CN110628914B

CN110628914B - LncRNA marker related to breast cancer, detection primer and application thereof

Info

Publication number: CN110628914B
Application number: CN201911075745.3A
Authority: CN
Inventors: 袁成良; 刘盈盈; 贾新建; 张乃丹; 刘朝红
Original assignee: Peoples Hospital of Deyang City
Current assignee: Peoples Hospital of Deyang City
Priority date: 2019-04-15
Filing date: 2019-11-06
Publication date: 2020-08-04
Anticipated expiration: 2039-11-06
Also published as: CN109825595A; CN110628914A

Abstract

The invention discloses a lncRNA marker related to breast cancer, and a detection primer and application thereof, wherein the lncRNA marker is L INC02422. the expression of L INC02422 in a breast cancer patient is found to be remarkably up-regulated for the first time through high-throughput sequencing, and QPCR further verifies that the expression of L INC02422 is remarkably up-regulated in the breast cancer, so that L INC02422 can be used as a biomarker for diagnosis and treatment of the breast cancer.

Description

LncRNA marker related to breast cancer, detection primer and application thereof

Technical Field

The invention belongs to the field of biological medicines, and relates to an lncRNA marker related to breast cancer, and a detection primer and application thereof, wherein the marker is L INC 02422.

Background

Breast cancer, the most common malignant tumor in women, has become a major health problem in China and even in the world. The incidence of breast cancer is rising year by year, and according to the increasing trend, the number of breast cancer patients in China can reach 250 ten thousands by 2021 year.

In recent years, the development of early diagnosis technology and the application of comprehensive treatment means obviously improve the survival rate of breast cancer compared with the past decades, but about 30 percent of early breast cancer patients relapse, 24 to 60 percent of patients have distant metastasis, and the relapse and metastasis of tumors seriously affect the life quality and survival time of the breast cancer patients and also bring great challenges to clinical work.

With the intensive genomic research and the application of high throughput sequencing technology, it has been surprisingly found that about 1.5% of the genomic sequences have the ability to encode proteins, while over 98% of the genomic sequences are transcribed in most regions, but the resulting RNA does not encode proteins. These non-coding sequences are considered "dark material" in the genome. Among these non-coding RNAs, a class of transcripts that are greater than 200 nucleotides in length and that do not have the ability to encode proteins is defined as long non-coding RNAs (incrnas). The discovery of long non-coding RNAs, which may originate from genomic sequences between two coding proteins, from regions such as introns or antisense strands of the gene encoding the protein, has led biologists to further recognize the complexity of the regulation of genomic transcription. More and more experimental data show that the long-chain non-coding RNA can play an important role in a plurality of processes such as epigenetics, transcriptional regulation, translation process, protein posttranslational modification and the like; in addition, many long-chain non-coding RNAs have relatively conserved secondary structures, and the expression of the long-chain non-coding RNAs has space-time specificity, and the molecular characteristics all suggest that the long-chain non-coding RNAs may have important biological functions and may play key roles in ontogeny, physiology and pathology. The study of long non-coding RNAs has become a hotspot in biology.

In organisms, long non-coding RNAs exert very complex biological functions and participate in different biological events. The current research finds that the long-chain non-coding RNA can regulate the expression of genes at multiple levels of epigenetic regulation, transcriptional regulation, post-translation and the like, and participate in multiple important life processes such as DNA damage response, X chromosome silencing, cell reprogramming, genome imprinting and the like. The abnormal expression of the long-chain non-coding RNA is related to the invasion and metastasis of various cancers such as breast cancer, leukemia, colon cancer, prostate cancer, liver cancer and the like. According to recent research at home and abroad, the lncRNA is expected to become a potential target for early diagnosis and treatment of the breast cancer.

Disclosure of Invention

In order to make up the defects of the prior art, the invention aims to provide a molecular marker related to the occurrence and development of breast cancer, wherein the marker can be used as a specific diagnosis marker of the breast cancer and applied to the early discovery of the breast cancer; meanwhile, the marker can be used as a specific molecular target of breast cancer and applied to personalized treatment 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 L INC02422 in preparation of a product for diagnosing breast cancer.

