A method of cancer screening

A technology for cancer screening, applied in the field of medical molecular biology

Active Publication Date: 2016-02-17
赖鸿政
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Problems solved by technology

[0008] Recent epidemiological studies have shown that the concentration of serum folate (a major source of methyl group) is associated with HPV infection and clearance; in the metabolism of the methyl cycle (methylcycle), the enzyme gene Genetic polymorphisms have also been reported to be associated with the development of cervical intraepithelial lesions; like the concept of supergene evolution, research on the relationship between DNA methylation and cervical cancer is also prevalent. The rapid increase of methylation research shows the possibility of using methylation as a cervical cancer screening; due to the characteristics of genetic and environmental interactions, the degree of methylation of tumor suppressor genes varies with different genes and different ethnic groups. Different diseases also have different methylatorphenotypes; however, the methylatorphenotypes of cervical cancer and their association with HPV genotypes are still unknown, and what specific genes in cervical cancer will be Methylation, and how many genes are needed to meet the needs of clinical application, these issues are still issues that need to be confirmed in the future
[0009] It can be seen that there are still many deficiencies in the above-mentioned commonly used cervical cancer screening methods, which need to be improved urgently

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Experimental program
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Embodiment 1

[0067] Embodiment one material and method

[0068] 1. Test materials

[0069]The test materials included a series of complete samples of cervical lesions, including normal samples (n=45), low-grade squamous cell intraepithelial lesions (LSIL, n=45), high-grade squamous cell intraepithelial lesions (HSIL, n=58 ), squamous cell carcinoma (squamous cell carcinoma, SCC, n=109); the test materials also included a series of complete ovarian tumor samples, including ovarian benign tumor samples (n=36), ovarian borderline tumor samples (n=6), Ovarian malignant tumor samples (n=122); all cervical and ovarian samples were obtained from the Gynecological Tumor Tissue Bank of Taipei Tri-Service General Hospital, and the genomic DNA of each sample was extracted with Qiagene DNA kit and quantified by PicoGreen fluorescence absorption method DNA, and the quality of the DNA was checked by gel electrophoresis.

[0070] In addition, the liver cell samples include normal liver cell samples (n=...

Embodiment 2

[0095] Example 2 Screening of cervical cancer methylation indicator genes

[0096]Using CpG island microarrays (CpGisland microarrays) to perform differential methylation heterozygosity (DMH) to screen out genes with high methylation in cervical squamous cell carcinoma (SCC); CpG island microarrays (CpGisland microarrays) The results showed that there were 216 differentially methylated sites between cervical cancer tissue samples and normal Pap smear samples. After removing sequence repeats, 26 CpG islands (promoter CGIs) of gene activator regions were obtained.

[0097] These gene activators were sequenced and analyzed, and 6 genes were selected, including: SOX1 (SEQ ID No: 1), PAX1 (SEQ ID No: 2), LMX1A (SEQ ID No: 3), NKX6-1 (SEQ ID No: 4 ), WT1 (SEQ ID No: 5) and ONECUT1 (SEQ ID No: 6), the detailed information of which is shown in Table 4; as can be seen from Table 4, these six genes are important transcription factors (transcription factors) in the development process, S...

Embodiment 3

[0103] Example 3 Correlation between DNA methylation and gene expression in cervical cancer cell lines

[0104] In order to confirm whether the expression of cervical cancer methylation pointer genes is regulated by DNA methylation, the DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) (SigmaChemicalCo.) was treated with 10 μM After 4 days in HeLa cervical cancer cell line, methylation-specific PCR (MSP) was used to check the demethylation of the above six gene activators; MSP primers ( U), and the MSP primer (M) that can specifically identify the sequence of the methylated gene for methylation-specific PCR (MSP), the results are as follows Figure 4A As shown, in the HeLa cervical cancer cell line (AZC-) not treated with 5'-aza-2'-deoxycytidine (AZC), all six target genes had methylation phenomenon (such as Figure 4A shown in column 1), and unmethylated genes (such as Figure 4A Shown in column 2); while in HeLa cervical cancer cell lines (AZC+) treated with 5'...

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Abstract

A cancer screening method comprises the following steps: (1) a tested body is provided; (2) the methylation state of a CpG sequence of at least one target gene of genome DNA of the tested body is detected, wherein the target gene comprises SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1; (3), whether the tested body has a cancer or a precancerous lesion is judged according to the methylation state of the target gene. A methylation state detection method adopts MSP (methylation-specific PCR), QMSP (quantitative methylation-specific PCR), BS (bisulfite sequencing), microarrays, mass spectrometer, DHPLC (denaturing high-performance liquid chromatography) and the like.

Description

technical field [0001] The invention relates to a cancer screening method, in particular to a cancer screening method using methylated DNA as a biomarker, and belongs to the field of medical molecular biology. Background technique [0002] Cervical cancer is one of the main causes of death for women in the world and in Taiwan, China. According to the statistics of the World Health Organization (WHO) in 2002, cervical cancer is the second cause of cancer death among women in the world, second only to breast cancer; Screening is the best way to prevent cervical cancer. There are two main methods of cervical cancer screening. One is the most common papsmear, and the other is HPV testing. ); Cervical smear is to take out the secretions of the cervix, and use a microscope to observe whether there are cancerous lesions in the exfoliated epithelial cells, so as to detect cervical cancer in the early stage; and HPV test is to use reverse transcription polymerization Enzyme chain re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/154
Inventor 赖鸿政
Owner 赖鸿政
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