CN116694768A - Application of human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening - Google Patents
Application of human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening Download PDFInfo
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Abstract
The invention discloses application of human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening. The present invention relates to the levels of interleukins 2, 4, 6, 10 and 17A, INF-gamma and TNF-alpha by analyzing the correlation of human HLA-B5801 with nasopharyngeal carcinoma. The results show that the HLA-B5801 allele frequency was higher in nasopharyngeal carcinoma patients than in non-tumor and other types of cancer groups. The HLA-B5801 positive group has higher VCA-IgA positive rate and lower levels of immune cytokines such as IL-2, 4,10, INF-gamma and TNF-alpha. The HLA-B5801 allele is a susceptibility gene of nasopharyngeal carcinoma, possibly related to lower level of cytokines causing the EB virus clearance disorder, and the susceptibility of nasopharyngeal carcinoma can be judged by detecting whether a patient carries the HLA-B5801 gene.
Description
Technical Field
The invention relates to the technical field of disease diagnosis markers, in particular to application of a human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening.
Background
Nasopharyngeal carcinoma (nasopharyngeal carcinoma, NPC) is one of the common malignant tumors in south China (guangdong, guangxi, hong kong, hunan, fujian and Jiangxi). EB virus infection plays an important role in the development of nasopharyngeal carcinoma, but not all patients infected with EB virus develop nasopharyngeal carcinoma, and the molecular mechanism is unknown. With the intensive research, genetic susceptibility was found to play a very important role in the development and progression of nasopharyngeal carcinoma.
HLA (human leukocyte antigen) is an expression product of the human major histocompatibility complex (Major Histocompatibility Complex, MHC), which is located on the short arm of chromosome 6 (6p21.3), full-length 3600kb. HLA allele systems are the most complex class of dominant polymorphic genetic systems, and a number of studies have shown that there is a significant correlation between certain HLA genes and susceptibility to cancer, such as lung cancer, thyroid cancer, etc. HLA genes can be classified into three classes, wherein there are thousands of alleles of HLA-B genes encoding HLA-B series antigens in class I molecules, one of which is HLA-B5801 allele. HLA-B5801 gene is currently mainly used for predicting allopurinol adverse reaction. HLA-B5801 is reported to be associated with severe adverse skin reactions (SCARs) caused by allopurinol, including allergic syndrome (HSS), stevens-johnson syndrome (SJS), toxic Epidermolysis Necrosis (TEN), and eosinophilia and systemic symptomatic Drug Response (DRESS). However, no report is currently available on the correlation between HLA-B5801 and nasopharyngeal carcinoma.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings in the prior art and provide the application of human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening.
The invention aims to provide an application of human leukocyte antigen HLA-B5801 gene in preparing nasopharyngeal carcinoma susceptibility screening products.
The invention also aims to provide application of the reagent for detecting the leukocyte antigen HLA-B5801 gene in preparing nasopharyngeal carcinoma susceptibility screening products.
The above object of the present invention is achieved by the following technical solutions:
the invention adopts polymerase chain reaction-sequence specific primer (PCR-SSP) technology to detect HLA-B5801 allele frequency of 806 samples, wherein 128 samples of nasopharyngeal carcinoma patients, 331 samples of cervical cancer patients, lung cancer patients, gastric cancer patients or colorectal cancer patients and 347 samples of non-tumor control samples, and discusses the relationship between HLA-B5801 and nasopharyngeal carcinoma susceptibility. The invention further analyzes the correlation between HLA-B5801, EBV VCA IgA and cytokine levels, including IL-2, IL-4, IL-6, IL-10, IL-17A, INF-gamma and TNF-alpha levels. The results show that the population positive for HLA-B5801 has a higher prevalence of nasopharyngeal carcinoma and may be associated with lower levels of immune-related cytokines that lead to epstein barr virus clearance. Namely, HLA-B5801 allele is susceptibility gene of nasopharyngeal carcinoma, HLA-B5801 allele can be used as susceptibility diagnosis marker of nasopharyngeal carcinoma, and can be used for screening and judging susceptibility of nasopharyngeal carcinoma by detecting whether patient carries HLA-B5801 gene.
Thus, the present invention provides the following uses for the human leukocyte antigen HLA-B5801 gene:
application of human leukocyte antigen HLA-B5801 gene as nasopharyngeal carcinoma susceptibility diagnosis marker in nasopharyngeal carcinoma susceptibility screening for non-disease diagnosis purpose.
Application of human leukocyte antigen HLA-B5801 gene as nasopharyngeal carcinoma susceptibility diagnosis marker in preparing nasopharyngeal carcinoma susceptibility screening product.