Further, the breast cancer is L tubular B type breast cancer.

Further, the product comprises reagents for detecting the expression level of L INC02422 in a sample by sequencing techniques, nucleic acid hybridization techniques, nucleic acid amplification techniques including polymerase chain reaction, reverse transcription polymerase chain reaction, transcription mediated amplification, ligase chain reaction, strand displacement amplification, and nucleic acid sequence based amplification.

Further, the reagent is selected from a probe specifically recognizing L INC02422, or

A primer for specifically amplifying L INC 02422.

Furthermore, the sequence of the primer for specifically amplifying L INC02422 is shown as SEQ ID NO. 1-2.

The invention provides a product for detecting L INC02422 expression level in vitro, which comprises a chip, a kit and a nucleic acid membrane strip.

Further, the chip comprises an oligonucleotide probe specifically recognizing L INC02422, the kit comprises a primer specifically amplifying L INC02422 or an oligonucleotide probe specifically recognizing L INC02422, and the nucleic acid membrane strip comprises an oligonucleotide probe specifically recognizing L INC 02422.

Further, the kit further comprises one or more substances selected from the group consisting of: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

The invention provides application of a product for detecting L INC02422 expression level in vitro in preparing a tool for diagnosing breast cancer.

Further, the breast cancer is L tubular B type breast cancer.

The invention provides application of L INC02422 in construction of a computational model for predicting breast cancer.

Further, the breast cancer is L tubular B type breast cancer.

The invention provides application of L INC02422 in preparation of a pharmaceutical composition for treating breast cancer.

Further, the breast cancer is L tubular B type breast cancer.

Further, the pharmaceutical composition comprises L INC02422 inhibitor which can reduce the expression level of L INC02422, wherein the inhibitor takes L INC02422 as a target sequence and can inhibit the expression level of L INC02422, and the inhibitor comprises shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microRNA, antisense nucleic acid, or a construct capable of expressing or forming the shRNA, the small interfering RNA, the dsRNA, the microRNA, the antisense nucleic acid and the like.

Further, the inhibitor is siRNA.

In a specific embodiment of the present invention, the siRNA has a sequence shown in SEQ ID NO.5-10, and in a more preferred embodiment, the siRNA has a sequence shown in SEQ ID NO. 7-8.

Drawings

FIG. 1 is a graph showing the expression of L INC02422 gene in breast cancer tissues by QPCR.

FIG. 2 is a graph of QPCR detection of siRNA silencing L INC 02422.

Figure 3 is a graph of the effect of L INC02422 on proliferation of breast cancer BT474 cells measured using CCK-8.

Figure 4 is a graph of the effect of L INC02422 on the migratory invasion capacity of breast cancer BT474 cells tested using a Transwell chamber.

Detailed Description

According to the invention, the expression of lncRNA in a breast cancer sample in a tumor tissue and a normal tissue is detected by a high-throughput method, and lncRNA with obvious expression difference is found, so that a better way and a method are found for diagnosis and targeted treatment of breast cancer, through screening, L INC02422 in the breast cancer is found for the first time, and the upregulation of L INC02422 is further verified by QPCR, and the difference has statistical significance.

L INC02422 gene is located on chromosome 12 with a gene ID of 105369723, including the L INC02422 gene and homologs, mutations, and isoforms thereof the term encompasses full-length, unprocessed L INC02422, as well as any form of L INC02422 that results from processing in a cell the term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of L INC02422 the term encompasses, for example, the L INC02422 gene, the gene sequence of human L INC02422 (NR _135029.1), as well as from any other vertebrate source.

It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level. The present invention may utilize any method known in the art for determining gene expression.

