EB virus related B cell lymphoma diagnostic kit based on GNAS methylation detection

Abstract

The invention discloses an EB virus related B cell lymphoma diagnostic kit based on GNAS methylation detection. The invention provides a new marker for diagnosis of EB virus related B cell lymphoma, provides a matched and verified special detection primer, and performs functional reverse verification of 5-aza-2 '-deoxycytidine drug intervention. According to the invention, the potential value of the marker in the aspect of curative effect prediction is defined, and a possible molecular basis is provided for implementing individualized demethylation treatment clinically.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular, to a diagnostic kit for EB virus-associated B-cell lymphoma based on GNAS methylation detection. Background Technology

[0002] EBV is an important tumor-associated virus associated with a variety of lymphoproliferative disorders. Its pathogenic mechanism is partly achieved by inducing abnormal methylation of the host's genomic DNA and silencing specific genes (such as tumor suppressor genes).

[0003] Although DNA methylation is known to occur extensively in EBV-related tumors, and proteins encoded by EBV itself, such as LMP2A, can affect the global methylation status, studies have identified a large number of hypermethylated genes in EBV-positive tumors (such as nasopharyngeal carcinoma and gastric cancer) using high-throughput technologies (such as methylation microarrays). However, systematic studies in lymphatic system diseases are lacking, and the methylation changes of most genes are not EBV-specific.

[0004] Some literature reports that genes such as p16 and DAPK are methylated in lymphoma, but these genes are also common in a variety of EBV-negative tumors, and their direct causal relationship with EBV infection is unclear.

[0005] 5-aza-2'-deoxycytidine, as a demethylating agent, has been shown to reverse gene silencing, but its application in EBV-positive lymphoma lacks effective predictive biomarkers to screen patients who may benefit.

[0006] Currently, there is a lack of specific molecular markers and corresponding detection methods that can effectively link EBV infection status with specific epigenetic changes in host cells and can be used for clinical diagnosis, differential diagnosis, and treatment guidance of EBV-associated B-cell lymphoma. The diagnosis of this type of disease currently relies on clinical manifestations, serological tests, imaging studies, and invasive biopsies, which suffer from insufficient specificity, difficulty in early diagnosis, and inconvenient monitoring of epigenetic therapy responses.

[0007] In summary, existing technologies have the following problems: (1) Weak correlation between disease and etiology: Many reported hypermethylated genes lack specific data in EBV-associated B-cell lymphoma, making it impossible to distinguish whether the epigenetic changes are driven by EBV or caused by other factors.

[0008] (2) Lack of functional and mechanism verification: Most studies remain at the level of correlation description and lack a complete chain of functional evidence in EBV positive / negative lymphoma cell lines, which is linked together by experiments such as gene expression, methylation status, and drug intervention.

[0009] (3) Lack of clinical translation tools: Existing research is mostly based on basic findings, and standardized reagents (such as specific MSP primers) and kits suitable for rapid and specific detection of clinical samples (such as peripheral blood) have not been developed, which limits their clinical application value.

[0010] (4) The therapeutic guidance significance is unclear: the association between these methylation markers and the prediction of the efficacy of demethylation drugs has not been explored. Summary of the Invention

[0011] The purpose of this invention is to provide a diagnostic kit for EB virus-associated B-cell lymphoma based on GNAS methylation detection, so as to solve the technical problems such as the difficulty in diagnosing EB virus-associated B-cell lymphoma in the prior art.

[0012] To achieve the above objectives, the present invention provides the application of GNAS methylation markers in the preparation of diagnostic reagents for EB virus-associated B-cell lymphoma. The marker is the methylation state of the GNAS (Gene ID: 2778) promoter region, which is a region containing key CpG islands. The nucleotide sequence of the promoter region is shown in SEQ ID NO.1.

[0013] As one of the preferred technical solutions, when the methylation level of the GNAS promoter region is upregulated, the subject is more likely to have EB virus-associated B-cell lymphoma.

