CN110541034B - Application of LINC01992 in breast cancer diagnosis and treatment - Google Patents

Abstract

The invention discloses application of LINC01992 in breast cancer diagnosis and treatment, and firstly discovers that LINC01992 is up-regulated in breast cancer by a high-throughput sequencing technology, namely whether a subject suffers from breast cancer can be judged by detecting the expression level of LINC 01992. The invention also discloses application of LINC01992 in constructing a calculation model for predicting breast cancer, and also discloses application of LINC01992 in preparing a medicament for treating breast cancer.

Description

Application of LINC01992 in breast cancer diagnosis and treatment

Technical Field

The invention belongs to the field of biological medicines, and relates to application of LINC01992 in breast cancer diagnosis and treatment.

Background

Breast cancer is one of the most common tumors in women. Statistics of tumors worldwide in 2012 in the united states show that women have the first and second incidence of breast cancer, respectively, with about 246660 new breast cancer patients each year, and the mortality rate is second only to respiratory tumors (Torre, l.a., et al, Global cancer statistics,2012, CACancer J Clin,2015.65(2): p 87-108.). Although China is one of the countries with low incidence of breast Cancer, in recent years, the incidence and mortality of breast Cancer still account for the first malignant tumors of women (Chen, W., et al., Cancer in China,2015.CA Cancer J Clin,2016.66(2): p 115-32.). The breast cancer is mainly treated by single or combined treatment modes such as operation, radiotherapy, chemotherapy, endocrine and targeted therapy. Although combination therapy reduces overall mortality, breast cancer is a highly heterogeneous tumor and mortality remains high. Breast cancer patients of the same clinical stage have large individual differences in sensitivity and efficacy to treatment even when they receive the same treatment regimen. Most patients with advanced breast cancer receive combined chemotherapy, but only about 50% benefit, while the other half suffer from the toxic side effects of chemotherapy. Therefore, it is very important to further refine typing to guide accurate treatment based on the existing molecular typing of breast cancer. Currently, there are four types of Molecular typing of breast cancer, including Luminal A type, Luminal B type, Her-2 overexpression type, and triple-negative type (Perou, C.M., et al, Molecular portals of human breast cancer. Nature.2000.406(6797): p.747-52.) among different patients with Molecular typing breast cancer, there are major differences in prognosis and therapeutic efficacy (Stathopoulos, G.P., et al, The role of Ki-67in The promotion and diagnosis of breast cancer tissue types, anticancer clones 2014.25(8): p.950-7.). Among them, the prognosis of Luminal type breast cancer patients is superior to other subtypes. 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.

The search for potential therapeutic targets for triple negative breast cancer has been one of the hot spots in the field of breast cancer research. The research shows that the prognosis of the triple negative breast cancer patient can be different due to different molecular markers. Molecular markers CK5/6, EGFR, etc. can be used to further sub-group the triple negative breast Cancer (Nielsen, T.O., et aL, immunological and clinical characterization of the basal-like subtype Cancer Res,2004.10(16): p.5367-74.). Among the different molecularly typed breast cancers, the Luminal A type is recognized as a relatively better type of prognosis, but some of them have poorer prognosis, so that we need to perform further sub-grouping on the Luminal A type breast cancer. So as to find out heterogeneous factors causing poor prognosis in Luminal A type breast cancer patients, thereby being capable of more accurately carrying out targeted therapy.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention is extensively and deeply researched, the expression of lncRNA in a breast cancer specimen in a tumor tissue and a normal tissue is detected by a high-throughput sequencing method, lncRNA with obvious expression difference is found, and the relation between the lncRNA and the occurrence of breast cancer is discussed, so that a better way and a better method are found for the diagnosis and the targeted therapy of the breast cancer.

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

the invention provides application of a reagent for detecting LINC01992 in preparation of a product for diagnosing breast cancer.

The present invention encompasses any method available in the art for detecting the expression of the intrinsic gene LINC 01992. 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).

Further, the reagent comprises a reagent for detecting the expression level of LINC01992 by reverse transcription PCR, real-time quantitative PCR, in-situ hybridization and a chip technology.

Further, the reagent for detecting the expression level of the LINC01992 by reverse transcription PCR at least comprises a pair of primers for specifically amplifying the LINC01992, the reagent for detecting the expression level of the LINC01992 by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying the LINC01992, the reagent for detecting the expression level of the LINC01992 by in situ hybridization comprises a probe for specifically recognizing the LINC01992, and the reagent for detecting the expression level of the LINC01992 by the chip technology comprises a probe for specifically recognizing the LINC 01992.

Further, a primer sequence of the specific amplification LINC01992 for detecting the expression level of LINC01992 by real-time quantitative PCR is shown in SEQ ID NO. 1-2.

Further, the product comprises a chip and a kit. The gene chip comprises a solid phase carrier and an oligonucleotide probe which is fixed on the solid phase carrier and specifically recognizes LINC01992, and the kit comprises a primer for specifically amplifying LINC01992 and the oligonucleotide probe or chip for specifically recognizing LINC 01992.

