Molecular marker of esophageal squamous carcinoma

A technology of esophageal squamous cell carcinoma and drugs, applied in the field of biomedicine, can solve problems such as limited value and difficulty in early diagnosis of esophageal squamous cell carcinoma

Active Publication Date: 2016-07-06
QINGDAO MEDINTELL BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods have their own advantages, they all have certain difficulties in the early diagnosis of esophageal squamous cell carcinoma.
[0006] With the in-depth research on the molecular mechanism of tumorigenesis and development, some molecular markers related to the occurrence and development of esophageal squ

Method used

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  • Molecular marker of esophageal squamous carcinoma
  • Molecular marker of esophageal squamous carcinoma
  • Molecular marker of esophageal squamous carcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Screening for gene markers associated with esophageal squamous cell carcinoma

[0064] 1. Sample collection

[0065] Paracancerous tissues and esophageal squamous cell carcinoma tissue samples were collected from 6 cases of esophageal squamous cell carcinoma. All the above specimens were obtained with the consent of the organizational ethics committee.

[0066] 2. Preparation of RNA samples ((use QIAGEN tissue RNA extraction kit for operation)

[0067] Take out the tissue samples frozen in liquid nitrogen, put the tissue samples into a pre-cooled mortar for grinding, and extract and isolate RNA according to the instructions in the kit. details as follows:

[0068] 1) Add Trizol and place at room temperature for 5 minutes;

[0069] 2) Add 0.2ml of chloroform, vibrate the centrifuge tube vigorously, mix well, and place it at room temperature for 5-10min;

[0070] 3) Centrifuge at 12000rpm for 15min, transfer the upper aqueous phase to another new centrifuge...

Embodiment 2

[0083] Example 2 QPCR sequencing to verify the differential expression of CCKBR gene

[0084] 1. Large-sample QPCR verification of differential expression of CCKBR gene. According to the sample collection method in Example 1, 50 cases of esophageal squamous cell carcinoma adjacent tissues and 50 cases of esophageal squamous cell carcinoma tissues were selected.

[0085] 2. The RNA extraction steps are the same as in Example 1.

[0086] 3. Reverse transcription:

[0087] Using a 25 μl reaction system, take 1 μg total RNA for each sample as template RNA, and add the following components to the PCR tube: DEPC water, 5× reverse transcription buffer, 10 mM dNTP, 0.1 mMDTT, 30 μM OligodT, 200 U / μl M-MLV, template RNA. Incubate at 42°C for 1 hour, then centrifuge briefly at 72°C for 10 minutes.

[0088] (3) QPCR amplification test

[0089] Primer design

[0090] The primer sequence of CCKBR gene is:

[0091] Forward primer: 5'-TTGGTTGCCAGTTTATAG-3' (SEQ ID NO.3)

[0092] Reve...

Embodiment 3

[0103] Overexpression of embodiment 3CCKBR gene

[0104] 1. Cell culture

[0105] Human esophageal squamous cell carcinoma cell line KYSE150 was incubated at 37°C and 5% CO in DMEM containing 10% fetal bovine serum and 1% P / S. 2 , Cultivated in an incubator with a relative humidity of 90%. Change the medium once every 2-3 days, and use 0.25% EDTA-containing trypsin for routine digestion and passage.

[0106] 2. Overexpression of CCKBR gene

[0107] 2.1 Construction of CCKBR gene expression vector

[0108] According to the coding sequence of CCKBR gene (as shown in SEQIDNO.1) design amplification primer, primer sequence is as follows:

[0109] Forward primer: 5'-CCGAAGCTTGCCACCATGGAGCTGCTAAAG-3' (SEQ ID NO.7)

[0110] Reverse primer: 5'-CGGGCGGCCGCGCCAGGGCCCAGTGTGCTA-3' (SEQ ID NO.8)

[0111] The coding sequence of the full-length CCKBR gene was amplified from the cDNA library of adult fetal brain (clontech company, product number: 638831), and the above cDNA sequence was...

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Abstract

The invention discloses a molecular marker of esophageal squamous carcinoma. According to the molecular marker, a high-throughput sequencing result is analyzed by using a bioinformatics method, the result shows a candidate gene CCKBR of esophageal squamous carcinoma, the CCKBR gene is in low expression in tissue of a patent suffering from esophageal squamous carcinoma, proliferation of esophageal squamous carcinoma cells can be inhibited by increasing the expression level of CCKBR, and the research result shows that the CCKBR gene and expression products of the CCKBR gene are potential therapeutic targets of esophageal squamous carcinoma.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a molecular marker of esophageal squamous cell carcinoma, specifically the molecular marker is a CCKBR gene. Background technique [0002] Esophageal cancer is a malignant tumor of the esophageal mucosal epithelium or esophageal glandular epithelium. It mainly includes two different clinicopathological types: esophageal squamous cell carcinoma and esophageal adenocarcinoma. The tumor pathogenesis and pathology of the two pathological types are different. Globally, the incidence and mortality of esophageal cancer rank ninth and eighth among malignant tumors, respectively. Esophageal cancer is one of the main malignant tumors in my country, the incidence rate ranks fifth among all kinds of malignant tumors, and the mortality rate ranks fourth. Different from esophageal adenocarcinoma, which frequently occurs in Europe and the United States, esophageal squamous cell carcinoma is the main ty...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/68G01N33/574A61K45/00A61P35/00
CPCA61K45/00C12Q1/6886C12Q2600/158G01N33/57407G01N33/68
Inventor 杨承刚孙耀兰李瑜
Owner QINGDAO MEDINTELL BIOMEDICAL CO LTD
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