[0014] As one of the preferred technical solutions, the diagnostic reagent includes a reagent for detecting the methylation level of the GNAS promoter region.

[0015] The present invention also provides the application of GNAS methylation markers in the preparation of screening reagents for the treatment of EB virus-associated B-cell lymphoma. The marker is the methylation state of the GNAS (Gene ID: 2778) promoter region, which is a region containing key CpG islands. The nucleotide sequence of the promoter region is shown in SEQ ID NO.1.

[0016] As one of the preferred technical solutions, the therapeutic drug screening reagent is a reagent for predicting the sensitivity of demethylated drugs.

[0017] As a further preferred technical solution, the demethylating drug is 5-aza-2'-deoxycytidine.

[0018] The present invention also provides diagnostic primers (MSP primers) for detecting GNAS methylation in EB virus-associated B-cell lymphoma, comprising a pair of methylated primers and a pair of unmethylated primers; Methylation primers: methylation-F (MF): 5'-CGGTTTATTAGGGTTTGCGTTATAGGTTCG-3', nucleotide sequence as shown in SEQ ID NO.2; methylation-R (MR): 5'-AAAACCACCTCCCCGCGAACTACGT-3', nucleotide sequence as shown in SEQ ID NO.3; Unmethylated primers (for control or to improve detection range): unmethylation-F (UF): 5'-TTGGTTTATTAGGGTTTGTGTTATAGGTTT-3', nucleotide sequence as shown in SEQ ID NO.4; unmethylation-R (UR): 5'-AAAACCACCTCCCCACAAACTACAT-3', nucleotide sequence as shown in SEQ ID NO.5.

[0019] This invention also provides the application of the aforementioned primers in the preparation of diagnostic kits for EB virus-associated B-cell lymphoma.

[0020] This invention also provides a diagnostic kit for EB virus-associated B-cell lymphoma based on GNAS methylation detection, comprising the aforementioned primers.

[0021] This invention also provides the application of the aforementioned primers in the preparation of screening reagents for the treatment of EB virus-associated B-cell lymphoma.

[0022] This invention also provides a screening reagent for the treatment of EB virus-associated B-cell lymphoma, comprising the aforementioned primers.

[0023] The present invention has the following beneficial effects: This invention provides a diagnostic kit for EBV-associated B-cell lymphoma based on GNAS methylation detection. This invention proposes a novel biomarker for the diagnosis of EBV-associated B-cell lymphoma, provides a matching, validated dedicated detection primer (MSP primer), and performs functional reverse validation of 5-aza-2'-deoxycytidine drug intervention. This invention clarifies the potential value of this biomarker in predicting treatment efficacy and provides a possible molecular basis for the clinical implementation of personalized demethylation therapy.

[0024] This invention focuses the study of methylation of target genes on the EBV-related B-cell lymphoma disease spectrum and establishes a direct and specific association with EBV infection through EBV-positive / negative cell lines and clinical sample controls.

[0025] This invention has the following advantages: 1. Diagnostic and Differential Diagnostic Effects: This invention reveals and verifies for the first time that hypermethylation of the target gene is a common and specific epigenetic feature of EBV-associated B-cell lymphoma. Using the primers provided by this invention, this marker can be detected with high specificity in peripheral blood samples from patients with EBV-associated B-cell lymphoma, which is helpful for the early diagnosis of the disease and its differentiation from EBV-negative lymphoma.

[0026] 2. Convenience and Stability of Detection: The MSP method is mature, low-cost, and fast, making it suitable for clinical application. This invention provides a set of experimentally validated, highly specific MSP primers suitable for clinical sample detection in this disease spectrum, solving the bridging problem from basic discovery to clinical application. The primers designed in this invention have been optimized, resulting in stable and reliable detection results.

[0027] 3. Treatment Guidance: This invention establishes the methylation status of this gene as a novel diagnostic biomarker and expands its potential application in predicting the efficacy of demethylating drugs such as 5-aza-2'-deoxycytidine. Experiments have demonstrated that EBV-positive lymphoma cells carrying this methylation marker are sensitive to 5-aza-2'-deoxycytidine treatment. This provides a basis for clinical translation, suggesting that patients positive for this biomarker may be more suitable for receiving or undergoing clinical trials of demethylation therapy, thus improving treatment precision.

