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Method and kit for diagnosing bladder cancer with urine

A technology of bladder cancer and detection kit, applied in the field of biomedical detection, can solve problems such as small sample size

Inactive Publication Date: 2012-01-11
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is feasible to extract the cellular DNA in the subject's urine sample to detect the methylation status of specific genes. The inventor's work related to early bladder cancer has applied for a patent in 2008, and the patent application number is 200710044106, but the previous work For a small sample size

Method used

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  • Method and kit for diagnosing bladder cancer with urine
  • Method and kit for diagnosing bladder cancer with urine
  • Method and kit for diagnosing bladder cancer with urine

Examples

Experimental program
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Effect test

Embodiment 1

[0106] Example 1. High-throughput methylation profiling at the genome level

[0107] The present invention conducts high-throughput genomic analysis on two cases of normal human bladder mucosa (obtained from Urology Department of Zhongshan Hospital) and two cases of bladder cancer cell lines (5637 (ATCC: HTB-9), T24 (ATCC-4)). Establishment of methylation profiles.

[0108] Extract the above-mentioned bladder cancer cells and normal tissues, and use MBD (methylation binding protein domain) affinity chromatography column to enrich the medium and low methylated DNA fragments step by step; step 2: the collected hypermethylated DNA fragments Add SOLEXA adapters and send to Shanghai Bohao Biotechnology Co., Ltd. for SOLEXA high-throughput sequencing. The bladder cancer tumor cell group and the normal bladder tissue group obtained 3 million reads respectively. After bioinformatics analysis, genome mapping, and the positioning information was uploaded to UCSC to establish a database...

Embodiment 2

[0109] Example 2, preliminary screening of methylation difference

[0110]Further screened from the above 1627 highly methylated regions associated with gene promoters in tumor cell lines to obtain the promoter-related regions of the top 104 genes with the largest difference in methylation value (sorted according to P value, select the region related to the normal bladder The top 104 genes with the highest degree of methylation compared to tissues). Including: NES, DLX4, C10orf114, C8orf84, COL25A1, CTSA, ESX1, GJD3, HNRNPF, HOPX, LAMA1, OXTR, PCSK6, RADIL, SNX31, RASD1, PAX6, SP8, TMEM163, GRID1, ISL2, BDNF, BEND4, BHLHE23, CYB5R2, CYP24A1, HS3ST3A1, MAFA, NKX6_1, NOS1, NPTX1, ONECUT1, PGR, HOXC4, SIM2, ADRA1A, ARPC1B, C1QL2, CDH8, CHRDL2, DPY19L2P2, E2F8, EVX1, FAM84B, LOC645323, MGC45800, MRGRPCRF, PGAM2, PHOX2A, PVT1, SFRP2, SLC1A2, DGKK, ACTA1, CCND1, CYP26B1, FGF3, FOXD3, LHX9, NEUROG2, OPRK1, PADI2, SLC6A4, BARHL1, CDO1, TUBB2B, IHH, TBX20, SLC46A2, PDZK1P1, FEZF2, B ...

Embodiment 3

[0119] Example 3, Further Screening for Differences in Methylation

[0120] The tested samples were bladder cancer cell lines 5637 (ATCC: HTB-9) and T24 (ATCC-4), 2 cases of normal bladder tissue, 8 cases of normal human urine samples (the urine sediment was obtained), bladder cancer There were 17 urine samples from patients. The urine samples were randomly selected, and the selection criteria were that the amount of genomic DNA was large enough to deal with multi-gene screening.

[0121] The specific regions of the 49 gene promoters screened in Example 2 were detected by methylation-specific PCR (MSP). The chemical modification of the sample with ammonium bisulfite and the methylation-specific PCR analysis method are the same as in Example 2.

[0122] see results image 3 . As a result, the inventors set the screening criteria as 4, that is, there were at least 4 cases of methylation in the urine samples of 17 bladder cancer patients, and the targets with a methylation rat...

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Abstract

The invention relates to a method and a kit for diagnosing bladder cancer with urine. The invention discloses a group of methylated sensitive genes, comprising ECEL1, KCNV1, LMX1A, PROX1, SLC6A20, TAL1, TMEM 26, and VAXI gene. In urine samples of bladder cancer patients, the specific CpG sites of the genes are showed the highest methylation levels. Accordingly, the genes are the biological marker of bladder cancer. The method and the kit can be used as the basic of designing diagnostic reagents of bladder cancer, and are suitable for auxiliary detection of cancer in hospitals, postoperative followup, screening of high risk group of bladder cancer in community health centers, and screening of common people and high risk practitioners of bladder cancer in physical examination center.

Description

technical field [0001] The present invention relates to biomedical detection; more specifically, the present invention relates to novel bladder cancer biomarkers (urine markers), and bladder cancer diagnostic reagents and kits based on the markers. Background technique [0002] The idea that cancer is considered a genetic disease has persisted in the field of oncology research for decades. With the successful completion of the Human Genome Project, the Cancer Genome Project has been launched in the United States. This project aims to map the detailed genetics of mutants and SNPs related to fifty common human cancers. Several recent systematic large-scale sequencing have confirmed that the number of somatic mutations in cancer tissues is significantly less than expected. important role in tumors. [0003] Epigenetics (Epigenomics) is a discipline that studies the heritable changes in gene function without DNA sequence changes, and ultimately leads to phenotypic changes. Ep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 余坚王红阳顾健人杨胜利赵仰星孙晋枫张红宇顾峻王韦何英华郭士成
Owner SHANGHAI INST OF ONCOLOGY
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