Methylation marker for detecting and diagnosing oral squamous cell carcinoma and application of methylation marker

Abstract

The invention discloses a methylation marker for detecting and diagnosing oral squamous cell carcinoma and application of the methylation marker. The methylation marker is the methylation level of COL11A2 gene. Compared with a normal group, the methylation level of the COL11A2 gene of an OSCC patient is remarkably improved. The marker can be used for detecting and diagnosing OSCC. According to the invention, the early diagnosis rate of OSCC can be effectively improved, a new means is provided for early diagnosis and treatment of OSCC, and the application has important significance in improving the survival rate of OSCC patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a methylation marker for detecting and diagnosing oral squamous cell carcinoma and an application thereof. Background Art

[0002] Oral squamous cell carcinoma (OSCC) is a malignant tumor that occurs in the oral mucosa, accounting for more than 90% of all oral and maxillofacial tumors. It is one of the most common malignant tumors worldwide, with high morbidity and mortality. Early diagnosis and treatment are crucial to improving the survival rate of OSCC patients. Traditional diagnostic methods include tissue biopsy and pathological examination, but these methods have the disadvantages of being highly invasive, taking a long time to diagnose, and having a high false-negative rate. Therefore, there is an urgent need to develop new, more accurate, and more convenient methods for detecting and diagnosing OSCC.

[0003] In recent years, DNA methylation technology has been widely used in the field of tumor diagnosis as a new biomarker detection method. DNA methylation refers to the process of covalently bonding a methyl group to the 5th carbon position of cytosine in the genomic CpG dinucleotide under the action of DNA methyltransferase. This modification does not change the sequence of DNA, but it can affect gene expression. Studies have shown that the DNA methylation pattern of tumor cells is significantly different from that of normal cells. The promoter region of certain genes is highly methylated, leading to gene silencing and promoting the occurrence and development of tumors. Therefore, detecting the methylation status of specific genes in tumor cells can be used as an important indicator for diagnosing tumors. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a methylation marker for the detection and diagnosis of oral squamous cell carcinoma, which is the methylation level of the COL11A2 gene. The methylation level of the COL11A2 gene is significantly higher in OSCC patients than in normal people, and the marker can be used to detect and diagnose OSCC.

[0005] The technical solution of the present invention is as follows:

[0006] In one aspect, the present invention provides a methylation marker for detection and / or diagnosis of oral squamous cell carcinoma, wherein the methylation marker is the methylation level of the COL11A2 gene.

[0007] In a second aspect, the present invention provides the use of the methylation marker in any one of the following (1) to (3):

[0008] (1) Use in the preparation of products for detecting and / or diagnosing oral squamous cell carcinoma;

[0009] (2) Its role in the preparation of products for predicting the efficacy of radiotherapy for oral squamous cell carcinoma;

[0010] (3) In predicting Tregs cells, neutrophils, B cells, CAFs cells, hematopoietic stem cells, CD4 + T cells, CD8 + Application of infiltration levels of T cells and / or NK cells.

[0011] Furthermore, studies have shown that the methylation level of the COL11A2 gene in oral squamous cell carcinoma samples is significantly higher than that in normal people. Therefore, the methylation level of the COL11A2 gene can be used to diagnose whether OSCC is present.

[0012] Further, the study found that when the methylation level of COL11A2 gene is around 0.23, the survival rate of patients with oral squamous cell carcinoma is higher; when the methylation level of COL11A2 gene is around 0.35, the survival rate of patients with oral squamous cell carcinoma is lower. Therefore, 0.23 and 0.35 can be used as indicators to predict the mortality rate of OSCC patients. It should be noted that 0.23 and 0.35 are not absolute values, and there can be reasonable fluctuations in actual application.

[0013] Furthermore, compared with those who did not receive radiotherapy, the survival rate of patients with oral squamous cell carcinoma was improved after radiotherapy when the COL11A2 methylation level was greater than 0.35.

[0014] Furthermore, if the methylation level of COL11A2 gene is low, it is predicted that the infiltration level of Tregs cells, neutrophils, B cells, CAFs cells, and hematopoietic stem cells is high; if the methylation level of COL11A2 gene is high, it is predicted that CD4 + T cells, CD8 + The infiltration levels of T cells and NK cells were high.