Application of detection reagent of human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening for non-disease diagnosis.
Application of detection reagent of human leukocyte antigen HLA-B5801 gene in preparing nasopharyngeal carcinoma susceptibility screening product.
Detection of HLA-B5801 gene in the present invention may be performed by conventional means in the art including, but not limited to, high throughput sequencing, small fragment replacement sequencing, PCR techniques, immunological methods, and the like.
Further, the detection reagent is a real-time fluorescent polymerase chain reaction reagent and comprises a set of HLA-B5801 genotype specific primers and a set of internal control gene primers. In particular from China century biotechnology Co.
The invention also provides a nasopharyngeal carcinoma susceptibility screening product, which comprises a reagent for detecting human leukocyte antigen HLA-B5801 genes.
Further, the product also includes a DNA extraction reagent.
The invention also provides application of the nasopharyngeal carcinoma susceptibility screening product in nasopharyngeal carcinoma susceptibility screening for non-disease diagnosis.
A screening method for susceptibility of nasopharyngeal carcinoma for the purpose of non-disease diagnosis is to detect the whole blood DNA of a patient by adopting a detection reagent of HLA-B5801 gene, and if HLA-B5801 gene is positive, the screening method is a susceptibility crowd of nasopharyngeal carcinoma.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses application of human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening. The study of the invention shows that HLA-B5801 allele is a susceptibility gene of nasopharyngeal carcinoma, and is possibly related to lower cytokine level causing EB virus clearance disorder, the HLA-B5801 allele can be used as a susceptibility diagnosis marker of nasopharyngeal carcinoma, and more modes can be provided for susceptibility screening of nasopharyngeal carcinoma by detecting whether a patient carries the HLA-B5801 gene or not.
Drawings
Figure 1 shows the gene frequencies of HLA-B5801 allele in non-tumor and different tumor groups. A: HLA-B5801 allele frequency in non-tumor and nasopharyngeal carcinoma groups; b: HLA-B5801 allele frequencies in non-tumor and lung cancer groups; c: HLA-B5801 allele frequencies in non-tumor and gastrointestinal and colorectal cancer groups; d: HLA-B5801 allele frequencies in non-tumor and breast cancer groups; e: HLA-B5801 allele frequencies in non-tumor and cervical cancer groups; f: HLA-B5801 allele frequencies between nasopharyngeal carcinoma and other tumor groups; * P <0.05, P <0.01. It can be seen that HLA-B5801 allele frequency is frequently up-regulated in nasopharyngeal carcinoma, and HLA-B5801 allele frequency increases the risk of nasopharyngeal carcinoma onset compared to lung, gastrointestinal and colorectal cancers, breast, cervical and non-tumor groups.
FIG. 2 is a correlation analysis of HLA-B5801 allele frequencies with EB virus VCA-IgA, IL-2, IL-4, IL-6, IL-10, IL-17A, INF-gamma and TNF-alpha in HLA-B5801 positive and HLA-B5801 negative nasopharyngeal carcinoma patients. A: comparing the EBV VCA IgA positive rates of HLA-B5801 positive group and HLA-B5801 negative group; b: comparison of IL-4 expression levels in EBV VCA-IgA positive patients and EBV VCA-IgA negative groups; c: comparison of IL-6 expression levels in EBV VCA-IgA positive patients and EBV VCA-IgA negative groups; d: comparison of IL-10 expression levels in EBV VCA-IgA positive patients and EBV VCA-IgA negative groups; e: comparison of IL-17A expression levels in EBV VCA-IgA positive patients and EBV VCA-IgA negative groups; f: comparing the expression level of INF-gamma in the EBV VCA-IgA positive patient and the EBV VCA-IgA negative group; g: comparison of the levels of TNF- α expression in EBV VCA-IgA positive patients and EBV VCA-IgA negative groups; h: comparison of IL-2 expression levels in EBV VCA-IgA positive patients and EBV VCA-IgA negative groups. * P <0.05, P <0.01. It can be seen that in nasopharyngeal carcinoma patients, HLA-B5801 allele frequencies are often positively correlated with EBV VCA IgA, while EBV VCA-IgA is often negatively correlated with IFN-gamma, IL-2, IL-4, IL-10 and IL-17A.
