, HLA-DQB1, genetic polymorphism, cytokines, hpv16, cervical cancer
Introduction
Cervical cancer is the most common malignancy in female reproductive system, which follows breast cancer and becomes second-largest causes of death in women with malignant disease [1]. In China, the morbidity and mortality of cervical cancer accounts for about one-third of the world’s morbidity and mortality [2-4].
Human leukocyte antigen (HLA) correlates with the occurrence of cervical cancer, especially for HLA II type DRB1 and DQB1 genes which closely related to the occurrence of cervical squamous cell carcinoma [5,6]. It has been reported that cytokines play an important role in cancer occurrence and development [7-9].
There were differences in the distribution of HLA II gene in patients with cervical cancer between different races and different regions. A lot of literatures had reported that DRB1 and DQB1 of HLA II gene were closely related to the cervical cancer with HPV16 infection [10,11]. Beskow et al. [10] had detected the HLA-DRB1 and HLA-DQB1 alleles in 440 cases of patients diagnosed with HPV16-infected cervical cancer and 476 cases of healthy individuals, and found that the frequency of HLA-DRB1*0801, HLA-DRB1*1501, HLA-DQB1*0402 and HLA-DQB1*0602 alleles increased, while the frequency of HLA-DRB1*0101, HLA-DRB1*1301, HLA-DQB1*0501 and HLA-DQB1*0603 alleles decreased; after correction, DQB1*0602 and DRB1*1501 were closely related to cervical cancer. Cuzick et al. [12] investigated the HLA genotyping in 116 cases of British women with HPV16-infected cervical cancer and found that HLA-DQB1*0301 (P = 0.02) and HLA-DRB1*0401 (P = 0.02) were the haploids susceptible to cervical cancer. Madeleine et al. [13] found that, HLA-DRB1*1501 and HLA-DQB1*0302 were the dominant genes. Guzalinuer et al. [14] suggested that, HLA-DRQB06 may be the susceptible gene of cervical cancer in Uygur women, which may increase the risk of cervical cancer in HPV16-positive women. The results of the study showed that, HLA-DRB1*07 was the dominant expressed gene, and its expression ratio was close to half. Madeleine et al. [13] found that HLA-DQB1*0301, HLA-DRB1*0301, and HLA-DQB1*0201 were associated with HPV16-positive squamous cell carcinoma (odds ratio was 0.5; 95% confidence interval 0.3~0.9).
In this study we analyzed the distribution of HLA-DRB1 and HLA-DQB1 alleles in HPV16 infected women with advanced cervical carcinoma, compared the levels of cytokines in people with different clinical stages and the healthy, and discussed the correlation between distribution of HLA-DRB1 and HLA-DQB1 allele and IFN-γ, IL-2, IL-6, IL-10.
Materials and methods
General information
137 cases of HPV16 infected patients with advanced cervical cancer (cervical cancer) in Binzhou People’s Hospital from January 2010 to March 2014 were enrolled in this study. The age ranged from 38 to 70, with a median age of 54 years. Cervical cancer stage (according to FIGO staging): 63 cases were at IIb stage, 60 cases were at IIIb stage, 3 case was at IIIA stage, 7 cases were at IVA stage. 4 case was at IVb stage. 175 cases of healthy women in the local were enrolled in the control group.
Methods
Whole blood genomic DNA extraction
5 mL of Cubital vein blood were collected from the cervical cancer patients before treatment and from the healthy controls (EDTA anticoagulant).Whole blood genomic DNA extraction kit was purchase form BaiTektronix Biotechnology Co., Ltd. (Beijing, China). The operation was in strict accordance with the instructions for DNA extraction. UV spectrophotometer was used for measuring the concentration and purity, and its A260/280 ratios were 1.8 to 1.9. Adjust the final concentration at 0.3~0.5 μg/μL and placed them at -20°C refrigerator for detection.
HLA-DRB1 and HLA-DQB1 gene subtype classification
Polymerase chain reaction/sequence specific primer method (PCR-SSP) was used. Specific steps refer to the reference [15].
Detection of cytokines
Plasma from blood samples were obtained by centrifuge and stored at -80°C refrigerator. IFN-γ, IL-2, IL-6, IL-10 levels were detected by ELISA method according to Li’s protocol [15].
Statistical analyses
Spss17.0 statistical software (Chicago, CA, USA) was used for analyzing the distribution of HLA-DRB1 and HLA-DQB1 allele; t test was used for cytokine concentrations in the two groups. Univariate analysis of variance was used for cytokine concentrations between HLA-DRB1 and HLA-DQB1 alleles. P < 0.05 was considered statistical significance.
Results
HLA-DRB1 expression in patients with cervical cancer
Eight HLA-DRB1 alleles were detected. The number of detected HLA-DRB1 alleles and composition ratio were showed in Table 1.