[0015] Beneficial effects of the present invention:

[0016] The present invention provides a new methylation marker for detecting and diagnosing oral squamous cell carcinoma and its application, wherein the methylation marker is the methylation level of the COL11A2 gene. Compared with the normal group, the methylation level of the COL11A2 gene in OSCC patients is significantly increased. By detecting the methylation marker of the present invention, oral squamous cell carcinoma can be detected quickly and accurately, and the early diagnosis rate of OSCC can be effectively improved, providing a new means for the early diagnosis and treatment of OSCC, which is of great significance to improving the survival rate of OSCC patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the Venn diagram of the Top 10 DMPs and Top 10 DMRs.

[0018] Figure 2 It is a smooth curve fitting diagram. The vertical axis is the logarithmic value of the relative risk (RR value), and the horizontal axis is the methylation level (β value).

[0019] Figure 3 This is a Cox survival curve. The horizontal axis is the survival time of patients with oral squamous cell carcinoma, and the vertical axis is the survival rate.

[0020] Figure 4 Violin plot for the association analysis between COL11A2 methylation and radiotherapy.

[0021] Figure 5 Violin plot of TME cell infiltration analysis. DETAILED DESCRIPTION

[0022] In order to better understand the technical content of the present invention, the present invention is further described below through specific embodiments.

[0023] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are conventional commercially available products unless otherwise specified.

[0024] Experimental example:

[0025] 1. Materials and Methods

[0026] 1.1 Research samples and data collection

[0027] Based on the existing head and neck squamous cell carcinoma (HNSC) methylation chip (Illumina methylation chip) data, the samples were filtered and oral tumor samples were selected for analysis (including 365 OSCC tumor tissue samples and 36 normal oral tissue samples); differential methylation sites and regions between normal and tumors were found and statistical information and visualization were performed. Methylation data is the methylation value (β value) of CpG sites in the entire genome. The beta mixed integer-quantile normalization (BMIQ) method was used to correct the methylation chip data, and the specific implementation was performed using the champ.norm function in the champ package.

[0028] 1.2 Differential methylation analysis

[0029] The methylation differences between OSCC tissues and normal oral tissues were analyzed by ChAMP packag. Differentially methylated sites (DMPs) were filtered by |log(foldchange)|≥0.02, p<0.05. Differentially methylated regions (DMRs) were represented by circlize. The intersection of the top 10 DMPs and the top 10 DMRs was analyzed to obtain collagen type XI alpha 2 chain (COL11A2). Figure 1 .

[0030] 1.3 Analysis of the association between COL11A2 methylation and overall survival

[0031] The smooth curve fitting of the overall survival rate of OSCC patients was drawn and it was found that COL11A2 methylation was associated with the overall survival rate of OSCC patients. The Kaplan-Meier (KM) curve was generated to describe the survival distribution, and the log rank test was used to calculate statistical significance. The Cox proportional hazard regression model was used to estimate the hazard ratio (HR) and 95% confidence interval (CI) between COL11A2 methylation and the overall survival rate of OSCC patients. All analyses were performed using Empower (www.empowerstats.com; X&YSolutions Inc.).

[0032] 1.4 Radiotherapy and COL11A2 methylation analysis

[0033] The beta value and 95% CI between COL11A2 methylation and radiotherapy were estimated using a multivariate regression model. For tumor microenvironment cells with significant differences between high and low methylation groups (p<0.05), the distribution differences between high and low methylation groups before and after radiotherapy were further analyzed, and the t-test was used to analyze the statistical differences.

[0034] 1.5 TME cell infiltration analysis

[0035] Immune infiltration analysis was used to identify differences in the tumor microenvironment (TME). The correlation between COL11A2 methylation and immune infiltration was comprehensively analyzed using the ESTIMATE algorithm, the Tumor Immune Estimation Resource (TIMER) database, CIBERSORT, MCPCOUNTER, and XCELL. All analyses were implemented in https: / / hiplot.com.cn / and https: / / omicshare.com / , and t-tests were used to analyze statistical differences.

[0036] 2. Experimental results and analysis

[0037] 2.1 Analysis of differentially methylated sites (DMPs) and differentially methylated regions (DMRs)

[0038] The champ.DMP function was used to perform methylation differential analysis on the corrected data to obtain differential analysis results of all methylation sites and analysis results of all significantly different methylation sites. The screening criteria for significant differences were logFC greater than or equal to 0.2 or less than or equal to -0.2, with a p value of 0.05 as the threshold, where those greater than 0.2 were considered hypermethylated sites, and those less than or equal to -0.2 were considered hypomethylated sites.