[0028] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description

[0029] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a blank control experiment using MSP primers. The leftmost element is the marker, U represents unmethylated primers, and M represents methylated primers.

[0030] Figure 2 These are the clinical sample MSP results, where U represents unmethylated lymphoma, M represents methylated lymphoma, 1 and 2 represent EBV-negative lymphoma, 3 represents EBV-positive infectious mononucleosis, 4 represents EBV-positive hemophagocytic syndrome, and 5-10 represent EBV-positive lymphoma.

[0031] Figure 3 To determine the mRNA and protein expression of GNAS in lymphoma cell lines and nasopharyngeal carcinoma cell lines latently infected with EBV.

[0032] Figure 4The expression of GNAS after chemically induced EBV reactivation is shown in the image from left to right: Akata-EBV, Raji, and Daudi.

[0033] Figure 5 To detect the methylation level of GNAS by MSP after chemically induced EBV reactivation, Raji and Daudi are shown from left to right.

[0034] Figure 6 The mRNA and protein expression levels of GNAS in Akata (left) and BJAB (right) cells after treatment with a chemical inducer.

[0035] Figure 7 The expression level of GNAS after treatment with 5-aza-2'-deoxycytidine was detected by RT-qPCR and WB experiments. The left cell is Daudi cells and the right cell is Raji cells.

[0036] Figure 8 Methylation levels of GNAS after treatment with 5-aza-2'-deoxycytidine were detected by MSP. Daudi cells are on the left and Raji cells are on the right.

[0037] Figure 9 The effect of different concentrations of the methylation inhibitor 5-aza-2'-deoxycytidine on GNAS expression. Detailed Implementation

[0038] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, the present invention can be implemented in many different ways as defined and covered by the claims.

[0039] Example 1 MSP primer design for GNAS (1) The promoter sequence (FASTA format) of the GNAS gene (Gene ID: 2778, transcript: NCBI Reference Sequence: NM_000516.7) was found through the NCBI website (National Center for Biotechnology Information), as shown in SEQ ID NO.1.

[0040] (2) Input the above starter region sequence using the Methprimer website (MethPrimer | DNA Methylation Primer Design), and set the relevant parameters: product size range (60-140bp), primer Tm value (55-75℃), primer length (20-30bp), and CpG sites contained in the primer (≥3). MSP primers are obtained. Select primer pairs for experiments based on actual conditions.

[0041] Methylation primers: methylation-F (MF): 5'-CGGTTTATTAGGGTTTGCGTTATAGGTTCG-3', nucleotide sequence as shown in SEQ ID NO.2; methylation-R (MR): 5'-AAAACCACCTCCCCGCGAACTACGT-3', nucleotide sequence as shown in SEQ ID NO.3; Unmethylated primers: unmethylation-F (UF): 5'-TTGGTTTATTAGGGTTTGTGTTATAGGTTT-3', nucleotide sequence as shown in SEQ ID NO.4; unmethylation-R (UR): 5'-AAAACCACCTCCCCACAAACTACAT-3', nucleotide sequence as shown in SEQ ID NO.5.

[0042] (3) MSP primer blank control: ddH2O was used as a template for a blank control experiment. Sulfite modification was performed according to the EZ DNA Methylation-Lightning Kit (Zymo Research, D5030T) instructions (program: 98°C for 8 minutes, 54°C for 60 minutes, 4°C for a maximum of 20 hours). The resulting product was stored at -20°C. 1 μl of the product was used as a template for PCR, followed by 1–2% agarose gel electrophoresis (110V, 45 min) and imaging.

[0043] The PCR reaction system (20 μL) consisted of: 7.5 μL of 2×Es Taq MasterMix (Dye) (Kangwei Century, CW0690M), 1 μL each of MF and MR primers (10 μM) / UF and UR primers (10 μM), 1 μL of template DNA (approximately 20 ng), and water added to a final volume of 20 μL.