The oligonucleotide probe specifically recognizing LINC01992 may be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. The length of the probe is not limited, and any length may be used as long as specific hybridization and specific binding to the target nucleotide sequence are achieved. The length of the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or more, even for the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and at most, usually not more than 30 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15 to 25 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs so as not to affect hybridization efficiency.

The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.

Further, the breast cancer is Luminal A type breast cancer.

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).

General methods for RNA extraction are well known in the art. In particular, RNA isolation can be performed using purification kits, buffer sets and proteases from commercial manufacturers, e.g., TIANGEN, following the manufacturer's instructions. Other commercially available RNA isolation kits include MASTERPURE^TMComplete DNA and RNA purification kits (Epicentre, Madison, Wis.) and Paraffin Block RNA isolation kits (Ambion, Austin, TX). For example, RNA Stat-60(Tel-Test, Friendshood, TX) can be used to isolate total RNA from tissue samples. For example, total RNA can be isolated from FFPE using a high purity FFPE RNA Microkit, cat # 04823125001(Roche Applied Science, Indianapolis, Ind.). For example, RNA prepared from tumors can be isolated by cesium chloride density gradient centrifugation. In addition, large numbers of tissue samples can be readily processed by using techniques well known to those skilled in the art.

The invention provides a product for diagnosing breast cancer, which comprises an agent for detecting the expression level of LINC 01992.

Further, the product comprises a chip or a kit, and the reagent for detecting the expression level of LINC01992 in the chip comprises a probe for specifically recognizing LINC01992 gene; the reagent for detecting the LINC01992 expression level in the kit comprises a primer for specifically amplifying the LINC01992 gene or a probe for specifically identifying the LINC01992 gene.

As one embodiment, the kit comprises a set of oligonucleotide primers sufficient to detect and/or quantify an intrinsic gene 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.

Further, the primer sequence of the specific amplification LINC01992 gene is shown in SEQ ID NO. 1-2.

The invention provides application of LINC01992 in constructing a calculation model for predicting breast cancer.

Further, the breast cancer is Luminal A type breast cancer.

The invention provides application of LINC01992 in preparing a medicine for treating breast cancer.

Further, the medicament includes an inhibitor of LINC01992, which has LINC01992 as a target sequence and is an agent capable of inhibiting the expression level of LINC01992, comprising: shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microRNA, antisense nucleic acid, or a construct capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microRNA, antisense nucleic acid, or the like.

As a preferred mode of the invention, the inhibitor is a small interfering RNA molecule specific to LINC 01992. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, which is the RNA interference (RNA interference) process. Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.

Further, the sequence of the siRNA is shown in SEQ ID NO. 5-8.

The invention provides a medicament for treating breast cancer, which comprises an inhibitor of LINC 01992. The inhibitor takes LINC01992 as a target sequence and can inhibit the expression level of LINC01992, and comprises the following components: shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microRNA, antisense nucleic acid, or a construct capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microRNA, antisense nucleic acid, or the like.

Further, the inhibitor is siRNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 5-8.

The medicament of the invention also comprises pharmaceutically acceptable carriers, including (but not limited to) diluents, binders, surfactants, humectants, adsorption carriers, lubricants, fillers and disintegrating agents.

In the present invention, the term "LINC 01992" is located on chromosome 17 and has a gene ID of 105371708, and includes the LINC01992 gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed LINC01992, as well as any form of LINC01992 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of LINC 01992. The term encompasses, for example, the LINC01992 gene, the gene sequence of human LINC01992 (NR _146896.1), and from any other vertebrate source.

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" is meant any sample of cells, tissues, or bodily fluids 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 LINC01992 gene is related to breast cancer for the first time, and whether the subject suffers from the breast cancer or not and the risk of suffering from the breast cancer can be judged by detecting the expression level of the LINC01992 gene in a sample of the subject, so that a clinician is guided to provide a prevention scheme or a treatment scheme for the subject.

The invention discovers the LINC01992 gene molecular marker related to the breast cancer, and the diagnosis is carried out by adopting the molecular marker, so that the diagnosis is more timely, more sensitive and more specific compared with the traditional diagnosis means.

Drawings

FIG. 1 is a graph showing the detection of the expression of LINC01992 gene in breast cancer tissue by QPCR.

FIG. 2 is a graph of the QPCR assay for silencing LINC01992 by siRNA.

FIG. 3 is a graph showing the effect of LINC01992 on the proliferation of MCF-7 cells measured by CCK.

FIG. 4 is a graph showing the effect of LINC01992 on the ability of MCF-7 to migrate and invade using a Transwell chamber, in which panel A shows cell migration and panel B shows cell invasion.

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

4 cancer tissues of Luminal A-type breast cancer and corresponding normal tissue samples (5 cm from the tumor margin) were collected, and subjected to high-throughput sequencing, all patients did not undergo chemotherapy, radiotherapy and endocrine treatment before surgery, all patients gave informed consent, all the samples were obtained with consent of the tissue ethics committee, and the patient information is shown in Table 1.

