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Biomarker for mass colorectal cancer screening

A biomarker, colorectal cancer technology, applied in biochemical equipment and methods, microbial determination/inspection, application, etc., can solve problems such as research on detection methods without RSPO2 gene methylation, and achieve fast processing and cost. Low, convenient and flexible sample collection effect

Active Publication Date: 2013-02-20
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, although there is a detection method for DNA gene methylation in the prior art, there is no specific research on the detection method for the methylation of the RSPO2 gene.

Method used

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  • Biomarker for mass colorectal cancer screening
  • Biomarker for mass colorectal cancer screening
  • Biomarker for mass colorectal cancer screening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Down-regulation of RSPO2 expression in colorectal cancer

[0038] Total cellular RNA was extracted by the Trizol method and reverse-transcribed into cDNA by the MLV-reverse transcriptase kit from Invitrogen Company, and all operations were performed according to the method recommended by the kit. Primers RSPO2-F and RSPO2-R for fluorescent quantitative PCR were designed according to the nucleotide sequence of human RSPO2. The primer sequences are as follows:

[0039]Primer RSPO2-F: 5'-ACAATACTGTGTCCAACCAT-3';

[0040] Primer RSPO2-R: 5'-TCCTCTTCTCCTTCGCCTTT-3';

[0041] Using GADPH as an internal reference, the amplification primers are:

[0042] GADPH-F: 5'-ACGGATTTGGTCGTATTGGGC-3';

[0043] GADPH-R: 5'-CTCGCTCCTGGAAGATGGTGAT-3'.

[0044] Using cDNA as a template, the RealMssterMix kit from TIANGEN Company was used for real-time fluorescent quantitative PCR reaction, and the operation was carried out according to the method recommended by the kit.

[0045] Specifi...

Embodiment 2

[0052] Identification of RSPO2 methylation as a potential biomarker in colorectal cancer

[0053] (1) The expression level of RSPO2 in intestinal cancer cells was up-regulated after 5-aza-2'-deoxycytidine treatment

[0054] The low expression of RSPO2 in colorectal cancer cells may be caused by epigenetic abnormalities. To distinguish whether it was due to gene methylation or histone modification, we used the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin, respectively. Intestinal cancer cells were treated with factor A and then the expression of RSPO2 mRNA in the cells was detected.

[0055] Specifically, 1×10 5 Cells / well were seeded in 6-well plates and cultured for 24 h. Experiment with the following groups:

[0056] The control group was the cells without drug addition in the same period;

[0057] test group:

[0058] ①5-Aza-dC group: treated with 2 μM 5-Aza-dC for 96 h,

[0059] ②TSA group: treated with 0....

Embodiment 3

[0083] Application of MSP to detect the methylation status of RSPO2 in colorectal cancer tissues

[0084] Genomic DNA extraction and sodium bisulfite modification were the same as described in Example 2. The MSP method was used to detect the status of RSPO2 methylation in colorectal cancer tissues and normal intestinal mucosal tissues. Specifically, using the modified DNA as a template, using methylated primers, RSPO2-M1 and RSPO2-M2, and unmethylated specific primers, RSPO2-U1 and RSPO2-U2, to perform PCR amplification to amplify the fragment Both were 139 bp in size.

[0085] The PCR reaction system is 20 μl: 10×PCR buffer 2 μl, Taq enzyme 0.5 μl, 10 mM dNTP 2 μl, DNA 60 ng, 10 mM primers 1 μl each, ddH 2 O was added to 20 μl. The PCR reaction conditions were: pre-denaturation at 94°C for 3 min, 37 cycles of 94°C for 30 s, annealing for 45 s, and 72°C for 40 s, and extension at 72°C for 10 min. PCR products were detected by 2% agarose gel electrophoresis.

[0086] MSP m...

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Abstract

The present invention discloses a biomarker for mass colorectal cancer screening, wherein the biomarker is RSPO2 gene. The present invention further discloses a RSPO2 gene methylation MSP detection method, which comprises the following steps: extracting genome DNA containing RSPO2 gene, and detecting purity and content; carrying out treatment modification by using sodium bisulfite; adopting the modified DNA as a template, designing RSPO2 methylation specific primers, carrying out PCR amplification, and then taking the PCR product to carry out electrophoresis detection; and analyzing a result, wherein a methylation positive result exists if a 139 bp band product is amplified through the RSPO2 methylation specific primers, ie., RSPO2 methylation exists. According to the present invention, RSPO2 is adopted as a biomarker for mass colorectal cancer screening, and an RSPO2 methylation status detection method is provided, such that whether patients suffer from colorectal cancer can be detected, and characteristics of no invasion, convenient and flexible sample collection, and the like are provided.

Description

technical field [0001] The invention relates to the technical field of colorectal cancer screening, in particular to a biomarker used for a large number of colorectal cancer screenings, that is, the methylation status of the biomarker in a DNA sample is detected to determine Likelihood of colorectal cancer. Background technique [0002] Colorectal cancer is one of the most common malignant tumors of the digestive tract in the world, and its morbidity and mortality both rank third among malignant tumors and are on the rise. The incidence of colorectal cancer in North America and Western European countries reached 44.4 / 100,000 and 42.9 / 100,000 respectively, and it has reached 13.29 / 100,000 in my country. Colorectal cancer usually undergoes 7 to 10 years of evolution from normal mucosal tissue, adenoma, and then to malignant tumors. The early cure rate of colorectal cancer can reach 90%, but the late cure rate is only 5%. Therefore, early screening and surgical resection of ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12Q1/68
Inventor 鲁新成路立婷廖婉琴武长杰邱孙全
Owner WENZHOU MEDICAL UNIV
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