Sieving and checking method for cancers

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

Active Publication Date: 2009-11-04
赖鸿政
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  • Abstract
  • Description
  • Claims
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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) is associated with HPV infection and clearance; in the metabolism of the methyl cycle, the enzyme The genetic polymorphisms (genetic polymorphisms) have also been reported to be related to the development of cervical intraepithelial lesions; like the concept of supergene evolution, the research on the relationship between DNA methylation and cervical cancer is also popular, and cervical cancer DNA methylation research in China is increasing rapidly, showing 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 will have different methylator phenotypes; however, the methylator phenotypes of cervical cancer and their association with HPV genotypes are still unknown, and what are the methylator phenotypes in cervical cancer Specific genes will be methylated, and how many genes are needed to meet the needs of clinical applications, 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 cancer samples (n=122); all cervical samples and ovarian samples were obtained from the Gynecological Tumor Tissue Bank of Taipei Tri-Service General Hospital. The DNA was quantified by the method, and the quality of the DNA was checked by gel electrophoresis.

[0070] In addition, the liver cell samples include normal liver cell samples (n=13), chronic hepatitis (n=15), liver cirrhosis (cirrhosis, n=40), liver cancer (hepatocellular ...

Embodiment 2

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

[0096]Using CpG island microarrays (CpG island microarrays) to carry out differential methylation heterozygous reaction (DMH) to screen out genes with high methylation in cervical squamous cell carcinoma (SCC); CpG island microarrays ( CpG island microarrays) showed that there were 216 sites with differential methylation between cervical cancer tissue samples and normal Pap smear samples. After removing sequence repeats, 26 gene activating sub-region CpG islands were obtained ( promoter CGIs).

[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 details of which are shown in Table 4; as can be seen from Table 4, these six genes are all developing Important transcription factors (transcription factors) in the process, SO...

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, DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) (Sigma Chemical Co. ) to treat the HeLa cervical cancer cell line for 4 days, and then check the demethylation of the above six gene activators by methylation-specific PCR (MSP); Primer (U), and the MSP primer (M) that can specifically identify methylated gene sequences 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'-aza-2'-deoxycytidine (AZC) for ...

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Abstract

The invention relates to a sieving and checking method for cancers, comprising the following steps: (1), providing a test body to be checked; (2), checking the CpG sequence methylation state of at least one target gene in the genome DNA of the test body to be checked, wherein at least one of the target genes comprises SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1; and (3), judging whether the test body has cancers or precancerous lesions or not according to the existence or inexistence of the methylation state of the target genes. The methylation state checking method comprises a methylation specific polymerase chain reaction, a quantitative methylation specific polymerase chain reaction, sulphite sequencing, micro-arrays, mass spectrograph analysis or denaturation high-efficiency 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 Pap smear (Pap smear) and the other is human papillomavirus (HPV) test. HPV testing); Pap 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 at an early stage; while HPV testing is to reverse Revers...

Claims

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Application Information

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