CN110592218B

CN110592218B - Biomarker for diagnosing and treating breast cancer

Info

Publication number: CN110592218B
Application number: CN201911006958.0A
Authority: CN
Inventors: 雷海; 贾新建; 罗雪; 易瑛
Original assignee: Peoples Hospital of Deyang City
Current assignee: Peoples Hospital of Deyang City
Priority date: 2019-10-22
Filing date: 2019-10-22
Publication date: 2020-09-15
Anticipated expiration: 2039-10-22
Also published as: CN110592218A

Abstract

The invention discloses a biomarker for diagnosing and treating breast cancer, which is lncRNA LINC 02343. According to the invention, the expression up-regulation of LINC02343 in breast cancer is found by detecting the differentially expressed genes in cancer tissues and normal tissues, and the fact that LINC02343 can be used as a detection target for diagnosing and treating breast cancer is prompted.

Description

Biomarker for diagnosing and treating breast cancer

Technical Field

The invention belongs to the field of biomedicine, and relates to a biomarker for diagnosing and treating breast cancer, wherein the biomarker is LINC 02343.

Background

With the increasing incidence of breast cancer, the breast cancer becomes a malignant tumor with the highest incidence rate in women, and the incidence rate of the breast cancer in China is still increasing at present. Systemic treatment has resulted in a significant reduction in the mortality rate of breast Cancer over the last decades, but the mortality rate remains secondary to the mortality rate of malignant tumors (Siegel R, Naishadham D, JemalA. Cancer statistics [ J ],2012.CA Cancer J Clin,2012,62(1): 10-29). According to the difference of gene expression profiles, the breast cancer is divided into different molecular subtypes at the mRNA level, mainly comprising triple negative breast cancer, HER-2 amplification type breast cancer, normal cell-like breast cancer, Luminal A type breast cancer and Luminal B type breast cancer. Currently, the approved molecular classification of breast cancer mainly comprises four types, Luminal A type breast cancer, Luminal B type breast cancer, triple negative type breast cancer and HER-2 overexpression type breast cancer. Although existing molecular typing provides a good basis for the treatment and prognosis evaluation of patients, therapeutic difficulties still cannot be solved for some of the breast cancer patients with high heterogeneity.

In recent years, with the progress of molecular biology and genomics, the study of metastasis and recurrence of malignant tumors from the molecular mechanism and molecular targeted therapy have become a hot research direction in oncology. More and more researchers have suggested that breast cancer is highly heterogeneous, and this difference in prognosis is thought to be due to the molecular difference of tumors, and that different molecular subtypes of breast cancer need to receive corresponding biological treatment as independent diseases (Sarlie T, Perou CM, Tibshirani R, et a1.Gene expression patterns of breast cancer disorders with clinical indications [ J ] Proc Natl Acad Sci USA 2001,98(19): 10869-74.). Therefore, at present, we need to find more accurate and more tumor markers and therapeutic targets, improve the traditional cancer assessment method, develop a personalized scheme for treating breast cancer, assess the curative effect, and predict the prognosis (Etzionir, urban N, Ramsey S, et al. the case for early detection [ J ]. NaI RevCancer,2003,3: 243-.

With the development of biotechnology and the intensive study of molecules, it was found that long non-coding RNA (LncRNA), which was previously called "dark substance", plays an important role in the development of tumors. lncRNA is RNA with length larger than 200bp, lack of open reading frame and can not code protein, and is related to proliferation, differentiation, metabolism, immunity and apoptosis of cells. The research on lncRNA related to the breast cancer has important significance for realizing further typing of the breast cancer and accurate medical treatment.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention detects the expression of lncRNA in a breast cancer specimen in a tumor tissue and a normal tissue through a high-throughput sequencing method, finds the lncRNA with obvious expression difference, and discusses the relationship between the lncRNA and the occurrence of breast cancer, thereby finding a better way and method for the diagnosis and the accurate targeted therapy of the breast cancer.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided an agent capable of detecting the level of LINC02343 in a sample.

"LINC 02343" is located on chromosome 13 with gene ID 105370158 and includes the LINC02343 gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed LINC02343, as well as any form of LINC02343 that results from processing in the cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of LINC 02343. The term encompasses, for example, the LINC02343 gene, the gene sequence of human LINC02343 (NR _146542.1), and from any other vertebrate source.

Further, the reagent comprises:

a probe that specifically recognizes LINC 02343; or

Primers specifically amplifying LINC 02343.

