Method for evaluating relevance between HPV methylation level and cervical lesion severity

Abstract

The invention discloses a method for evaluating relevance between HPV methylation level and cervical lesion severity. The method comprises the steps of sample collection, DNA extraction, DNA methylation database establishment, HPV virus capture sequencing, methylation data analysis and characteristic methylation site screening ability evaluation. According to the method for evaluating the relevance between the HPV methylation level and the cervical lesion severity, all methylation sites are screened out on the HPV16 whole genome level, and relevance analysis is carried out on the HPV16 whole genome methylation level, the methylation level of a single CpG site and the cervical lesion development degree; the CpG site which can be used as a classifier for distinguishing the cervical lesions is searched, and the correlation between the CpG site and the occurrence and progress of the cervical lesions is explored, so that the correlation between the HPV methylation level and the severity of the cervical lesions is evaluated.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for evaluating the correlation between HPV methylation level and the severity of cervical lesions. Background Art

[0002] Cervical lesions are a common female reproductive system disease, and their severity tends to increase during the development process; HPV (human papillomavirus) infection is the main pathogenic factor of cervical lesions and cervical cancer, among which HPV16 is one of the most common carcinogenic types.

[0003] In recent years, many studies have shown that HPV16 infection can lead to abnormal methylation of genomic DNA. Methylation is an important epigenetic modification, and its abnormality can affect gene expression and regulation, thereby affecting cell proliferation, differentiation and transformation, thereby promoting or inhibiting the occurrence and progression of tumors. These changes play an important role in the occurrence, development and prognosis of cancer. Although some studies have explored the association between HPV16 methylation levels and the severity of cervical lesions, these studies mainly focus on the sequences of some elements of the HPV16 viral genome. Only a few studies have evaluated the methylation status of all CpG sites in the entire HPV genome, and only focused on the analysis of HPV methylation. There are still many unknowns and deficiencies in its exact role in cervical lesions and disease grading diagnosis.

[0004] In view of the above situation, it is necessary to screen more HPV16 methylation sites and conduct in-depth research on their role in the development of cervical lesions, so as to provide new biomarkers for the early diagnosis, lesion grading, treatment and prognosis evaluation of cervical lesions. Summary of the invention

[0005] The purpose of the present invention is to provide an evaluation method for the correlation between HPV methylation levels and the severity of cervical lesions in response to the deficiencies of the prior art. The evaluation method for the correlation between HPV methylation levels and the severity of cervical lesions screens out all methylation sites at the HPV16 whole genome level, and performs a correlation analysis between the HPV16 whole genome methylation level and the methylation level of a single CpG site and the degree of cervical lesion development, in order to find CpG sites that can be used as classifiers for distinguishing cervical lesions and explore their correlation with the occurrence and progression of cervical lesions, thereby evaluating the correlation between HPV methylation levels and the severity of cervical lesions.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions.

[0007] A method for evaluating the correlation between HPV methylation level and the severity of cervical lesions includes the following steps, specifically: Step a, sample collection: Collect cervical exfoliated cell samples from women who are HPV16 positive; Step b, DNA extraction: extracting whole genome DNA from the cervical exfoliated cell sample collected in step a using a DNA extraction kit; Step c: DNA methylation library construction, which specifically includes: Step c1, performing sulfite conversion on the DND extracted in step b, and ensuring that the unmethylated cytosine is successfully converted to the corresponding methylated state; Step c2, the methylated DNA is randomly fragmented using an ultrasonic shearing device to ensure the uniformity and coverage of the library; Step c3, methylation library construction, purification, and quality inspection; Step d: HPV virus capture and sequencing, and specifically includes: Step d1, designing and purchasing an RNA probe complementary to the positive strand of HPV16 converted by sulfite; Step d2, hybridization capture; Step d3, sequencing the captured DNA methylation library by high-throughput sequencing technology; Step e: methylation data analysis and evaluation of characteristic methylation site screening capabilities, specifically including: Step e1, processing of raw data of HPV16 methylation sequencing; Step e2, calculation and analysis of methylation levels; Step e3, confirming the biomarkers of differentially methylated sites; Step e4: ROC curve analysis to evaluate the biomarker classification performance.