[0044] Reaction program: 94°C for 2 minutes; 35 cycles (94°C for 30 seconds, 50°C for 30 seconds, 72°C for 3 seconds); 72°C for 2 minutes, 12°C for 2 minutes.

[0045] Evaluation of effectiveness: Whether the bands of methylated primers and unmethylated primers appear as a single bright band at the expected size (63 bp for methylated product and 64 bp for unmethylated product).

[0046] Experimental results: No single bright band was observed with either methylated or unmethylated primers, proving that the primers were usable.

[0047] Example 2 Clinical sample cohort MSP validation of GNAS methylation status 2.1 Experimental objective: To investigate whether the hypermethylation state of GNAS is prevalent in clinical samples.

[0048] 2.2 Experimental Methods: Ten clinical samples from EBV-related diseases were collected (including one case of EBV-positive infectious mononucleosis, one case of EBV-positive hemophagocytic lymphohistiocytosis, six cases of EBV-positive lymphoma, and two cases of EBV-negative lymphoma). The samples were centrifuged at 3500 rpm for 10 min, and three distinct layers were observed upon removal. The supernatant was collected to obtain plasma as the source for circulating cell-free DNA extraction (CWhipro Circulating Nucleic Acid Kit, CW2603S) for bisulfite-MSP detection.

[0049] 2.3 Result evaluation: Observe the presence / brightness of GNAS methylated and unmethylated bands in different samples.

[0050] 2.4 Experimental Results: In the EBV-positive lymphoma group, the positive detection rate of target gene methylation was 100%, while non-methylated bands were still present in EBV-negative lymphoma patients (negative control) and EBV-positive infectious mononucleosis and hemophagocytic syndrome (general EBV-positive sample control), indicating that the degree of methylation was lower than that in EBV-positive lymphoma.

[0051] Example 3 Cell line models validate that GNAS expression is regulated by EBV 3.1 Test Objective: To investigate the regulation of GNAS by EBV latency. 3.1.1 Test methods (RT-qPCR, WB): Cell lines: EBV-positive cell lines: Akata-EBV, HK1-EBV, and HONE1-EBV EBV-negative cell lines: Akata, HK1, HONE1 The HK1-EBV and HONE1-EBV cell lines were provided by Professor Cao Shihua of the University of Hong Kong, PMID: 20091869; Daudi [1] (PMID: 4168541) This cell line was donated by Professor Zou Yizhou of Central South University. HONE1 [2] (PMID: 2556716), HK1 [3](PMID: 6259064), BJAB [4] (PMID: 179629), Raji [5] (PMID:14086209), Akata [6] (PMID:1647567) and Akata-EBV [7] (PMID: 8057484) This cell line was donated by Professor Cao Ya from Central South University.

[0052] 3.1.2 Specific steps: Obtain EBV-positive cell lines (Akata-EBV, HONE1-EBV, and HK1-EBV) and EBV-negative cell lines (Akata, HONE1, and HK1). The cell lines were cultured in RPMI-1640 (Sango BBI, E600028-0500) complete medium containing 10% fetal bovine serum (FBS, Gibco). The cell pellets of the above cell lines were collected, and total RNA (TRIZol method) and total protein (lysed on ice with RIPA:PMSF=100:1 lysis buffer) were extracted simultaneously.

[0053] (1) Quantitative reverse transcription of 1 μg RNA (Vazyme, R233), dilute the obtained cDNA with water (e.g., 100 times), and perform RT-qPCR according to the RT-qPCR system (Vazyme, Q711).