In a specific embodiment of the invention, the primer sequence for specifically amplifying LINC02343 is shown as SEQ ID NO. 1-2.

In a second aspect the invention provides a kit comprising the reagents of the first aspect of the invention.

As a non-limiting embodiment, the kit comprises a set of oligonucleotide primers sufficient to detect and/or quantify the intrinsic genes of the invention. The oligonucleotide primers may be provided in lyophilized or reconstituted form, or may be provided as a set of nucleotide sequences. In one embodiment, the primers are provided in the form of a microplate (microplate), wherein each primer set occupies a well (or wells, as in the case of a replicate) in the microplate. The microplate may further comprise primers sufficient to detect one or more housekeeping genes as described below. The kit may further comprise reagents and instructions sufficient to amplify the expression product of the gene of the invention.

To facilitate quick access, for example, for comparison, review, recovery, and/or modification, the molecular signatures/expression profiles are typically recorded in a database. Most typically, the database is a relational database accessible by computer equipment, although other forms may be used, such as manually accessed index files as profiles for photographs, analog or digital imaging readings, spreadsheets, and the like. Whether the expression pattern originally recorded is analog or digital in nature, the expression pattern, expression profile (collective expression pattern), and molecular signature (correlated expression pattern) are stored in digital form and accessed through a database. Typically, the database is compiled and maintained at a central facility, and can be accessed locally and/or remotely.

In a third aspect, the invention provides a chip comprising a reagent according to the first aspect of the invention. Further, the kit may also comprise a computer readable medium.

"chip," also referred to as an "array," refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.

A "microarray" is an ordered array of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The matrix may be a solid matrix, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid matrix, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any permutation thereof.

A fourth aspect of the present invention provides a nucleic acid membrane strip comprising the reagent according to the first aspect of the present invention.

The nucleic acid membrane strip comprises a substrate and oligonucleotide probes fixed on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.

A fifth aspect of the invention provides a composition comprising an inhibitor of LINC 02343. The inhibitor comprises an interfering molecule which targets LINC02343 and can inhibit LINC02343 gene expression or gene transcription, and comprises: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

Further, the inhibitor is siRNA.

As a preferred embodiment, the sequence of the siRNA is shown in SEQ ID NO. 5-10.

A sixth aspect of the present invention provides a method of screening a candidate drug for the treatment of breast cancer, the method comprising:

treating a culture system expressing or containing LINC02343 with a substance to be screened; and

detecting the expression level of LINC02343 in said system;

wherein, if the substance to be screened can inhibit the level of LINC02343, the substance to be screened is indicated to be a candidate drug for treating breast cancer.

A seventh aspect of the invention provides a use as claimed in any one of:

a. the reagent of the first aspect of the invention, the kit of the second aspect of the invention, the chip of the third aspect of the invention and the nucleic acid membrane strip of the fourth aspect of the invention are applied to the preparation of products for early diagnosis of breast cancer;

b. the use of a composition according to the fifth aspect of the invention in the manufacture of a medicament for the treatment of breast cancer;

c. use of a method according to the sixth aspect of the invention for screening a candidate for treatment of breast cancer;

the application of LINC02343 in preparing medicine for treating breast cancer;

use of linc02343 in the construction of a computational model for the diagnosis of breast cancer.

Further, the breast cancer in the application of the seventh aspect of the present invention is a Luminal a breast cancer.

The present invention encompasses any art-available method for detecting the expression of the intrinsic gene LINC 02343. By "detecting expression" is meant determining the amount or presence of an RNA transcript of an intrinsic gene or an expression product thereof. Methods of detecting intrinsic gene expression, i.e., gene expression profiling, of the present disclosure include methods based on polynucleotide hybridization analysis, methods based on polynucleotide sequencing. These methods generally detect the expression products of the intrinsic genes described herein (e.g., lncRNA).

Many expression detection methods use isolated RNA. The starting material is typically total RNA isolated from a biological sample, e.g. from a tumor or tumor cell line, respectively, and a corresponding normal tissue or cell line. If the source of the RNA is a primary tumor, RNA (e.g., mRNA) can be extracted from a frozen or preserved paraffin-embedded and fixed (e.g., formalin-fixed) tissue sample (e.g., a pathologist-directed tissue core sample).

