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CN-122012718-A - Epstein-Barr virus-related B cell lymphoma diagnostic kit based on GNAS methylation detection

CN122012718ACN 122012718 ACN122012718 ACN 122012718ACN-122012718-A

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. The invention defines the potential value of the marker in the aspect of curative effect prediction, and provides a possible molecular basis for clinically implementing the personalized demethylation treatment.

Inventors

  • LU JIANHONG
  • Qin Qingshuang
  • CAO PENGFEI
  • YANG LI
  • YANG JING
  • XIN YUJIE
  • JIANG MINGJUAN
  • ZHOU YUANDONG

Assignees

  • 中南大学

Dates

Publication Date
20260512
Application Date
20260319

Claims (10)

  1. The application of the GNAS methylation marker in preparing an EB virus related B cell lymphoma diagnostic reagent is characterized in that the marker is the methylation state of a GNAS promoter region, the promoter region is a region containing a key CpG island, and the nucleotide sequence of the promoter region is shown as SEQ ID NO. 1.
  2. 2. The use according to claim 1, characterized in that when the methylation level of the GNAS promoter region is up-regulated, the subject has an increased likelihood of having epstein barr virus associated B-cell lymphoma.
  3. 3. The use according to claim 1, characterized in that the diagnostic reagent comprises a reagent for detecting the methylation level of the GNAS promoter region.
  4. The application of the GNAS methylation marker in preparing a screening reagent of an EB virus-related B cell lymphoma therapeutic drug is characterized in that the marker is the methylation state of a GNAS promoter region, the promoter region is a region containing a key CpG island, and the nucleotide sequence of the promoter region is shown as SEQ ID NO. 1.
  5. 5. The use of claim 4, wherein the therapeutic screening agent is an agent that predicts the susceptibility of a demethylated drug.
  6. 6. An epstein barr virus-associated B cell lymphoma diagnostic primer for detecting GNAS methylation, comprising a pair of methylated primers and a pair of unmethylated primers; Methylation primers: The nucleotide sequence of methyl-F5'-CGGTTTATTAGGGTTTGCGTTATAGGTTCG-3' is shown as SEQ ID NO. 2; the nucleotide sequence of methyl-R5'-AAAACCACCTCCCCGCGAACTACGT-3' is shown as SEQ ID NO. 3; Unmethylated primer: unmethylation-F is 5'-TTGGTTTATTAGGGTTTGTGTTATAGGTTT-3', and the nucleotide sequence is shown in SEQ ID NO. 4; unmethylation-R is 5'-AAAACCACCTCCCCACAAACTACAT-3', and the nucleotide sequence is shown in SEQ ID NO. 5.
  7. 7. The use of the primer of claim 6 for preparing diagnosis kit of EB virus related B cell lymphoma.
  8. An epstein barr virus-associated B cell lymphoma diagnostic kit comprising the primer of claim 6.
  9. 9. The use of the primer of claim 6 in preparing a reagent for screening a medicament for treating epstein barr virus-associated B cell lymphoma.
  10. An eb virus-associated B cell lymphoma therapeutic screening reagent comprising the primer according to claim 6.

Description

Epstein-Barr virus-related B cell lymphoma diagnostic kit based on GNAS methylation detection Technical Field The invention relates to the technical field of biological medicine, in particular to an EB virus-related B cell lymphoma diagnostic kit based on GNAS methylation detection. Background EBV is an important tumor-associated virus, associated with a variety of lymphoproliferative diseases. The pathogenesis of this is accomplished in part by inducing abnormal methylation of host genomic DNA to silence specific genes (e.g., cancer suppressor genes). Although DNA methylation is known to occur extensively in EBV-associated tumors, and proteins encoded by EBV itself, such as LMP2A, can affect global methylation status. There are studies to identify a large number of highly methylated genes in EBV positive tumors (e.g. nasopharyngeal carcinoma, gastric carcinoma) by high throughput techniques (e.g. methylation chips), but there is a lack of systematic studies in lymphatic system diseases, and methylation changes of most genes are not EBV specific. Some documents report methylation of genes such as p16, DAPK, etc. in lymphomas, but these genes are also common in various EBV-negative tumors, and the direct causal relationship with EBV infection is not clear. 5-Aza-2' -deoxycytidine as a demethylating drug has been shown to reverse gene silencing, but its use in EBV positive lymphomas lacks effective predictive biomarkers to screen potentially benefited patients. In the prior art, a specific molecular marker which can effectively correlate the EBV infection state with the specific epigenetic change of host cells and can be used for clinical diagnosis, differential diagnosis and treatment guidance of EB virus-related B cell lymphomas and a matched detection method are lacking. At present, diagnosis of the diseases depends on clinical manifestations, serology, imaging and invasive biopsy, and has the problems of insufficient specificity, difficult early diagnosis, incapability of conveniently monitoring epigenetic therapeutic response and the like. In summary, the prior art has the following problems: (1) The disease has weak association with the etiology, and many hypermethylation genes reported lack specific data in EB virus-associated B cell lymphomas, and cannot distinguish between EBV driving or epigenetic changes due to other factors. (2) The lack of functional and mechanical verification, in which most studies remain in the description of the correlation, and the lack of complete functional evidence chains in series through experiments of gene expression, methylation status, drug intervention, etc., in EBV positive/negative lymphoma cell lines. (3) The existing research is mostly based on the discovery that the standardized reagent (such as specific MSP primer) and the kit which are suitable for the rapid and specific detection of clinical samples (such as peripheral blood) are not developed, so that the clinical application value of the kit is limited. (4) The significance of the treatment guidance is not clear, and the correlation between the methylation markers and the treatment effect prediction of the demethylated drugs is not explored. Disclosure of Invention The invention aims to provide an EB virus-related B cell lymphoma diagnosis kit based on GNAS methylation detection, so as to solve the technical problems of difficult diagnosis of the EB virus-related B cell lymphoma and the like in the prior art. In order to achieve the above purpose, the invention provides application of a GNAS methylation marker in preparing an EB virus related B cell lymphoma diagnostic reagent, wherein the marker is the methylation state of a GNAS (Gene ID: 2778) promoter region, the promoter region is a region containing key CpG islands, and the nucleotide sequence of the promoter region is shown as SEQ ID NO. 1. As one of the preferred embodiments, when the methylation level of the GNAS promoter region is up-regulated, the subject has an increased likelihood of having epstein barr virus-associated B cell lymphoma. As one of the preferred embodiments, the diagnostic reagent comprises a reagent for detecting the methylation level of the GNAS promoter region. The invention also provides application of the GNAS methylation marker in preparation of a screening reagent of the EB virus-related B cell lymphoma therapeutic drug, wherein the marker is the methylation state of a GNAS (Gene ID: 2778) promoter region, the promoter region is a region containing a key CpG island, and the nucleotide sequence of the promoter region is shown as SEQ ID NO. 1. As one of the preferred embodiments, the therapeutic drug screening agent is an agent that predicts the sensitivity of the demethylated drug. As a further preferred embodiment, the demethylating agent is 5-aza-2' -deoxycytidine. The invention also provides an EB virus-associated B cell lymphoma diagnostic primer (MSP primer) for detecting GNAS methylation, comprising a pair of methyla