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Triple negative breast cancer marker COL4A2 and application thereof

A triple-negative breast cancer, marker technology, applied in the field of molecular biology and tumor drugs

Active Publication Date: 2015-04-22
PEKING UNIV SHENZHEN HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very few studies on triple-negative breast cancer markers at present, and the purpose of the present invention is to discover new triple-negative breast cancer markers

Method used

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  • Triple negative breast cancer marker COL4A2 and application thereof
  • Triple negative breast cancer marker COL4A2 and application thereof
  • Triple negative breast cancer marker COL4A2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Real-time PCR detection of COL4A2 gene mRNA expression in each breast cancer cell

[0020] 1. Experimental materials:

[0021] Individual cells and their media:

[0022] (1)MDA-MB-231: L15+10%FBS

[0023] (2) MCF-7: DMEM-H+10%FBS+0.01mg / ml insulin

[0024] (3) BT-474: RPMI1640++10%FBS+0.01mg / ml insulin

[0025] (4)SK-BR-3: RPMI1640++10%FBS

[0026] 2. Experimental method:

[0027] 2.1 Extraction of total cellular RNA:

[0028] Collect about 1 × 10 cultured cells after transfection 6 One, wash twice with PBS; add 1ml of pre-cooled TRNzol to the centrifuge tube, mix well, place at room temperature for 10min, centrifuge at 10000rpm for 10min at 4°C, take the supernatant and transfer to a new centrifuge tube; add 0.2ml of chloroform , cover the tube cap, shake vigorously for 15 seconds, and place at room temperature for 3 minutes; centrifuge at 10,000 rpm for 10 minutes at 4°C, take the upper colorless aqueous phase, transfer to a new centrifuge tube, add a...

Embodiment 2

[0045] Example 2 Western blot detection of COL4A2 protein expression in each breast cancer cell

[0046] 1. Experimental method

[0047] 1.1 Extraction of target protein:

[0048]After the cell culture was completed, the cell culture medium in the culture plate was discarded, washed twice with 0.9% NaCl pre-cooled at 4°C, the NaCl was blotted dry, and the cell culture plate was placed on ice. Add 100 μl of RIPA cell lysate pre-added with protease inhibitors to each well, and lyse the cells on ice for 3 min. Scrape the lysed cells with a cell scraper, transfer the cell lysate to a pre-cooled 1.5ml EP tube on ice, and centrifuge at 12000rpm at 4°C for 20min. After transfer and centrifugation, transfer the supernatant to a new pre-cooled 1.5ml EP tube and measure the protein concentration.

[0049] 1.2 Determination of protein concentration:

[0050] According to the number of standards and samples, prepare an appropriate amount of BCA working solution with 50 volumes of BCA ...

Embodiment 3

[0073] Construction of embodiment 3 interference plasmid

[0074] 3.1 Design and synthesis of COL4A2 gene interference sequence

[0075] According to the gene sequence of COL4A2 (Gene ID: 1284) in GenBank, 6 interference sequences and 1 negative control sequence were designed. The interference sequences and their nomenclature are shown in Table 8:

[0076] Table 8 Design of interference sequences

[0077] serial number

sequence naming

sequence

SEQ ID NO 1

Si-1

GGGTGTGAAGAAGTTTGAT

SEQ ID NO 2

Si-2

GCCTTATGCACTGCCTAAA

SEQ ID NO 3

Si-3

GGGGTGAACCTGGAGAGCC

SEQ ID NO 4

Si-4

GCCAAGACCAAGGAGAATG

SEQ ID NO 5

Si-5

GGAATGCAGATGTACAGAA

SEQ ID NO 6

Si-6

GGCAACAGAGGACTTGGTT

SEQ ID NO 7

Neg

GCAGATAGGTAGGCGTTAT

[0078] 3.2 COL4A2 interference sequence primer design

[0079] The primer has a HindIII restriction site at the 5' end and a BamHI restriction site at t...

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Abstract

The invention relates to a triple negative breast cancer marker COL4A2 and application thereof. By means of comparison researches on different cell lines, a specifically expressed gene COL4A2 is discovered in the triple negative breast cancer cell line MDA-MB-231, the sequence interfering with expression of the COL4A2 gene is further designed and synthesized and is respectively constructed to plasmid vectors to transfect the cell MDA-MB-231, and a sequence capable of efficiently interfering with expression of the COL4A2 gene is successfully obtained. The triple negative breast cancer marker COL4A2 and the siRNA efficiently interfering with the marker gene have an important clinical application value.

Description

technical field [0001] The invention relates to the fields of molecular biology and tumor medicine, in particular to a triple-negative breast cancer marker COL4A2 and its application in the diagnosis of triple-negative breast cancer. Background technique [0002] Breast cancer is the most common and deadly malignancy in women. Nearly 1.3 million women worldwide suffer from breast cancer every year, and more than 400,000 women die of breast cancer metastasis and recurrence. Different types of breast cancer can have significantly different biological characteristics and clinical manifestations. Therefore, classifying a patient's breast cancer has become an important component for determining treatment options. At present, immunohistochemical methods are mainly used clinically, and breast cancer is divided into four molecular subtypes according to the detection results of estrogen receptor (ER), progesterone receptor (PR) and HER-2. : luminal A type (ER positive or PR positi...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12Q1/68A61K48/00A61P35/00
Inventor 何劲松陈伟财叶伟亮孙婕俞莉敏
Owner PEKING UNIV SHENZHEN HOSPITAL
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