The term "differential expression" as used herein means a difference in the expression level of L ncRNA, as determined by the method described herein and understood by those skilled in the art, of the RNA of one or more biomarkers of the invention and/or one or more splice variants of said biomarker incRNA, in one sample compared to the expression level of the same one or more biomarkers in a second sample the differential expression may be determined as described herein and as understood by those skilled in the art the term "differential expression" or "change in expression level" means an increase or decrease in the measurable expression level of a given biomarker in a sample compared to the measurable expression level of a given biomarker in a second sample group if the measured amount of RNA is an increase or decrease in the measurable expression level of the given biomarker, e.g., a p-1, a p-2, or a p-1, a p-2, a p-1, a-2, a p-1, a-2, a p-1, a-2, a p-1, a-2, a p-1, a.

By "differential expression increase" or "up-regulation" is meant that gene expression (expressed as RNA) shows an increase of at least 10% or more, e.g., 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more or 1.1-fold, 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold or more, of the gene relative to a control.

By "differential expression reduction" or "down-regulation" is meant a gene whose expression (as measured by RNA expression) exhibits a reduction in gene expression relative to a control of at least 10% or more, e.g., 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or less than 1.0-fold, 0.8-fold, 0.6-fold, 0.4-fold, 0.2-fold, 0.1-fold or less. For example, an up-regulated gene includes a gene that has an increased level of expression of RNA in a sample isolated from an individual characterized as having breast cancer, as compared to the expression of RNA isolated from a normal individual. For example, a down-regulated gene includes a gene that has a reduced level of RNA expression in a sample isolated from an individual characterized as having breast cancer, as compared to a sample isolated from a normal individual.

Detection techniques

The lncrnas of the invention are detected using a variety of nucleic acid techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification techniques.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

Another illustrative, non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high throughput sequencing), which is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.

Illustrative, non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

Southern and Northern blots were used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from the sample is fragmented, separated by electrophoresis on a matrix gel, and then transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Detecting the hybridization probes bound to the filter. A variation of this procedure is a reverse Northern blot, in which the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from the tissue and labeled.

Illustrative, non-limiting examples of nucleic acid amplification techniques include, but are not limited to, Polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), ligase chain reaction (L CR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). one of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA to DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction, commonly known as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of the target nucleic acid sequence, transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of the target nucleic acid sequence under conditions of substantially constant temperature, ionic strength, and pH, where multiple RNA copies of the target sequence autocatalytically generate additional copies; the ligase chain reaction of L CR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid; other amplification methods include, for example, nucleic acid sequence-based amplification, commonly known as NASBA, amplification of the probe molecule itself using RNA replicase (commonly known as Q β replicase), transcription-based amplification methods, and self-sustained sequence amplification.

Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.

Chip, nucleic acid membrane strip and kit

The invention provides a product for detecting the expression level of L INC02422 gene in a sample, which comprises (but is not limited to) a chip, a nucleic acid membrane strip or a kit, wherein the chip comprises a solid phase carrier and oligonucleotide probes orderly fixed on the solid phase carrier, and the oligonucleotide probes specifically correspond to part or all of the sequence represented by L INC 02422.

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.

The invention provides a nucleic acid membrane strip which comprises a substrate and an oligonucleotide probe which is fixed on the substrate and is used for L INC02422, wherein the substrate can be any substrate which is suitable for fixing the oligonucleotide probe, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass sheet, a silica gel wafer, a micro magnetic bead and the like.

The present invention provides a kit useful for detecting the expression of L INC02422, the kit further comprising one or more substances selected from the group consisting of a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent.

The components of the kit may be packaged in aqueous medium or in lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, pet bottle, syringe, or other container in which a component may be placed and, preferably, suitably aliquoted. Where more than one component is present in the kit, the kit will also typically comprise a second, third or other additional container in which the additional components are separately disposed. However, different combinations of components may be contained in one vial. The kit of the invention will also typically include a container for holding the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.

The present invention provides for the use of L INC02422 in the preparation of a computational model for predicting breast cancer, as will be appreciated by the skilled artisan, measurements of two or more markers can be used to improve diagnostic problems in an investigation.