In one embodiment of the invention, a breast cancer subtype is assessed by assessing the expression pattern or profile of the intrinsic genes described herein in one or more subject samples. For the purposes of this discussion, the term subject, or subject sample, refers to an individual, regardless of its health and/or disease state. The subject may be a subject, a study participant, a control subject, a screening subject, or any other class of individual from which a sample is obtained and evaluated in the context of the present invention. Accordingly, a subject may be diagnosed with breast cancer, may present with one or more symptoms of breast cancer, or may be receiving treatment or therapy for breast cancer as a causative factor for breast cancer, such as a family (genetic) or medical history (medical) factor, and the like. Alternatively, the subject may be healthy with respect to any of the above factors or criteria. It should be understood that the term "healthy" as used herein is with respect to breast cancer status, as the term "healthy" cannot be defined as corresponding to any absolute assessment or status. Thus, for any particular disease or disease criteria, an individual defined as healthy may be diagnosed as having, or exhibiting, virtually any other disease or diseases criteria, including one or more cancers other than breast cancer. However, the healthy control preferably does not have any cancer.

In particular embodiments, a method of predicting an intrinsic subtype of breast cancer includes collecting a biological sample comprising cancer cells or tissue, such as a breast tissue sample or a primary breast tumor tissue sample. By "biological sample" or "specimen" is meant any sample of cells, tissue, or body fluid in which intrinsic gene expression can be detected. Examples of such biological samples include, but are not limited to, biopsies and smears. Body fluids that may be used in the present invention include blood, lymph, urine, saliva, nipple aspirate, gynecological fluid, or any other bodily exudate or derivative thereof. Blood may include whole blood, plasma, serum or any blood derivative. In some embodiments, the biological sample comprises breast cells, particularly breast tissue from a biopsy, such as a breast tumor tissue sample. Biological samples can be obtained from a subject by a variety of techniques, including, for example, by scraping or swabbing an area, by aspiration of cells or bodily fluids using a needle, or by removal of a tissue sample (i.e., biopsy). Methods for collecting various biological samples are well known in the art. In some embodiments, the breast tissue sample is obtained by, for example, a fine needle aspiration biopsy, a needle biopsy, or an excisional biopsy. Fixatives and staining solutions may be applied to cells or tissues to preserve the sample and facilitate examination. Biological samples, particularly breast tissue samples, can be transferred to a slide for magnified viewing. In one embodiment, the biological sample is a formalin-fixed, paraffin-embedded breast tissue sample, particularly a primary breast tumor sample. In various embodiments, the tissue sample is obtained from a pathologist-directed tissue core sample.

The invention has the advantages and beneficial effects that:

the invention discovers that the expression level of the LINC02343 gene is related to breast cancer for the first time, the LINC02343 is up-regulated in a breast cancer patient, and whether the subject suffers from the breast cancer and the risk of suffering from the breast cancer can be judged by detecting the expression level of the LINC02343 in a sample of the subject, so that a clinician is guided to provide a prevention scheme or a treatment scheme for the subject, and meanwhile, the diagnosis is carried out by adopting a molecular marker, so that the kit has timeliness, sensitivity and specificity.

Drawings

FIG. 1 is a graph showing the detection of the expression of LINC02343 gene in breast cancer tissues by QPCR.

FIG. 2 is a graph of QPCR used to detect siRNA silencing LINC 02343.

FIG. 3 is a graph showing the effect of LINC02343 on the proliferation of MCF-7 cells measured by CCK.

FIG. 4 is a graph showing the effect of LINC02343 on the migratory invasion capacity of MCF-7 cells measured using a Transwell chamber, in which panel A is a cell migration map and panel B is a cell invasion map.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1 screening of Gene markers associated with Breast cancer

1. Sample collection

Cancer tissues and corresponding normal tissue samples (5 cm from the tumor margin) of 4 Luminal type A breast cancers were collected and subjected to high-throughput sequencing, and all patients were not subjected to chemotherapy, radiotherapy and endocrine treatment before operation.

2. Preparation and Mass analysis of RNA samples

RNA was extracted from tissues using Takara RNA extraction kit (Code No.9767) as follows:

1) fresh or cryogenically frozen animal tissue samples were quickly transferred to a liquid nitrogen pre-cooled mortar and the tissue ground with a pestle with liquid nitrogen added continuously until ground to a powder. The sample, which was ground to a powder, was added to a 1.5ml sterile centrifuge tube containing lysis Buffer RL and repeatedly pipetted until no significant precipitation occurred in the lysate.

2) The lysate was centrifuged at 12,000rpm at 4 ℃ for 5 min.

3) Carefully aspirate the supernatant into a new 1.5ml RNase Free Tube.