[0039] The results showed that there were 41,843 differential sites among a total of 396,065 methylation sites, 19,090 hypermethylated sites, and 22,753 hypomethylated sites.

[0040] The ChAMP package was used to analyze the methylation regions of two tissue types (normal tissue and tumor tissue). 10117 DMRs were screened out with p value less than or equal to 0.05 and minimum differential methylation sites greater than or equal to 7 as conditions. The distribution results of DMR on chromosomes showed that the most were on chromosome 1, with 1003, and the least were on chromosome 9, with 58. The distribution range of DMR was as small as 109bp and as large as 26103bp, with 2834 exceeding 2000bp. The number of CpGs contained in DMR ranged from 7 to 249, with 54 exceeding 50 and 8 exceeding 100 cpgs.

[0041] By analyzing the intersection of Top 10DMPs and Top 10DMRs, we found that COL11A2 gene is a key gene for OSCC, and there are significant differences in the methylation levels of COL11A2 between OSCC tissues and normal tissues.

[0042] 2.2 Results of association analysis between COL11A2 methylation and overall survival

[0043] Smooth curve fitting showed that COL11A2 methylation showed a nonlinear relationship with patient prognosis ( Figure 2 ). The log-likelihood ratio test found that the breakpoint at β = 0.35 may have a certain significance. COL11A2 differential methylation was negatively nonlinearly correlated with the overall survival rate of OSCC patients, and the mortality rate of patients was the highest when β = 0.35. Cox regression analysis chart ( Figure 3 ) showed that the overall survival rate was decreased in the COL11A2 hypermethylation group compared with the COL11A2 hypomethylation group.

[0044] 2.3 Radiotherapy and COL11A2 methylation analysis results

[0045] The results of the association analysis between COL11A2 methylation and radiotherapy showed that the overall survival rate of people with COL11A2 methylation β>0.35 who had received radiotherapy was significantly improved. Compared with the COL11A2 low methylation group, the COL11A2 high methylation group had a higher sensitivity to radiotherapy ( Figure 4 ). The COL11A2 high methylation group has a strong sensitivity to radiotherapy. This type of patients is suitable for radiotherapy and can improve survival through radiotherapy. COL11A2 methylation level can be used as a potential predictive indicator for radiotherapy.

[0046] 2.4 TME cell infiltration analysis results

[0047] Violin plot( Figure 5 ) showed that the infiltration levels of Tregs, neutrophils, B cells, CAFs, and hematopoietic stem cells (HSC) in the low methylation group were higher than those in the high methylation group, while CD4 + T cells, CD8 + The opposite is true for T cells and NK cells.

[0048] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A methylation marker for the detection and / or diagnosis of oral squamous cell carcinoma, characterized in that: The methylation marker is the methylation level of the COL11A2 gene.

2. Use of the methylation marker according to claim 1 in any one of the following (1) to (3): (1) Use in the preparation of products for detecting and / or diagnosing oral squamous cell carcinoma; (2) Its role in the preparation of products for predicting the efficacy of radiotherapy for oral squamous cell carcinoma; (3) In predicting Tregs cells, neutrophils, B cells, CAFs cells, hematopoietic stem cells, CD4 + T cells, CD8 + Application of infiltration levels of T cells and / or NK cells.

3. The use according to claim 2, characterized in that: In (1), the methylation level of COL11A2 gene in oral squamous cell carcinoma samples was significantly higher than that in normal subjects.

4. The use according to claim 2, characterized in that: In (1), if the methylation level of COL11A2 gene is 0.23, the survival rate of patients with oral squamous cell carcinoma is high; if the methylation level of COL11A2 gene is 0.35, the survival rate of patients with oral squamous cell carcinoma is low.

5. The use according to claim 2, characterized in that: In (2), compared with not receiving radiotherapy, after radiotherapy, the COL11A2 methylation level was greater than 0.35, and the survival rate of patients with oral squamous cell carcinoma was improved.

6. The use according to claim 2, characterized in that: In (3), if the methylation level of COL11A2 gene is low, it is predicted that the infiltration level of Tregs cells, neutrophils, B cells, CAFs cells, and hematopoietic stem cells is high; if the methylation level of COL11A2 gene is high, it is predicted that CD4 + T cells, CD8 + The infiltration levels of T cells and NK cells were high.

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