In the present invention, the step of associating a marker level with a certain likelihood or risk may be carried out and carried out in different ways. Preferably, the measured concentrations of the gene and one or more other markers are mathematically combined and the combined value is correlated to the underlying diagnostic problem. The determination of marker values may be combined by any suitable prior art mathematical method.

Preferably, the mathematical algorithm applied in the marker combination is a logarithmic function. Preferably, the result of applying such a mathematical algorithm or such a logarithmic function is a single value. Such values can be readily correlated with, for example, an individual's risk for breast cancer or with other diagnostic uses of interest that help in assessing breast cancer patients, based on underlying diagnostic questions. In a preferred manner, such a logarithmic function is obtained as follows: a) classifying individuals into groups, e.g., normal humans, individuals at risk for breast cancer, patients with breast cancer, etc., b) identifying markers that differ significantly between these groups by univariate analysis, c) logistic regression analysis to assess independent difference values of the markers that can be used to assess these different groups, and d) constructing a logistic function to combine the independent difference values. In this type of analysis, the markers are no longer independent, but represent a combination of markers.

Suitable statistical methods are, for example, Discriminant Analysis (DA) (i.e., linear, quadratic, regular DA), Kernel methods (i.e., SVM), non-parametric methods (i.e., k-nearest neighbor classifier), P L S (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest methods, boosting/bagging methods), generalized linear models (i.e., logarithmic regression), principal component-based methods (i.e., SIMCA), generalized superposition models, fuzzy logic-based methods, neural network and genetic algorithm-based methods.

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 cases of L samples of cancer tissue and corresponding normal tissue (5 cm from the tumor margin) of the type B breast cancer were collected and subjected to high throughput sequencing, all patients underwent no prior chemotherapy, no prior radiation therapy, and no prior endocrine therapy, all patients gave informed consent, all samples were obtained with consent from the institutional review board, and the patient information is shown in Table 1.

TABLE 1 sample information

2. Preparation and Mass analysis of RNA samples

Extraction of tissue Total RNA Using TRIZO L method

1) Cutting tissue with scissors, adding 1ml Trizol, and shaking on oscillator for 1 min; standing at room temperature for 10min to completely decompose nucleoprotein.

2) Adding 200 μ l chloroform (chloroform), covering the tube, shaking vigorously for 15s, and standing at room temperature for 10 min.

3) Centrifuge at 11000rpm for 15min at 4 ℃.

4) Transferring the water sample layer into a new centrifuge tube, and adding 500 mul of isopropanol; after the mixture was inverted and mixed, the mixture was left standing at room temperature for 10 min.

5) Centrifuge at 11000rpm for 15min at 4 ℃.

6) The liquid was carefully aspirated off with a gun, the precipitate was left at the bottom of the tube, 1ml of 75% ethanol was added, the mixture was shaken on a shaker for 5s, and the precipitate was washed once.

7) Centrifuge at 8000rpm for 5min at 4 ℃.

8) Carefully removing the supernatant, drying the precipitate for 10min, and adding appropriate amount of water to dissolve the precipitate for 10 min.

9) 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 2100 Bioanalyzer 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 compared the expression difference of lncRNA between the control and disease groups, and the screening criteria for the difference-shifted lncRNA were | log2FC | >1 and pvalue < 0.05.

5. Results

Sequencing data are shown in table 2, bioinformatics analysis finds that L INC02422 is significantly up-regulated in breast cancer patients, suggesting that L INC02422 can be used as a possible detection target for early diagnosis of breast cancer.

TABLE 2 sequencing data

Example 2 QPCR sequencing validation L differential expression of INC02422 Gene

1. Large-sample QPCR validation of differential expression of L INC02422 gene was performed on 25 luminal B breast cancer tissue samples and normal tissue samples collected according to the collection protocol of example 1.

2. RNA extraction

Tissue RNA was extracted using Trizol as a specific procedure in example 1.

3. Reverse transcription: the operation was carried out using a reverse transcription kit (Takara code: DRR047A) of TAKARA.