[0054] (2) Quantify protein concentration using BCA method. Take an equal amount of protein for SDS-PAGE electrophoresis. Transfer the protein to a PVDF membrane and block it with 5-7% (w / v) skim milk TBST solution for 1-2 h. Incubate overnight at 4°C with anti-GNAS antibody (Proteintech Group, 10150-2-AP) and anti-GAPDH antibody (Proteintech Group, 10494-1-AP). After washing the membrane 15 min × 3 times, incubate it with the corresponding HRP-labeled secondary antibody (ABclonal, AS014) at 37°C for 1 h. After washing the membrane 15 min × 3 times, use an ECL chemiluminescence kit (e.g., Vazyme, E433) to develop the image on a chemiluminescence imaging system (e.g., SageCapture™ iChemi chemiluminescence imager).

[0055] 3.1.3 Results Evaluation: Using GAPDH as an internal control, the GNAS expression levels of EBV-positive cell lines were compared with those of EBV-negative cell lines. The grayscale values ​​of protein levels were semi-quantitatively analyzed using image analysis software (such as ImageJ).

[0056] 3.1.4 Experimental results: RT-qPCR results showed that GNAS expression in EBV-positive cell lines was downregulated by more than 30% compared with the mRNA level in EBV-negative cell lines, and the protein level was also downregulated.

[0057] RT-qPCR showed that gene expression was significantly downregulated (30% or more), and Western blot analysis also revealed that GNAS expression was significantly downregulated.

[0058] Figure 3 This study investigated the mRNA and protein expression of GNAS in EBV-laced lymphoma and nasopharyngeal carcinoma cell lines. The expression of GNAS mRNA and protein in Aktata, HK1, and HONE1 cells and their corresponding EBV-infected cell lines (Aktata-EBV, HK1-EBV, and HONE1-EBV) was detected by RT-qPCR and Western blotting. P < 0.05 P < 0.01, P < 0.001.

[0059] 3.2 Test Objective: To investigate the regulation of GNAS expression by EBV lysis phase. 3.2.1 Test methods (RT-qPCR, WB, and MSP): Drug preparation: TPA (Beyotime, S1819) was diluted with DMSO to a stock solution of 200 ng / μl. NaB (MedChemExpress, HY-B0350A) was prepared with sterile purified water to a stock solution of 1 M. All prepared drugs were filtered through a 0.22 μm disposable syringe filter (Biosharp, BS-PES25-22-S). All prepared drugs were stored at -20°C. When treating cells, ensure that the final DMSO concentration does not affect cell viability (typically below 0.1% (v / v)).

[0060] Daudi and Raji cell lines were transferred to six-well plates, and EBV lysis was induced by adding TPA (200 ng / ml) and NaB (2.5 mM / ml) at final concentrations. An equal volume of DMSO was set up as a control group. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 48 h. The cell pellet was collected and RNA, protein and genomic DNA were extracted.

[0061] (1) Quantitative reverse transcription of 1 μg RNA (Vazyme, R233), dilute the obtained cDNA with water (e.g., 100 times), and perform RT-qPCR according to the RT-qPCR system (Vazyme, Q711).

[0062] (2) Quantify protein concentration using the BCA method. Take an equal amount of protein for SDS-PAGE electrophoresis. Transfer the protein to a PVDF membrane and block it with TBST solution of 5-7% (w / v) skim milk for 1-2 h. Incubate overnight at 4°C with anti-GNAS antibody (Proteintech Group, 10150-2-AP), anti-BZLF1 antibody (Santa Cruz Biotech, sc-53904), and anti-GAPDH antibody (Proteintech Group, 10494-1-AP). After washing the membrane 15 min × 3 times, incubate it with the corresponding HRP-labeled secondary antibody (ABclonal, AS014 / AS003) at 37°C for 1 h. After washing the membrane 15 min × 3 times, use an ECL chemiluminescence kit (e.g., Vazyme, E433) and a chemiluminescence imaging system (e.g., SageCapture™ iChemi chemiluminescence imager) for development.

[0063] (3) Genomic DNA was extracted according to the Universal Genomic DNA Kit (Kangwei Century, CW2298M).