TABLE 1 sample information

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 2, bioinformatics analysis finds that the expression of LINC01992 in breast cancer patients is remarkably up-regulated, and suggests that LINC01992 can be used as a detection target for early diagnosis of breast cancer.

TABLE 2 sequencing data

Example 2 QPCR sequencing verification of differential expression of LINC01992 Gene

1. Large-sample QPCR validation of differential LINC01992 gene expression was performed on 25 Luminal type A breast cancer and normal tissue samples collected according to the collection protocol 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 LINC01992 and GADPH, and the primer sequences are shown in table 3.

TABLE 3 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 4. In the Thermal Cycler

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

TABLE 4 QPCR reaction System and reaction conditions

4. Results

The QPCR results are shown in fig. 1, LINC01992 is up-regulated in breast cancer tissue compared to normal tissue, the difference is statistically significant (P <0.05), consistent with the high throughput sequencing results, wherein LINC01992 expression is up-regulated in 27 samples of all samples, and the breast cancer tissue samples account for 24 samples and the normal tissue samples account for 3 samples, while LINC01992 is not significantly changed in 23 samples, and the breast cancer tissue samples account for 1 sample and the normal tissue samples account for 22 samples, suggesting that whether the subject suffers from breast cancer can be determined by detecting the level of LINC01992, and when the level of LINC01992 is significantly increased, the subject suffers from breast cancer or has a risk of suffering from breast cancer, the shRNA and siRNA targeting LINC01992 can be designed by the relationship between LINC01992 and breast cancer, thereby treating breast cancer.

Example 3 expression of LINC01992 in a breast cancer cell line

1. Cell culture

Culturing the Luminal A-type breast cancer MCF-7 cell line in 5% CO₂Culturing in a constant temperature incubator at 37 deg.C, adding 10% fetal calf serum and 1% of all cell culture mediaAnd P/S. 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-LINC01992 used in the application are purchased from Shanghai Ji code pharmaceutical technology Co., Ltd, and are shown in sequences siRNA-1 and siRNA-2 of silencing LINC 01992:

sequence of siRNA-1:

the sense strand is 5'-UACCAUAUCUUAUACUCUGAG-3' (SEQ ID NO.5)

The antisense strand is 5'-CAGAGUAUAAGAUAUGGUAUA-3' (SEQ ID NO.6)

sequence of siRNA-2:

the sense strand is 5'-UUUGUUGAUCAUGUUUCUGGG-3' (SEQ ID NO.7)

The antisense strand is 5'-CAGAAACAUGAUCAACAAAGA-3' (SEQ ID NO.8)

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 (siRNA-1, siRNA-2), wherein the siRNA of the negative control group has no homology with the sequence of the LINC01992 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 expression level of LINC01992

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 and siRNA-2 micromolecules have good silencing effects on LINC01992, wherein the silencing effect of the siRNA-1 is most obvious (P is less than 0.05) (figure 2), so the siRNA-1 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 LINC01992 gene on proliferation of breast cancer cells

The breast cancer cells transfected by siRNA-1 are used as an experimental group, the cells transfected by siRNA-NC are used as a control group, the cells are added into a 96-well plate, the number of the cells added into each well is 3000, and each group is provided with 5 multiple wells. 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 siRNA-1 transfection in the experimental group is obviously lower than that of the control group (figure 3), which indicates that LINC01992 influences the proliferation of the breast cancer cells.

Example 5 Transwell Chamber examination of the Effect of LINC01992 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 (figure 4A) and invading (figure 4B) in the group of transfected siRNA-1 is obviously reduced, which indicates that the migration and invasion abilities of the cells are obviously inhibited (P <0.05) compared with those of a control group, and LINC01992 is related to 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.

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Claims (7)

1. Application of a reagent for detecting LINC01992 in preparing a product for diagnosing breast cancer.

2. The use according to claim 1, wherein the reagents comprise reagents for detecting the expression level of LINC01992 by reverse transcription PCR, real-time quantitative PCR, in situ hybridization, chip technology.

3. The use according to claim 2, wherein the reagents for detecting the expression level of LINC01992 by reverse transcription PCR comprise at least one pair of primers for specifically amplifying LINC01992, the reagents for detecting the expression level of LINC01992 by real-time quantitative PCR comprise at least one pair of primers for specifically amplifying LINC01992, the reagents for detecting the expression level of LINC01992 by in situ hybridization comprise probes specifically recognizing LINC01992, and the reagents for detecting the expression level of LINC01992 by chip technology comprise probes specifically recognizing LINC 01992.

4. The use according to any one of claims 1 to 3, wherein the product comprises a chip, a kit.

Application of LINC01992 in constructing a calculation model for predicting breast cancer.

Use of an inhibitor of LINC01992 in the manufacture of a medicament for the treatment of breast cancer, wherein the inhibitor is siRNA.

7. The use of claim 6, wherein the siRNA has a sequence as shown in SEQ ID No. 5-8.

Images