FIG. 3 is a comparison of the expression levels of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF- α and INF- γ in HLA-B5801 positive and HLA-B5801 negative nasopharyngeal carcinoma patients. * P <0.05, P <0.01. It was shown that HLA-B5801 positive nasopharyngeal carcinoma patients generally have lower IL-2, IL-4, IL-10, TNF- α and IFN- γ levels.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1
1. Method of
(1) Patient sample and inclusion criteria
From 2018 to 2021, a total of 806 patients in Qingyuan people hospitals participated in three independent cohort studies; all patients were in the Han nationality. In cohort 1, 128 patients were first diagnosed with NPC by pathology. Cohort 2 included 331 other cancer patients, including 42 cervical cancers, 74 lung cancers, 140 breast cancers, and 75 gastrointestinal and colorectal cancers. In cohort 3, 347 non-tumor individuals were included in the control group. Excluding autoimmune diseases, there is no blood relationship between all patients. The research scheme is approved by the ethical committee of Qingyuan people's hospital (Qingyuan China) in Qingyuan City, an affiliated sixth hospital of Guangzhou medical science.
(2) DNA extraction
DNA was extracted from whole blood using a DNA extraction kit (Biotechnology Co., ltd., china Xiamen).
(3) HLA-B5801 allele frequency analysis
Real-time fluorescent polymerase chain reaction was used to detect HLA-B5801 gene. The kit used (century biosciences in China) included a set of HLA-B5801 genotype specific primers and a set of internal control gene primers. Two separate polymerase chain reactions, including a test reaction and an internal control reaction, were performed simultaneously for each sample. In the presence of HLA-B5801, the highly specific primers will propagate the gene and initiate the PCR reaction, ultimately binding internal controls and determining HLA-B5801 results.
(4) Interleukin 2, 4, 6, 10, 17A, INF-gamma and TNF-alpha assays
Two microspheres (4 μm and 8 μm in size, with seven different fluorescent lights) were detected using a liquid chip-bound double antibody sandwich technique, and the microspheres (China Co., ltd.) were captured with antibodies directed against INF-gamma, IL-2, IL-4, IL-6, IL-10, IL-17A and TNF-alpha. Next, PE-labeled streptavidin (SA-PE) was added and levels of INF-gamma, IL-2, IL-4, IL-6, IL-10, IL-17A and TNF-alpha were obtained by detecting immune complex fluorescence.
(5) Statistical analysis
Statistical analysis was performed using SPSS 13.0 software (SPSS Inc. of Chicago, ill.) and GraphPad Prism (GraphPad-software, inc. of Lajoba, calif.). Chi-square test was used to detect HLA-B5801 allele frequencies and EBV VCA IgA positives. The differences between cytokine expression levels were assessed by Student t-test. Statistically significant is set to P <0.05, P <0.01, P < 0.001P <0.05 is considered statistically significant.
2. Results
(1) HLA-B5801 allele increases nasopharyngeal carcinoma incidence risk
HLA-B5801 was detected by PCR-SSP in 128 nasopharyngeal carcinoma patients, 331 other tumor patients and 347 controls. As a result, as shown in fig. 1, the HLA-B5801 allele positive frequency was higher in the nasopharyngeal carcinoma group than in the normal control group (fig. 1A) and the other tumor group (fig. 1F). While there was no significant difference in HLA-B5801 allele frequency between the other five tumor groups and the control group (fig. 1B-E). It can be seen that HLA-B5801 allele frequency was frequently up-regulated in nasopharyngeal carcinoma, and HLA-B5801 allele frequency increased the risk of nasopharyngeal carcinoma onset compared to lung (fig. 1B), gastrointestinal and colorectal (fig. 1C), breast (fig. 1D), cervical (fig. 1E), and non-tumor groups.
(2) HLA-B5801 is associated with higher levels of EB virus VCA-IgA, which is inversely related to IFN-gamma, IL-2, IL-4, IL-10 and IL-17A
In view of the important role of epstein barr virus VCA-IgA in nasopharyngeal carcinoma, the present invention evaluates the correlation between HLA-B5801 allele positive frequency and epstein barr virus VCA IgA, and finds that the VCA-IgA positive rate of HLA-B5801 allele positive group is higher than that of HLA-B5801 allele negative group (fig. 2A). To further investigate the possible factors affecting the VCA-IgA positive rate of the nasopharyngeal carcinoma group, seven cytokines including INF-gamma, IL-2, IL-4, IL-6, IL-10, IL-17A and TNF-alpha were analyzed by fluorescence flow cytometry. The results showed that, as shown in FIGS. 2B-H, the levels of IL-2 (FIG. 2H) and IL-4 (FIG. 2B) were lower in the EBV VCA-IgA positive group, no significant difference in the IL-6 levels in the EBV VCA-IgA positive group and the EBV VCA-IgA negative group (FIG. 2C), the IL-10 (FIG. 2D), IL-17A (FIG. 2E) and INF-gamma (FIG. 2F) levels were lower in the EBV VCA-IgA positive group, and no significant difference in the TNF-alpha levels between the EBV VCA IgA positive group and the EBV VCA-IgA negative group (FIG. 2G), i.e., the levels of INF-gamma, IL-2, IL-4, IL-10 and IL-17A were lower in the VCA-IgA positive nasopharyngeal carcinoma patients than in the VCA-IgA-negative nasopharyngeal carcinoma patients. It can be seen that in nasopharyngeal carcinoma patients, HLA-B5801 allele frequencies are positively correlated with EBV VCA IgA, while EBV VCA-IgA is negatively correlated with IFN-gamma, IL-2, IL-4, IL-10 and IL-17A.