[0064] 3.2.2 Results Evaluation: Using GAPDH as an internal control, the expression levels of GNAS in the TPA+NaB treatment group relative to the DMSO group were compared by RT-qPCR and Western blotting. Genomic DNA was extracted from cells, modified with sulfite, and subjected to MSP, 1-2% agarose gel electrophoresis, and imaging.

[0065] 3.2.3 Experimental Results: After treatment, the mRNA expression level of the target gene in the experimental group was downregulated by approximately 20% compared to the control group; the protein level was also significantly downregulated. Simultaneously, MSP detection showed only methylation-specific bands, while unmethylated bands essentially disappeared.

[0066] RT-qPCR showed that gene expression was significantly downregulated (downregulated by 20% or more), and Western blot analysis revealed that GNAS expression was also significantly downregulated. MSP showed that only methylated bands were present, while unmethylated bands were weakened or disappeared.

[0067] Figure 4 To investigate GNAS expression after chemically induced EBV reactivation, Akata-EBV, Raji, and Daudi cells were treated with DMSO / TPA (200 ng / ml) and 2.5 mM NaB for 48 hours. GNAS expression levels were then detected by RT-qPCR and Western blotting.

[0068] Figure 5To detect GNAS methylation levels by MSP assay after chemically induced EBV reactivation, Raji and Daudi cells were treated with DMSO / TPA (200 ng / ml) and 2.5 mM NaB for 48 hours, and GNAS methylation levels were detected by MSP assay.

[0069] 3.3 Test Objective: To investigate the effects of chemically induced drugs TPA and NaB on GNAS expression. 3.3.1 Test methods (RT-qPCR and WB) Akata and BJAB cell lines were transferred to six-well plates and treated with TPA (200 ng / ml) and NaB (2.5 mM / ml) at final concentrations. An equal volume of DMSO was used as a control group. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 48 h. The cell pellet was collected and RNA and protein were extracted.

[0070] (1) Quantitative reverse transcription of 1 μg RNA (Vazyme, R233), dilute the obtained cDNA with water (e.g., 100 times), and perform RT-qPCR according to the RT-qPCR system (Vazyme, Q711).

[0071] (2) Quantify protein concentration using the BCA method. Take an equal amount of protein for SDS-PAGE electrophoresis. Transfer the protein to a PVDF membrane and block it with 5-7% (w / v) skim milk TBST solution for 1-2 h. Incubate overnight at 4°C with anti-GNAS antibody (Proteintech Group, 10150-2-AP) and anti-GAPDH antibody (Proteintech Group, 10494-1-AP). After washing the membrane 15 min × 3 times, incubate it with the corresponding HRP-labeled secondary antibody (ABclonal, AS014 / AS003) at 37°C for 1 h. After washing the membrane 15 min × 3 times, use an ECL chemiluminescence kit (e.g., Vazyme, E433) to develop the image on a chemiluminescence imaging system (e.g., SageCapture™ iChemi chemiluminescence imager).

[0072] 3.3.2 Evaluation of results: Using GAPDH as an internal reference, the expression level of GNAS in the TPA+NaB treatment group was compared with that in the DMSO group by RT-qPCR and WB.

[0073] 3.3.3 Experimental results: After treatment, there was no significant difference in the expression level of the target gene mRNA in the experimental group compared with that in the control group; there was also no significant difference in the protein level.

[0074] Evaluation of efficacy: Using GAPDH as an internal reference, RT-qPCR and WB were used to compare the GNAS expression levels of the TPA+NaB treatment group relative to the DMSO group.

[0075] Results: After treatment, there was no significant difference in the mRNA expression level of the target gene in the experimental group compared with that in the control group; there was also no significant difference in the protein level, thus excluding the influence of the chemical inducing drugs TPA and NaB on GNAS.

[0076] Figure 6 The mRNA and protein expression levels of GNAS in Akata (left) and BJAB (right) cells after treatment with chemical inducers. The expression levels of GNAS in Akata and BJAB cells after treatment with chemical inducers were determined by RT-qPCR and Western blotting after 48 hours of treatment with DMSO / TPA (200 ng / ml) and 2.5 mM NaB.