1) Removal of genomic DNA

5 × gDNA Eraser B. mu.ffer 2.0. mu.l, gDNA Eraser 1.0. mu.l, total RNA 1. mu.g, RNase Free ddH₂O to make the total volume to 10 μ l, heating in water bath at 42 deg.C for 2 min.

2) Reverse transcription reaction

Will be provided with

Buffer 2 4.0μl，

RT Enzyme Mix I 1.0μl，RTPrimer Mix 1.0μl，RNase Free ddH₂O4.0. mu.l was added to the above test tube and mixed together to give 20. mu.l, which was then heated in a water bath at 37 ℃ for 15min and 85 ℃ for 5 s.

4. QPCR amplification

1) Primer design

Primers are designed according to gene sequences of L INC02422 and GADPH, and specific primer sequences are as follows:

l INC02422 gene:

the forward primer is 5'-AATAGATTCATTGGAGAGT-3' (SEQ ID NO. 1);

the reverse primer was 5'-CCTAAGTCTACTGGTAAC-3' (SEQ ID NO. 2).

GAPDH gene:

the forward primer is 5'-AATCCCATCACCATCTTCCAG-3' (SEQ ID NO. 3);

the reverse primer was 5'-GAGCCCCAGCCTTCTCCAT-3' (SEQ ID NO. 4).

2) QPCR amplification assay

By using

Premix Ex Taq^TMII (Takara Code: DRR081) kit is configured with a PCR reaction system in a Thermal Cycler

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

Prepare 25. mu.l reaction:

premix Ex TaqTM II (2 ×) 12.5. mu.l, forward (reverse) primers 1. mu.l each, DNA template 2. mu.l, and sterile distilled water 8.5. mu.l.

The reaction conditions are 95 ℃ for 30s, (95 ℃ for 5s, 60 ℃ for 30s) × 40

5. Results

The QPCR results are shown in figure 1, compared with normal tissues, the expression of L INC02422 is up-regulated in breast cancer tissues, the difference is statistically significant (P <0.05), and the result is consistent with the high-throughput sequencing result, which suggests that L INC02422 can be used as a biomarker for the diagnosis and treatment of breast cancer.

Of these, L INC02422 was upregulated in 25 samples, with no significant difference in 25 samples, 23 of the 25 upregulated samples were cancer tissue samples, and 2 were paracancer tissue samples, as shown in table 3.

TABLE 3 Positive in disease

Example 3L expression of INC02422 in a Breast cancer cell line

1. Cell culture

The BT474 cell line of L tubular B-type breast cancer was cultured in DMEM medium containing 10% fetal bovine serum (Gibco Co.) in 5% CO₂And culturing at 37 deg.C in a constant temperature incubator.

2. Transfection

General siRNA-NC, siRNA-L INC02422 used in the present application was purchased from Shanghai Ji code pharmaceutical technology, Inc., and the sequence of siRNA1-3 silencing L INC02422 is shown below.

sequence of siRNA 1:

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

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

sequence of siRNA 2:

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

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

sequence of siRNA 3:

the sense strand is 5'-UCUAAGUCGGAUUAAGCUGUG-3' (SEQ ID NO.9)

The antisense strand is 5'-CAGCUUAAUCCGACUUAGAAA-3' (SEQ ID NO.10)

L ipofectamin of Invitrogen corporation was used^TM2000 kit, L INC02422 siRNA was transfected into breast cancer BT474 cells in logarithmic phase of growth, cells previously seeded in 6-well plates in an incubator were prepared before cell transfection, cells in 6-well plates were changed and cultured continuously 24h after transfection, and the experiment was divided into 3 groups, a control group (BT474), a negative control group (siRNA-NC) and an experimental group (siRNA 1-3).

4. QPCR detection of L INC02422 expression level

1) Extraction of RNA

At 48h after cell transfection, cellular RNA was extracted using Trizol method.

2) QPCR detection procedure as in example 2

5. Results

As shown in fig. 2, the expression level of L INC02422 was not significantly different (P >0.05) between the control group (BT474) and the negative control group (siRNA-NC), and the experimental group was able to significantly reduce the expression level of L INC02422 with statistical significance (P <0.05) compared to the control group and the negative control group, in which the effect of siRNA2 was the most significant, so siRNA2 was selected for the subsequent experiments.