4) 70% ethanol, which had an equal volume to the liquid, was added and the solution was mixed well using a pipette gun.

5) The mixture was immediately transferred to RNA Spin Column in its entirety.

6) Centrifuge at 12,000rpm for 1min and discard the filtrate. The RNA Spin Column was placed back into the 2ml collection tube.

7) Mu.l of Buffer RWA was added to the RNA Spin Column, centrifuged at 12,000rpm for 30s, and the filtrate was discarded.

8) Mu.l of Buffer RWB was added to the RNA Spin Column, centrifuged at 12,000rpm for 30s, and the filtrate was discarded.

9) Repeat step 8).

10) The RNA Spin Column was re-mounted on a 2ml collection tube and centrifuged at 12,000rpm for 2 min.

11) Placing RNA Spin Column on 1.5ml RNase-Free collection tube, and adding 50-200 μ l RNase Free dH to the center of RNA Spin Column membrane₂Treating water with O or 0.1% DEPC, and standing at room temperature for 5 min.

12) The RNA was eluted by centrifugation at 12,000rpm for 2 min.

13) And detecting the concentration of the RNA, and identifying the yield and purity of the RNA.

3. construction and sequencing of cDNA libraries

1) Total RNA DNase I digestion: digesting DNA fragments existing in a Total RNA sample by using DNase I, purifying and recovering reaction products by using magnetic beads, and finally dissolving the reaction products in DEPC water;

2) removing rRNA: taking a digested Total RNA sample, removing rRNA by using a Ribo-Zero kit of Epicentre, detecting Agilent 2100 after removing the rRNA, and verifying the rRNA removing effect;

3) RNA disruption: taking the sample in the previous step, adding a breaking Buffer, and placing the sample in a PCR instrument for thermal breaking till 140-;

4) reverse transcription one-strand synthesis: adding a proper amount of primers into the broken sample, fully and uniformly mixing, reacting for a certain time at a proper temperature of a Thermomixer to open a secondary structure and combine with the primers, adding a one-chain synthesis reaction system Mix prepared in advance, and synthesizing one-chain cDNA on a PCR instrument according to a corresponding procedure;

5) synthesis of reverse transcription duplex: preparing a double-chain synthesis reaction system, reacting on a Thermomixer at a proper temperature for a certain time to synthesize double-chain cDNA with dUTP, and purifying and recovering reaction products by using magnetic beads;

6) and (3) repairing the tail end: preparing a tail end repairing reaction system, reacting in a Thermomixer at a proper temperature for a certain time, repairing the viscous tail end of a cDNA double-chain obtained by reverse transcription under the action of enzyme, purifying and recovering a tail end repairing product by using magnetic beads, and finally dissolving a sample in EB Solution;

7) 3' end of cDNA plus "A": preparing an A reaction system, reacting in a Thermomixer at a proper temperature for a certain time, and adding A basic groups to the 3' end of a product cDNA with repaired end under the action of enzyme;

8) ligation of cDNA 5' adapter: preparing a joint connection reaction system, reacting in a Thermomixer at a proper temperature for a certain time, connecting a joint with the A base under the action of enzyme, and purifying and recovering a product by using magnetic beads;

9) UNG digested cDNA double strand: preparing a UNG digestion reaction system, digesting two strands in double-stranded DNA by UNG enzyme, and purifying and recovering a product by using magnetic beads;

10) PCR reaction and product recovery: preparing a PCR reaction system, selecting a proper PCR reaction program, amplifying the product obtained in the previous step, carrying out magnetic bead purification and recovery on the PCR product, dissolving the recovered product in EB solution, and labeling.

11) And (3) detecting the quality of the library: the library quality was checked using Agilent 2100Bioanalyzer and ABI StepOneplus Real-Time PCR System;

12) and (3) machine sequencing: and (4) detecting a qualified library, adding NaOH to denature the library into a single chain, and diluting the single chain to a certain computer-loading concentration according to the expected computer-loading data quantity. The denatured diluted library was added to the FlowCell, hybridized to the linker on the FlowCell, bridge PCR amplification was done on cBot, and finally sequenced using Illumina Hiseq x-ten platform.