[0008] Among them, in step a, during the process of collecting HPV16-positive female cervical exfoliated cell samples, the subjects are first subjected to cervical biopsy and divided into three groups according to the histological diagnosis results: squamous cell carcinoma (SCC) group, high-grade cervical intraepithelial neoplasia (CIN2+) group, and normal cervical epithelium or cervicitis without atypical hyperplasia (Nor) group, and the grouping data are entered as clinical data.

[0009] Among them, in step a, the collected HPV16-positive female cervical exfoliated cell samples are stored in a refrigerator at minus 80 degrees Celsius, the specimens are classified and sorted into a warehouse, and the corresponding clinical data are entered at the same time.

[0010] Wherein, in the step b, the quality and concentration of the extracted DNA are tested using an ultraviolet spectrophotometer to ensure that the quality of the extracted DNA meets the requirements of subsequent experiments.

[0011] Compared with the prior art, the present invention has the following beneficial effects, specifically: the method for evaluating the correlation between HPV methylation level and the severity of cervical lesions of the present invention screens out all methylation sites at the HPV16 whole genome level, and performs correlation analysis between the HPV16 whole genome methylation level and the methylation level of a single CpG site and the degree of development of cervical lesions, in order to find CpG sites that can be used as classifiers for distinguishing cervical lesions and explore their correlation with the occurrence and progression of cervical lesions, thereby evaluating the correlation between HPV methylation level and the severity of cervical lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention is further described below with the aid of the accompanying drawings. However, the embodiments in the accompanying drawings do not constitute any limitation to the present invention.

[0013] Figure 1 It is a schematic diagram of the process of the present invention.

[0014] Figure 2 A collection of differentially methylated sites to distinguish different stages of cervical lesions.

[0015] Figure 3 Diagram for the evaluation of the diagnostic performance of methylation biomarkers in distinguishing cervical lesions from normal cervix. DETAILED DESCRIPTION

[0016] The present invention is described below in conjunction with specific implementation methods.

[0017] Embodiment 1, as Figure 1 As shown, a method for evaluating the correlation between HPV methylation level and the severity of cervical lesions includes the following steps, specifically: Step a, sample collection: Collect cervical exfoliated cell samples from women who are HPV16 positive; Step b, DNA extraction: extracting whole genome DNA from the cervical exfoliated cell sample collected in step a using a DNA extraction kit; Step c: DNA methylation library construction, which specifically includes: Step c1, performing sulfite conversion on the DND extracted in step b, and ensuring that the unmethylated cytosine is successfully converted to the corresponding methylated state; Step c2, the methylated DNA is randomly fragmented using an ultrasonic shearing device to ensure the uniformity and coverage of the library; Step c3, methylation library construction, purification, and quality inspection; Step d: HPV virus capture and sequencing, and specifically includes: Step d1, designing and purchasing an RNA probe complementary to the positive strand of HPV16 converted by sulfite; Step d2, hybridization capture; Step d3, sequencing the captured DNA methylation library by high-throughput sequencing technology; Step e: methylation data analysis and evaluation of characteristic methylation site screening capabilities, specifically including: Step e1, processing of raw data of HPV16 methylation sequencing; Step e2, calculation and analysis of methylation levels; Step e3: Figure 2 As shown, differentially methylated sites confirm biomarkers; Step e4: Figure 3 As shown, ROC curve analysis evaluated the biomarker classification performance.

[0018] It should be pointed out that in step a, when collecting cervical exfoliated cell samples from HPV16-positive women, the subjects were first subjected to cervical biopsy and divided into three groups according to the histological diagnosis results: squamous cell carcinoma (SCC) group, high-grade cervical intraepithelial neoplasia (CIN2+) group, and normal cervical epithelium or cervicitis without atypical hyperplasia (Nor) group, and the grouping data were entered as clinical data.

