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TMEM176A gene promoter region DNA methylation detection

A technology for TMEM176A and gene promoter region, applied in the kit containing the primer pair, non-methylated primer pair, methylated primer pair for detecting the methylation state of TMEM176A gene promoter region, can solve the problem of esophagus Cancer epigenetic changes and functional studies have not yet been reported, and have achieved far-reaching clinical significance and promotion value, good stability, and easy operation.

Active Publication Date: 2016-03-16
GENERAL HOSPITAL OF PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In summary, there are still many deficiencies in the current functional research on TMEM176A, and its epigenetic changes and functional research in esophageal cancer have not been reported yet.

Method used

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  • TMEM176A gene promoter region DNA methylation detection
  • TMEM176A gene promoter region DNA methylation detection
  • TMEM176A gene promoter region DNA methylation detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Template preparation (extraction of genomic DNA and sulfuration modification process)

[0044] Preparation of DNA: Obtain esophageal cancer, colorectal cancer specimens and normal tissue specimens mentioned above. In this example, 8 cases of esophageal cancer (EC1-EC8), 8 cases of colorectal cancer (CRC1-CRC8), 4 cases of normal esophagus (EN1-EN4), and 8 cases of normal colorectum (CN1-CN8) were treated with phenol - The method of chloroform extraction was used to extract genomic DNA respectively, and the absorbance (A) value was measured by an ultraviolet spectrophotometer to determine its content and purity.

[0045] Sulfite modification: refer to herman (J.G.Herman, J.R.Graff, S.Myohanen, B.D.NelkinandS.B.Baylin, Methylation-specificPCR: novelPCRassayformethylationstatusofCpGislands, Proc.Natl.Acad.Sci.USA93(1996), 9821–9826.), etc. method of reporting. Take the genomic DNA prepared above, take 2ug DNA accurately after dilution, add deionized water to a final v...

Embodiment 2

[0071] Example 2 Clinical Specimen Detection

[0072] 103 clinical specimens of esophageal cancer, 96 clinical specimens of colorectal cancer, 4 normal esophageal tissue specimens, and 8 normal colorectal tissue specimens were collected. Perform MS-PCR amplification, template preparation, PCR amplification system and conditions, and detection of amplified products are the same as those in Implementation 1. For the detection results, please refer to The following table :

[0073] Classification Number of cases Number of M (methylation) cases Number of cases of U (no methylation) Methylation positive rate Esophageal cancer 103 63 40 61.2％ normal esophagus 4 0 4 0 colorectal cancer 96 51 45 53.13％ normal colorectal tissue 8 0 8 0

Embodiment 3

[0074] Embodiment 3 sensitivity experiment

[0075] The DNA of the esophageal cancer cell line K410 (100% methylation in the promoter region of the TMEM176A gene) was mixed with the DNA of normal esophageal tissue cells (100% non-methylation in the promoter region of the TMEM176A gene) in proportion, and the sulfuration modification was carried out (the method was the same as that of Embodiment 1), then carry out MS-PCR. PCR products were subjected to 2% agarose gel electrophoresis, measured by ultraviolet transmission analyzer and photographed.

[0076] Grouping: Group 1: 100% DNA of esophageal cancer cell line K410+0% DNA of normal esophageal tissue cells

[0077] Group 2: 50% DNA of esophageal cancer cell line K410+50% DNA of normal esophageal tissue cells

[0078] Group 3: 5% DNA of esophageal cancer cell line K410+95% DNA of normal esophageal tissue cells

[0079] Group 4: 1% DNA of esophageal cancer cell line K410 + 99% DNA of normal esophageal tissue cells

[0080] ...

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Abstract

According to the present invention, TMEM176A gene is firstly adopted as a target gene, and the promoter region in esophageal cancer cells and colon cancer cells presents the high methylation state through a MSP technology; primers and a kit for detecting cell TMEM176A gene promoter region methylation state are provided, wherein the primers are a pair of methylation primers, or a pair of methylation primers and a pair of non-methylation primers; with the primers and the kit, the esophageal cancer detection specificity is good, and the sensitivity is high and achieves 0.5%, wherein the esophageal cancer detection specificity is 61.2%, the colorectal cancer detection specificity is 53.13%, and five cancer cells in 1000 cells can be detected; and the application of the kit to detect the TMEM176A gene promoter region DNA methylation state can be adopted as the powerful tool for digestive tumor diagnosis, treatment effect observation, prognosis determination, minimal residual disease detection and the like, and the advantages of easy operation, good stability, far-reaching clinical significance and promotion are provided.

Description

technical field [0001] The present invention relates to methylation detection technology, in particular to the detection of DNA methylation in the promoter region of TMEM176A gene, especially a pair of methylation primers for detecting the methylation state of the promoter region of TMEM176A gene, further comprising Unmethylated primer pair. Meanwhile, the present invention also relates to a kit containing the pair of primers. Background technique [0002] Esophageal cancer is one of the most lethal malignant tumors worldwide, ranking eighth among common tumors and fourth among digestive tract tumors. Despite advances in clinical oncology, esophageal cancer remains one of the leading causes of cancer-related mortality. Esophageal cancer mainly includes squamous cell carcinoma of the esophagus and adenocarcinoma of the esophagus, and its incidence rate has great regional differences. The high-incidence areas mainly include southern and eastern Africa, central and eastern As...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/154
Inventor 郭明洲令狐恩强张游韩英杰
Owner GENERAL HOSPITAL OF PLA
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