4. Bioinformatics analysis

1) Carrying out trim on 5 'and 3' sections of reads by using cutadapt, wherein bases with the mass of less than 20 are removed from trim, and more than 10% of reads with N are deleted;

2) hisat2 was aligned to the reference genome. The reference genome is from the Ensembl database, genome version GRCh38, and the gene annotation information is Ensemble 92;

3) stringtie quantifies the expression quantity of lncRNA and outputs the expression quantity in a standardized way;

4) the edgeR package compares the expression difference of lncRNA of a control group and a disease group, and the screening standard of the lncRNA with the difference is | log₂FC|>1 and pvalue<0.05。

5. Results

Sequencing data are shown in table 1, bioinformatics analysis finds that the expression of LINC02343 is significantly up-regulated in breast cancer patients, and suggests that LINC02343 may be applied to early diagnosis of breast cancer as a detection target.

TABLE 1 sequencing data

Example 2 QPCR sequencing verification of differential expression of LINC02343 Gene

1. Large sample QPCR validation of differential expression of the LINC02343 gene was performed on 25 Luminal type A breast cancer tissue samples and normal tissue samples collected according to the collection method of example 1.

2. RNA extraction

The Takara RNA extraction kit (Code No.9767) extracts RNA from tissues, the specific steps are described in example 1.

3、QPCR

Primers were designed based on the gene sequences of LINC02343 and GADPH, and the primer sequences are shown in table 2.

TABLE 2 amplification primers

TaKaRa One Step TB Green^TMPrime Script^TMThe RT-PCR kit (Code No. RR066A) was used for PCR reaction, and the reaction system and reaction conditions are shown in Table 3. In the Thermal Cycler

PCR amplification is carried out on a RealTime System amplification instrument, after the reaction is finished, the amplification curve and the dissolution curve of the RealTime PCR are confirmed, and relative quantification is carried out by a delta CT method.

TABLE 3 QPCR reaction System and reaction conditions

4. Results

The QPCR result is shown in FIG. 1, compared with the normal tissue, the expression of LINC02343 in the Luminal A type breast cancer tissue is up-regulated, and the difference has statistical significance (P <0.05), which is consistent with the high-throughput sequencing result. The expression of LINC02343 is up-regulated in 23 cancer tissues, 2 are not significantly changed, the expression of LINC02343 is up-regulated in 1 normal tissue, and the expression of LINC02343 is not significantly changed in the rest 24 normal tissues, so that whether a subject suffers from breast cancer or not can be judged by detecting the level of LINC02343, when the level of LINC02343 is significantly increased, the subject suffers from breast cancer or has the risk of suffering from breast cancer, shRNA and siRNA targeting LINC02343 can be designed through the relation between LINC02343 and breast cancer so as to treat the breast cancer, and meanwhile, a calculation model for predicting the breast cancer can be constructed based on the relation between LINC02343 and the breast cancer.

Example 3 expression of LINC02343 in breast cancer cell lines

1. Cell culture

Culturing the Luminal A-type breast cancer MCF-7 cell line in 5% CO₂The cells were cultured in a 37 ℃ incubator with 10% fetal bovine serum and 1% P/S in all cell culture media. The solution was changed 1 time 2-3 days, and cells were passaged by conventional digestion with 0.25% EDTA-containing trypsin at a ratio of 1: 3.

2. siRNA sequences

The siRNA-NC and siRNA-LINC02343 used in the application are purchased from Shanghai Ji code pharmaceutical technology Co., Ltd, and the sequences of the silent LINC02343 are shown in Table 4.

TABLE 4 siRNA sequences

3. Transfection

Lipofectamin from Invitrogen was used^TM2000 reagents were used for cell transfection and the experiments were divided into three groups: a control group (MCF-7), a negative control group (siRNA-NC) and an experimental group (siRNA1, siRNA2, siRNA3), wherein the siRNA of the negative control group has no homology with the sequence of the LINC02343 gene.

Before cell transfection, cells planted in a 6-well plate in advance in an incubator are prepared, the transfected cells are washed twice by using serum-free OPTI-MEM transfection solution, 1500 mu l of OPTI-MEM transfection solution is added into each well, the 6-well plate is placed back to the cell incubator for continuous culture, and the medium contains serum during transfection. siRNA diluted with serum-free OPTI-MEM and Lipofectamine^TMAnd (3) uniformly mixing the reagents, incubating for 15min, adding the incubated transfection complex into cells, culturing in an incubator, discarding the transfection solution after 6h, and putting the cells back into the incubator for continuous culture.

4. QPCR detection of the expression level of LINC02343

1) Extraction of RNA

Total RNA in the cultured cells was extracted using Takara RNA extraction kit (Code No. 9767).