Table 1.
The distribution of HLA-DRB1 allele in HPV1 infected patients with advanced cervical cancer (2n = 274)
| HLA-DRB1 gene | Number of alleles | Frequency (%) |
|---|---|---|
| DRB1*01 | 28 | 10.4 |
| DRB1*03 | 28 | 10.4 |
| DRB1*04 | 30 | 11.2 |
| DRB1*07 | 119 | 44.2 |
| DRB1*09 | 21 | 7.8 |
| DRB1*11 | 21 | 7.8 |
| DRB1*12 | 8 | 2.9 |
| DRB1*13 | 14 | 5.2 |
HLA-DQB1 expression in patients with cervical cancer
Four HLA-DQB1 alleles were detected in 137 cases of patients in the whole group. The number of detected HLA-DQB1 alleles and composition ratio were showed in Table 2.
Table 2.
The distribution of HLA-DQB1 allele in HPV16 infected patients with advanced cervical cancer (2n = 274)
| HLA-DQB1-1 gene | Number of alleles | Frequency (%) |
|---|---|---|
| DQB1*02 | 156 | 56.93 |
| DQB1*03 | 94 | 34.31 |
| DQB1*05 | 12 | 8.76 |
| DQB1*06 | 9 | 6.57 |
Comparisons of CK concentrations in HLA-DRB1 alleles
Excluding small sample size of HLA-DRB1*12 and HLA-DRB1*13 allele groups, the CK levels in HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*07, HLA-DRB1*09, and HLA-DRB1*11 allele groups were compared; ANOVA analysis showed that: There were no significant differences among alleles in IFN-γ, IL-2, IL-6, and IL-10 (Table 3).
Table 3.
Comparison of CK concentration between each allele of HLA-DRB1 (pg/mL, Mean ± SD)
| CK | HLA-DRB1*01 (n = 28) | HLA-DRB1*03 (n = 28) | HLA-DRB1*04 (n = 30) | HLA-DRB1*07 (n = 119) | HLA-DRB1*09 (n = 21) | HLA-DRB1*11 (n = 21) | HLA-DRB1*12 (n = 8) | HLA-DRB1*13 (n = 14) |
|---|---|---|---|---|---|---|---|---|
| IFN-γ | 114.1 ± 48.3 | 124.4 ± 57.6 | 103.3 ± 68.4 | 125.4 ± 78.1 | 128.5 ± 65.1 | 121.6 ± 50.3 | 120.1 ± 60.1 | 124.5 ± 50.1 |
| IL-2 | 23.4 ± 12.3 | 24.8 ± 5.9 | 20.3 ± 10.4 | 18.1 ± 8.7 | 19.0 ± 9.8 | 21.6 ± 10.6 | 22.0 ± 9.1 | 21.4 ± 10.3 |
| IL-6 | 98.9 ± 7.5 | 100.4 ± 28.5 | 107.1 ± 23.8 | 115.1 ± 19.0 | 120.3 ± 18.5 | 111.9 ± 18.3 | 122.3 ± 18.0 | 116.3 ± 18.2 |
| IL-10 | 105.4 ± 18.7 | 104.5 ± 17.5 | 113.8 ± 19.0 | 121.3 ± 18.4 | 113.5 ± 15.4 | 111.8 ± 17.3 | 119.3 ± 15.1 | 114.2 ± 17.2 |
Comparisons of CK concentrations in HLA-DQB1 alleles
Excluding small sample size of HLA-DQB1*05 and HLA-DQB1*06 groups, the CK levels in HLA-DQB1*02 and HLA-DQB1*03 groups were compared by ANOVA analysis, and it showed that: There were no significant differences between the alleles in IFN-γ, IL-2, IL-6, and IL-10, as shown in Table 4.
Table 4.