[0077] Example 4 Effects of intervention with the methylation inhibitor 5-aza-2'-deoxycytidine on GNAS expression (RT-qPCR, WB, and MSP) 4.1 Test objective: To investigate whether the methylation status of GNAS is altered after treatment with the methylation inhibitor 5-aza-2'-deoxycytidine.

[0078] 4.1.1 Test methods (RT-qPCR, WB, and MSP): Cells were transferred to six-well plates at an appropriate density and treated with 2 and 2.5 μM 5-aza-2'-deoxycytidine, respectively. An equal volume of DMSO was used as a control group. Cells were cultured for 48 hours at 37°C and 5% CO2. Cell pellet was collected, and RNA, protein, and genomic DNA were extracted.

[0079] 4.1.2 Results Evaluation: Using GAPDH as an internal control, the expression levels of GNAS in the 5-aza-2'-deoxycytidine treatment group were compared with those in the DMSO treatment group. Genomic DNA was extracted from cells, modified with sulfite, and subjected to MSP, 1-2% agarose gel electrophoresis, and imaging.

[0080] 4.1.3 Experimental Results: After treatment, the mRNA expression level of the target gene in the experimental group was upregulated by approximately 2-fold compared to the control group; the protein level was also significantly upregulated. Simultaneously, MSP detection showed that methylation-specific bands were weakened or even almost completely disappeared.

[0081] Figure 7To detect the expression level of GNAS after treatment with 5-aza-2'-deoxycytidine using RT-qPCR and Western blotting, the left image shows Daudi cells treated with 2.5 μM 5-aza-2'-deoxycytidine (DEC) for 48 h; the right image shows Raji cells treated with 2 μM DEC for 48 h. Cells were collected and RNA and protein were extracted. The expression level of GNAS was detected by RT-qPCR and Western blotting.

[0082] Figure 8 Methylation levels of GNAS were detected by MSP. The left image shows Daudi cells treated with 2.5 μM 5-aza-2'-deoxycytidine (DEC) for 48 h; the right image shows Raji cells treated with 2 μM DEC for 48 h. Cells were collected and gDNA was extracted, and MSP was used to detect GNAS methylation levels.

[0083] Example 5 Effects of different concentration gradients of the methylation inhibitor 5-aza-2'-deoxycytidine on GNAS expression 5.1 Purpose of the test: To investigate the effects of different concentration gradients of 5-aza-2'-deoxycytidine on GNAS expression. 5.1.1 Test Method (WB): Cells were transferred to six-well plates at an appropriate density and treated with 1, 2, 2.5, and 5 μM 5-aza-2'-deoxycytidine. A control group with an equal volume of DMSO was included. Cells were cultured for 48 hours in a cell culture incubator at 37°C and 5% CO2. After 24 hours of treatment with 1 μM 5-aza-2'-deoxycytidine, the drug-containing medium was discarded, and the cells were cultured in drug-free medium for 3 days. Cell pellets were collected, and proteins were extracted.

[0084] 5.1.2 Results Evaluation: Using GAPDH as an internal reference, the expression level of GNAS in the 5-aza-2'-deoxycytidine treatment group was compared with that in the DMSO treatment group.

[0085] 5.1.3 Experimental results: After treatment, the protein level of the target gene in the experimental group was significantly upregulated.