2) QPCR detection procedure as in example 2

5. Results

The siRNA 1-3 small molecules have a good silencing effect on LINC02343, wherein the silencing effect of siRNA2 is the most significant (P <0.05) (FIG. 2), so that siRNA2 is selected as a silencing sequence to be applied to subsequent functional verification, and all cell experiments in the experiment are repeated for 3 times.

Example 4 CCK-8 method for examining the Effect of LINC02343 gene on proliferation of breast cancer cells

The breast cancer cells transfected with siRNA2 were used as experimental groups and siRNA-NC transfected cells were used as control groups, and the cells were added to a 96-well plate at 3000 cells per well, with 5 duplicate wells per group. The method is used for detecting the detection time points of 24h, 48h, 72h and 96h respectively. Adding 10 mul of CCK-8 detection solution into the cell wells every 24h, continuously putting the 96-well plate into a cell culture box for incubation for about 4h, detecting the absorbance value of each well at the wavelength of 450nm by using a microplate reader, recording data, and continuously measuring for 96 h. And drawing a growth curve according to the average value of the detected OD values.

The growth curve results show that the proliferation capacity of the cells after transfection of siRNA2 in the experimental group is obviously lower than that of the control group (FIG. 3), which indicates that LINC02343 affects the proliferation of breast cancer cells, and that the proliferation capacity of the breast cancer cells can be changed by changing the expression level of LINC 02343.

Example 5 Transwell Chamber assay the effect of LINC02343 on cell migration and invasion

1. Transwell cell preparation

Melting the Matrigel in an ice bath under aseptic condition, diluting the Matrigel glue according to the proportion of 1:8, slowly adding the Matrigel glue to the bottom of an upper chamber of a Transwell, spreading the Matrigel glue, and quickly transferring the Matrigel glue into a cell culture box at 37 ℃ for incubation until the Matrigel glue is solidified into a gel shape.

2. In the migration experiment, the amount of 5 × 10 added in the upper chamber⁴Adding 600. mu.l of culture medium containing 10% fetal calf serum into the lower chamber, setting 3 multiple wells in each group, and adding 1 × 10 in number into the upper chamber paved with Matrigel gel in invasion experiment⁵Adding 600 mul of culture medium containing 10% fetal calf serum into the lower chamber, and arranging 3 compound holes in each group; the Transewll chamber was placed in a cell culture chamber and the culture was continued for 24 h.

3. Dyeing process

The Transwell was removed and washed 2 times with PBS, fixed with paraformaldehyde, stained with crystal violet, stained for 20min at room temperature, rinsed 2 times with PBS, placed under a fluorescent microscope for observation and counted.

4. Results

The results of Transwell experiments show that the number of cells migrating and invading in the group transfected with siRNA2 (FIG. 4A) is reduced, which indicates that the migration and invasion abilities of the cells are inhibited (P <0.05) compared with the control group, and suggests that LINC02343 can be used for treating the migration and invasion of breast cancer cells.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> Deyang city people hospital

<120> biomarker for diagnosing and treating breast cancer

<160>10

<170>SIPOSequenceListing 1.0

<210>1

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

ttaatctacc ttctgtga 18

<210>2

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

caactatatg accttctg 18

<210>3

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

aatcccatca ccatcttcca g 21

<210>4

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gagccccagc cttctccat 19

<210>5

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

acaauucccu ucaguuuccc u 21

<210>6

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

ggaaacugaa gggaauugug u 21

<210>7

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

agugaaagug cucuguauga u 21

<210>8

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

cauacagagc acuuucacug c 21

<210>9

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

auugucauuu auuacacucu g 21

<210>10

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gaguguaaua aaugacaaug a 21

Claims

1. A method of screening for a candidate drug for the treatment of breast cancer, the method comprising:

detecting the expression level of LINC02343 in said system;

wherein, if the substance to be screened can inhibit the expression level of LINC02343, the substance to be screened is indicated to be a candidate drug for treating breast cancer.

2. Application of a reagent, a kit, a chip and a nucleic acid membrane strip for detecting the expression level of LINC02343 in a sample in preparing a product for early diagnosis of breast cancer.

3. Use of the method of claim 1 for screening for a candidate drug for the treatment of breast cancer.

Use of an inhibitor of LINC02343 in the manufacture of a medicament for the treatment of breast cancer, wherein the inhibitor is siRNA.

5. The use of claim 2, wherein said agent comprises:

a probe that specifically recognizes LINC02343 gene; or

A primer for specifically amplifying LINC02343 gene.

6. The use of claim 5, wherein the primer sequence for specifically amplifying the LINC02343 gene is shown as SEQ ID No. 1-2.