Comparison of CK concentration between each allele of HLA-DQB1 (pg/mL, Mean ± SD)
| CK | HLA-DRB1*02 (n = 156) | HLA-DRB1*03 (n = 94) | DQB1*05 (n = 12) | DQB1*06 (n = 9) |
|---|---|---|---|---|
| IFN-γ | 124.5 ± 18.5 | 118.4 ± 17.6 | 119.2 ± 11.8 | 112.1 ± 17.4 |
| IL-2 | 13.4 ± 8.7 | 15.8 ± 8.9 | 13.0 ± 8.9 | 14.2 ± 8.1 |
| IL-6 | 108.5 ± 48.8 | 113.9 ± 38.4 | 113.5 ± 42.1 | 115.4 ± 44.1 |
| IL-10 | 135.6 ± 22.5 | 128.3 ± 27.4 | 132.3 ± 21.4 | 129.5 ± 23.2 |
Discussion
Cytokine is the intercellular communication tool, and a component of the host’s immune response to foreign pathogens. Interferon-γ (IFN-γ) is the only member of the family of type II interferon, mainly generated by T lymphocytes and NK cells, having antiviral, antitumor and immunomodulatory effects. Studies have shown that in malignant melanoma, gastric cancer, lung cancer, glioblastoma, nasopharyngeal cancer, colorectal cancer, cervical cancer and head and neck cancer, IFN-γ levels decreased [16]. Soong et al. [17] found that IFN-γ receptor agonists may improve the immune response and enhance the anti-tumor effects. Wang et al. [18] reported that due to lymph node metastases in patients with advanced cervical cancer, immune microenvironment was destroyed, resulting in immune escape; the study observed that IL-6 and IL-10 levels were higher than the normal level, and IFN-γ level was lower than normal level. The results of the study showed that in patients with different clinical stages of cervical cancer, the IFN-γ levels were significantly lower than that in the control group, consistent with the findings of Lippitz et al. [19] and Wang et al. [18]. IL-2 cytokine is mainly produced by T lymphocytes, playing a regulatory role in immune response, which can enhance immune activity. It can induce the differentiation of activated killer cells (LAK), natural killer cells (NK) and cytotoxic T cell (CTL) to play anti-tumor effects. Paradkar et al. [20] reported that, in peripheral blood of women with HPV infection, IL-2 levels in lymphocytes decreased compared with the normal group. Valle-Mendiola et al. [21] reported that high doses of IL-2 could inhibit the proliferation of cervical cancer cells.
The results of the study showed that IL-2 in cervical cancer group was lower than that in the healthy control group (P < 0.05), which was consistent with the literature [20,21]. IL-6 is a typical cytokine with multiple biological functions; IL-6 could be generated by B cells, T cells, monocytes, fibroblasts, endothelial cells, astrocytes and microglia, which plays an important role in cell immunity, inflammation and hematopoietic regulation. The clinical studies of Guo et al. [22] showed that in patients with advanced multiple myeloma, non-small cell lung cancer, colorectal cancer, renal cell carcinoma, prostate cancer, breast cancer and ovarian cancer, serum IL-6 increased, so blocking IL-6 signaling has become as a potential cancer therapeutic strategy. Wang et al. [18] found that IL-6 was not expressed or lowly expressed in normal cervical tissue, and highly-expressed in cervical squamous cell carcinoma; IL-6 expression rate in patients with cervical squamous cell carcinoma was significantly higher than that in normal group (P < 0.01). This study showed that in IIb and IIIb cervical cancer group, serum levels were significantly higher than those in the healthy control group (all P < 0.05), which was similar to the results of Wang et al. [18]. IL-10 is a cytokine with immunosuppressive activity, which can inhibit the generation of IL-2, INF-γ and other factors; literature [23] reported that IL-10 interacted with HPV by inhibiting immune function to promote the formation and development of cervical cancer. Related studies showed that IL-10 was overexpressed in many human tumors, such as renal cancer, gastrointestinal cancer and melanoma. Therefore, inhibition of IL-10 overexpression may play an anti-tumor effect [24]. The results of the study showed that IL-10 levels in IIb and IIIb groups were significantly higher than that in the control group, and the differences were statistically significant. This was consistent with the literature [23,24]. Comprehensive analysis of the results showed that, compared with the healthy control group, IFN-γ and IL-2 in women with HPV16-infected IIb and IIIb cervical cancer were lower than the normal level, while IL-6 and IL-10 were higher than the normal level, and the differences were statistically significant; this means that these cytokines may play an important role in the occurrence and development of cervical cancer.
We hypothesized that there are differences between HLA-DRB1 and HLA-DQB1 alleles in cytokines, and compared the CK concentrations of HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*07, HLA-DRB1*09 and HLA-DRB1*11; one-way ANOVA analysis showed that there were no significant differences among the alleles. The CK concentrations of HLA-DQB1*02 and HLA-DQB1*03 alleles were compared by one-way ANOVA analysis, which showed no significant difference in CK concentration between the alleles. Comprehensive analysis showed it may be related to the small sample size or that there may be no difference in CK itself between HLA-DRB1 and HLA-DQB1 alleles.
In summary, HLA-DRB1*07, HLA-DQB1*02 and HLA-DQB1*03 may be the superior genes in HPV16 infected women with middle or advanced cervical cancer; there were statistically significant differences in IFN-γ, IL-2, IL-6 and IL-10 levels between patients with cervical cancer and healthy individuals; but the relevance between alleles (such as HLA-DRB1 and HLA-DQB1) in advanced cervical cancer with HPV16 infection and cytokine levels, such as IFN-, IL-2, IL-6 and IL-10, still need to be further studied by expanding the sample size.
Disclosure of conflict of interest
None.
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