[0086] Figure 9 To investigate the effect of different concentrations of the methylation inhibitor 5-aza-2'-deoxycytidine on GNAS expression. Daudi and Raji cells were treated with 1 μM MDEC for 24 h, then the drug-containing medium was discarded, and the cells were cultured in normal medium for another 3 days. Daudi and Raji cells were treated with 2 μM, 2.5 μM, and 5 μM MDEC for 48 h, after which cells were collected for protein extraction, and GNAS expression was detected by Western blotting.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0088] References [1] KLEIN E, KLEIN G, NADKARNI JS, et al. Surgace IgM specificity oncells derived from a Burkitt's lymphoma [J]. Lancet (London, England), 1967,2(7525): 1068-70. [2] GLASER R, ZHANG HY, YAO KT, et al. Two epithelial tumor celllines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus that werederived from nasopharyngeal carcinomas [J]. Proceedings of the National Academy of Sciences of the United States of America, 1989, 86(23): 9524-8. [3] HUANG DP, HO JH, POON YF, et al. Establishment of a cell line(NPC / HK1) from a differentiated squamous carcinoma of the nasopharynx [J]. International journal of cancer, 1980, 26(2): 127-32. [4] MENEZES J, LEIBOLD W, KLEIN G, et al. Establishment andcharacterization of an Epstein-Barr virus (EBC)-negative lymphoblastoid Bcell line (BJA-B) from an exceptional, EBV-genome-negative African Burkitt'slymphoma [J]. Biomedicine / [publiee pour l'AAICIG], 1975, 22(4): 276-84. [5] PULVERTAFT J V. CYTOLOGY OF BURKITT'S TUMOUR (AFRICAN LYMPHOMA)[J]. Lancet (London, England), 1964, 1(7327): 238-40. [6] TAKADA K, HORINOUCHI K, ONO Y, et al. An Epstein-Barr virus-producer line Akata: establishment of the cell line and analysis of viral DNA[J]. Virus genes, 1991, 5(2): 147-56. [7] SHIMIZU N, TANABE-TOCHIKURA A, KUROIWA Y, et al. Isolation ofEpstein-Barr virus (EBV)-negative cell clones from the EBV-positive Burkitt'slymphoma (BL) line Akata: malignant phenotypes of BL cells are dependent onEBV [J]. Journal of virology, 1994, 68(9): 6069-73.

Claims

1. The application of GNAS methylation markers in the preparation of diagnostic reagents for EB virus-associated B-cell lymphoma, characterized in that, The marker is the methylation state of the GNAS promoter region, which is a region containing key CpG islands, and the nucleotide sequence of the promoter region is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, When the methylation level of the GNAS promoter region is upregulated, the likelihood of the subject having EBV-associated B-cell lymphoma increases.

3. The application according to claim 1, characterized in that, The diagnostic reagents include those for detecting the methylation level of the GNAS promoter region.

4. The application of GNAS methylation markers in the preparation of screening reagents for EB virus-associated B-cell lymphoma treatment, characterized in that, The marker is the methylation state of the GNAS promoter region, which is a region containing key CpG islands, and the nucleotide sequence of the promoter region is shown in SEQ ID NO.

1.

5. The application according to claim 4, characterized in that, The therapeutic drug screening reagent is a reagent for predicting the sensitivity of demethylated drugs.

6. A diagnostic primer for detecting GNAS methylation in EBV-associated B-cell lymphoma, characterized in that, It includes a pair of methylating primers and a pair of unmethylating primers; Methylation primers: Methylation-F: 5'-CGGTTTATTAGGGTTTGCGTTATAGGTTCG-3', nucleotide sequence as shown in SEQ ID NO. 2; methylation-R: 5'-AAAACCACCTCCCCGCGAACTACGT-3', nucleotide sequence as shown in SEQ ID NO.3; Unmethylated primers: unmethylation-F: 5'-TTGGTTTATTAGGGTTTGTGTTATAGGTTT-3', nucleotide sequence as shown in SEQ ID NO. 4; unmethylation-R: 5'-AAAACCACCTCCCCACAAACTACAT-3', nucleotide sequence as shown in SEQ ID NO.

5.

7. The use of the primers described in claim 6 in the preparation of a diagnostic kit for EB virus-associated B-cell lymphoma.

8. A diagnostic kit for EB virus-associated B-cell lymphoma, characterized in that it comprises the primers as described in claim 6.

9. The use of the primers of claim 6 in the preparation of screening reagents for the treatment of EB virus-associated B-cell lymphoma.

10. A screening reagent for therapeutic drugs in EB virus-associated B-cell lymphoma, characterized in that, It contains the primers described in claim 6.

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