[0019] Through the above steps, the evaluation method of the correlation between HPV methylation level and severity of cervical lesions in the first embodiment has the following advantages, specifically: 1. By screening all methylation sites at the HPV16 whole genome level and performing correlation analysis on the HPV16 whole genome methylation level and the methylation level of a single CpG site and the degree of cervical lesion development, we sought CpG sites that can be used as classifiers for distinguishing cervical lesions and explored their correlation with the occurrence and progression of cervical lesions, thereby evaluating the correlation between HPV methylation levels and the severity of cervical lesions, and expanding our understanding of the role of HPV methylation in the development of cervical cancer; 2. The differential methylation site combinations finally confirmed by the present invention were comprehensively evaluated for diagnostic utility through ROC analysis, and the results showed that these combinations showed significant potential as biomarkers in clinical diagnosis. By developing biomarkers for the diagnosis of cervical lesions, this biomarker can be used as a basis for judging the grading of cervical lesions in clinical applications, thereby improving the early diagnosis rate of cervical lesions and the evaluation of treatment effects.

[0020] Embodiment 2. The difference between Embodiment 2 and Embodiment 1 is that: in step a, the collected HPV16-positive female cervical exfoliated cell samples are stored in a refrigerator at minus 80 degrees Celsius, the specimens are classified and sorted into a warehouse, and the corresponding clinical data are entered at the same time.

[0021] Embodiment 3: The difference between Embodiment 3 and Embodiment 1 is that: in step b, an ultraviolet spectrophotometer is used to detect the quality and concentration of the extracted DNA to ensure that the quality of the extracted DNA meets the requirements of subsequent experiments.

[0022] The above contents are only preferred embodiments of the present invention. For ordinary technicians in this field, according to the concept of the present invention, there will be changes in the specific implementation methods and application scopes. The content of this specification should not be understood as limiting the present invention.

Claims

1. A method for evaluating the correlation between HPV methylation level and the severity of cervical lesions, characterized in that: The following steps are included, specifically: Step a, sample collection: Collect cervical exfoliated cell samples from women who are HPV16 positive; Step b, DNA extraction: extracting whole genome DNA from the cervical exfoliated cell sample collected in step a using a DNA extraction kit; Step c: DNA methylation library construction, which specifically includes: Step c1, performing sulfite conversion on the DND extracted in step b, and ensuring that the unmethylated cytosine is successfully converted to the corresponding methylated state; Step c2, the methylated DNA is randomly fragmented using an ultrasonic shearing device to ensure the uniformity and coverage of the library; Step c3, methylation library construction, purification, and quality inspection; Step d: HPV virus capture and sequencing, and specifically includes: Step d1, designing and purchasing an RNA probe complementary to the positive strand of HPV16 converted by sulfite; Step d2, hybridization capture; Step d3, sequencing the captured DNA methylation library by high-throughput sequencing technology; Step e: methylation data analysis and evaluation of characteristic methylation site screening capabilities, specifically including: Step e1, processing of raw data of HPV16 methylation sequencing; Step e2, calculation and analysis of methylation levels; Step e3, confirming the biomarkers of differentially methylated sites; Step e4: ROC curve analysis to evaluate the biomarker classification performance.

2. The method for evaluating the correlation between HPV methylation level and severity of cervical lesions according to claim 1, characterized in that: In step a, during the process of collecting cervical exfoliated cell samples from HPV16-positive women, the subjects were first subjected to cervical biopsy and divided into three groups according to the histological diagnosis results: squamous cell carcinoma (SCC) group, high-grade cervical intraepithelial neoplasia (CIN2+) group, and normal cervical epithelium or cervicitis without atypical hyperplasia (Nor) group, and the grouping data was entered as clinical data.

3. The method for evaluating the correlation between HPV methylation level and severity of cervical lesions according to claim 1, characterized in that: In step a, the collected HPV16-positive female cervical exfoliated cell samples are stored in a refrigerator at minus 80 degrees Celsius, the specimens are classified and sorted into a warehouse, and the corresponding clinical data are entered at the same time.

4. The method for evaluating the correlation between HPV methylation level and severity of cervical lesions according to claim 1, characterized in that: In step b, the quality and concentration of the extracted DNA are tested using an ultraviolet spectrophotometer to ensure that the quality of the extracted DNA meets the requirements of subsequent experiments.

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