Example 4 CCK-8 assay to determine the Effect of L INC02422 Gene on Breast cancer cell proliferation

The breast cancer cells transfected with siRNA2 were used as experimental groups, and the cells transfected with siRNA-NC were used as control groups, and the cells were added to a 96-well plate, wherein the number of cells added per well was 5000, and 5 duplicate wells were provided for each group. The method is used for detecting the detection time points of 24h, 48h, 72h and 96h respectively.

During detection, 10 mul of CCK-8 detection solution is added into a cell hole, a 96-well plate is continuously placed into a cell culture box for incubation for about 4h, an enzyme-labeling instrument is used for detecting the absorbance value of each hole at the wavelength of 450nm and recording data, and a growth curve is drawn according to the average value of detected OD values.

The growth curve results show that the proliferation capacity of the cells after siRNA transfection in the experimental group is obviously lower than that of the control group (FIG. 3), which indicates that L INC02422 influences the proliferation of the breast cancer cells, and the proliferation capacity of the breast cancer cells can be changed by changing the expression level of L INC 02422.

Example 5 Transwell Chamber assay L Effect of INC02422 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. The upper chamber is added with 1 × 10⁵The cell suspension (100. mu.l) was added to the lower chamber in 600. mu.l of a medium containing 10% fetal bovine serum, each group was provided with 3 multiple wells, and cultured in a constant temperature incubator at 37 ℃ for 24 hours.

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

Results of Transwell experiments are shown in fig. 4, the cell migration and invasion number of the experimental group transfected with siRNA are significantly reduced compared with the control group (P <0.05), which indicates that the expression level of L INC02422 is related to the migration and invasion of breast cancer cells, and L INC02422 is expected to be a target for treating breast cancer.

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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<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

aaaacacgac cuuccuuucu a 21

<210>6

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

gaaaggaagg ucguguuuuc a 21

<210>7

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

uagguuuaac uauuugcuca a 21

<210>8

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gagcaaauag uuaaaccuag g 21

<210>9

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

ucuaagucgg auuaagcugu g 21

<210>10

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

cagcuuaauc cgacuuagaa a 21

Claims

1. Application of a reagent for detecting L INC02422 in preparation of a product for diagnosing breast cancer.

2. The use of claim 1, wherein the product comprises reagents for detecting the expression level of L INC02422 gene in a sample by sequencing, nucleic acid hybridization, or nucleic acid amplification techniques.

3. The use according to claim 1, wherein the agent is selected from the group consisting of:

a probe specifically recognizing L INC02422, or

A primer for specifically amplifying L INC 02422.

4. The use according to claim 3, wherein the primer sequence of specific amplification L INC02422 is shown as SEQ ID No. 1-2.

5. A product for detecting L INC02422 expression level in vitro, which comprises a chip, a kit and a nucleic acid membrane strip.

6. The product of claim 5, wherein the chip comprises an oligonucleotide probe that specifically recognizes L INC02422, the kit comprises a primer that specifically amplifies L INC02422 or an oligonucleotide probe that specifically recognizes L INC02422, and the nucleic acid membrane strip comprises an oligonucleotide probe that specifically recognizes L INC 02422.

7. The product according to claim 6, wherein the primer sequence of the specific amplification L INC02422 is shown as SEQ ID No. 1-2.

8. The product of claim 7, wherein the kit further comprises one or more substances selected from the group consisting of: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

9. Use of a product according to any one of claims 5 to 8 in the manufacture of a means for diagnosing breast cancer.

Use of L INC02422 for constructing a computational model predictive of breast cancer.

11, L INC02422 for use in the preparation of a medicament for the treatment of breast cancer.

12. The use of claim 11, wherein the medicament comprises an inhibitor of L INC 02422.

13. The use of claim 12, wherein the inhibitor is an siRNA.