(3) Immunocytokine levels in HLA-B5801 positive nasopharyngeal carcinoma patients tend to be lower than in HLA-B5801 negative patients
The above results indicate that HLA-B5801 may be a susceptibility factor for nasopharyngeal carcinoma, and that HLA-B5801 is positively correlated with EBV VCA-IgA. To investigate the possible relationship between HLA-B5801 allele positive frequency, EBV VCA IgA and immunocytokines, fluorescence flow cytometry was used to analyze the levels of INF-gamma, IL-2, IL-4, IL-6, IL-10, IL-17A and TNF-alpha in nasopharyngeal carcinoma patients. As a result, as shown in fig. 3, lower IL-2 (fig. 3A), IL-4 (fig. 3B), IL-10 (fig. 3D), TNF- α (fig. 3F) and IFN- γ (fig. 3G) levels were observed in HLA-B5801 positive nasopharyngeal carcinoma patients, but there was no significant difference in IL-6 (fig. 3C) and IL-17A (fig. 3E) levels between HLA-B5801 positive and negative nasopharyngeal carcinoma patients, i.e., IL-2, IL-4, IL-10, TNF- α and IFN- γ levels were generally lower in HLA-B5801 positive nasopharyngeal carcinoma patients. It can be seen that patients with HLA-B5801 allele positive nasopharyngeal carcinoma are more likely to have lower levels of immune-related cytokines. Specifically, HLA-B5801 positive nasopharyngeal carcinoma patients have lower IL-2, IL-4, IL-10, TNF- α and IFN- γ levels than HLA-B5801 negative nasopharyngeal carcinoma patients.
Therefore, the detection reagent of human leukocyte antigen HLA-B5801 gene can be used for detecting the whole blood DNA of a patient, and if HLA-B5801 gene is positive, the patient is a nasopharyngeal carcinoma susceptible population.
Claims (10)
1. Application of human leukocyte antigen HLA-B5801 gene as nasopharyngeal carcinoma susceptibility diagnosis marker in nasopharyngeal carcinoma susceptibility screening for non-disease diagnosis purpose.
2. Application of human leukocyte antigen HLA-B5801 gene as nasopharyngeal carcinoma susceptibility diagnosis marker in preparing nasopharyngeal carcinoma susceptibility screening product.
3. Application of detection reagent of human leukocyte antigen HLA-B5801 gene in nasopharyngeal carcinoma susceptibility screening for non-disease diagnosis.
4. Application of detection reagent of human leukocyte antigen HLA-B5801 gene in preparing nasopharyngeal carcinoma susceptibility screening product.
5. The use according to claim 3 or 4, wherein the detection reagent is a real-time fluorescent polymerase chain reaction reagent comprising a set of HLA-B5801 genotype specific primers and a set of internal control gene primers.
6. A nasopharyngeal carcinoma susceptibility screening product, comprising a reagent for detecting a human leukocyte antigen HLA-B5801 gene.
7. The nasopharyngeal carcinoma susceptibility screening product of claim 6, further comprising a DNA extraction reagent.
8. The nasopharyngeal carcinoma susceptibility screening product of claim 6, wherein the product is a kit.
9. Use of the nasopharyngeal carcinoma susceptibility screening product according to any one of claims 6 to 8 in nasopharyngeal carcinoma susceptibility screening for non-disease diagnosis purposes.
10. A screening method for susceptibility of nasopharyngeal carcinoma for non-disease diagnosis purpose is characterized in that a detection reagent of human leukocyte antigen HLA-B5801 gene is adopted to detect the DNA of whole blood of a patient, and if HLA-B5801 gene is positive, the patient is a susceptible population of nasopharyngeal carcinoma.
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