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Application of gene used as biomarker in colon adenocarcinoma

A colon adenocarcinoma and genetic technology, applied in the field of biomedicine, can solve the problems of poor patient compliance, early diagnosis of colon adenocarcinoma, low sensitivity and specificity, etc.

Inactive Publication Date: 2017-09-19
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinically, the diagnosis of colon adenocarcinoma mainly relies on laboratory tests such as fecal occult blood (FOBT) test, cytological diagnosis, histopathological examination, serum carcinoembryonic antigen (CEA) determination, fiber optic colonoscopy, imaging diagnosis such as colon Air-barium double-contrast radiography, CT scan, MRI, ultrasound section imaging diagnosis, nuclide diagnosis, and colonoscopy are the most reliable methods for the diagnosis of colon adenocarcinoma, but they are invasive, expensive, and poor patient compliance; CT and ultrasound examinations are difficult to detect Small lesions, the early diagnosis of colon adenocarcinoma is limited; CEA is a widely used serum marker in clinical practice, its sensitivity and specificity are low, and it is mainly used for treatment monitoring of patients with colon adenocarcinoma

Method used

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  • Application of gene used as biomarker in colon adenocarcinoma
  • Application of gene used as biomarker in colon adenocarcinoma
  • Application of gene used as biomarker in colon adenocarcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1 Screening for Gene Markers Related to Colon Adenocarcinoma

[0091] 1. Sample collection

[0092] The cancer tissues and paracancerous tissues of 8 patients with early colon adenocarcinoma were collected. All patients had not received radiotherapy and chemotherapy before surgery, and all patients gave informed consent. All the above specimens were obtained with the approval of the organizational ethics committee.

[0093] 2. Preparation of RNA samples

[0094] Tissue RNA was extracted using QIAGEN tissue RNA extraction kit, and the operation was performed according to the specific steps in the manual. Nanodrop2000 was used to detect the concentration and purity of the extracted RNA, agarose gel electrophoresis was used to detect RNA integrity, and Agilent2100 was used to determine the RIN value. Concentration ≥ 200ng / μL, OD260 / 280 between 1.8 and 2.2.

[0095] 3. Remove rRNA

[0096] Ribosomal RNA was removed from total RNA using the Ribo-Zero kit.

[009...

Embodiment 2

[0105] Example 2 QPCR sequencing to verify the differential expression of ARFGEF3 gene

[0106] 1. Large-sample QPCR verification of differential expression of ARFGEF3 gene. According to the sample collection method in Example 1, 60 colon adenocarcinoma tissue samples and 60 paracancerous tissue samples were collected.

[0107] 2. RNA extraction The steps are the same as in Example 1.

[0108] 3. QPCR detection:

[0109] (1) Reverse transcription reaction

[0110] Use 25μl reaction system, take 1μg total RNA for each sample as template RNA, and add the following components to PCR tubes: DEPC water, 5× reverse expression buffer, 10mM dNTP, 0.1mM DTT, 30μM Oligo dT, 200U / μl M-MLV, template RNA.

[0111] According to the reverse transcription reaction conditions in RNA PCR Kit (AMV) Ver.3.0.

[0112] (2) Polymerase chain reaction

[0113] Amplification primers for the ARFGEF3 gene and the housekeeping gene GAPDH were designed and synthesized by Sangon. The primer sequences ...

Embodiment 3

[0120] Example 3 Silencing of ARFGEF3 Gene

[0121] 1. Cell culture

[0122] Human colon adenocarcinoma cell line SW480, at 37°C, 5% CO 2 , cultured in an incubator with a relative humidity of 90%, using medium RPMI-1640 containing 10% fetal bovine serum and 1% P / S. Use well-grown cells for experiments.

[0123] 2. siRNA design

[0124] siRNA sequence against ARFGEF3 gene:

[0125] Negative control siRNA sequence (siRNA-NC):

[0126] Sense strand: 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO.5),

[0127] Antisense strand: 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO.6);

[0128] siRNA1:

[0129] Sense strand: 5'-AUCAGAAUCUGUUUCCAUGGA-3' (SEQ ID NO.7),

[0130] Antisense strand: 5'-CAUGGAAACAGAUUCUGAUGA-3' (SEQ ID NO.8);

[0131] siRNA2:

[0132] Sense strand: 5'-ACUAUACCUAGAUCCAUAGGA-3' (SEQ ID NO.9),

[0133] Antisense strand: 5'-CUAUGGAUCUAGGUAUAGUGA-3' (SEQ ID NO.10);

[0134] siRNA3:

[0135] The sense strand is 5'-UUGACAAACUUCAUAAGGCAC-3' (SEQ ID NO.11),

[0136] The antise...

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Abstract

The invention discloses application of a gene used as a biomarker in colon adenocarcinoma. It first discovered that proliferation, migration and invasion of colon adenocarcinoma cells can be changed by increasing expression of an ARFGEF3 gene in a patient suffering from the colon adenocarcinoma and reducing the expression level of ARFGEF3, and therefore, ARFGEF3 can be used for developing a product for early diagnosis of the colon adenocarcinoma and a drug for treating the colon adenocarcinoma. The application of the gene used as the biomarker in the colon adenocarcinoma provides a theory and experiment foundation for implementation of the precision medicine.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to the application of genes as biomarkers in colon adenocarcinoma, in particular to the application of ARFGEF3 as a biomarker in colon adenocarcinoma. Background technique [0002] Colon cancer is the most common malignant tumor in developed countries such as Western Europe and North America, and it is also one of the nine common malignant tumors in my country. The incidence of colon cancer in China is increasing year by year. In my country, among the deaths due to colon cancer, males rank fifth among malignant tumor deaths, and females rank sixth. According to clinical data, about 40% to 50% of patients with colon cancer will die due to recurrence or deterioration after surgery. Colon adenocarcinoma is the most common type of colon cancer, and its occurrence and development involve multiple complex pathological processes such as the environment, genes, cell types, cell signaling pathways,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/68G01N33/574A61K45/00A61P35/00
Inventor 吴东李骥李景南钱家鸣杨承刚
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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