CN110628914A - LncRNA marker related to breast cancer, detection primer and application thereof - Google Patents

Abstract

The invention discloses an lncRNA marker related to breast cancer, and a detection primer and application thereof, wherein the lncRNA marker is LINC 02422. According to the invention, the expression of LINC02422 in a breast cancer patient is found to be remarkably up-regulated for the first time through high-throughput sequencing, QPCR further verifies that the expression of LINC02422 in the breast cancer is remarkably up-regulated, and the fact that LINC02422 can be used as a biomarker for diagnosis and treatment of the breast cancer is suggested.

Description

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

Technical Field

The invention belongs to the field of biomedicine, and relates to an lncRNA marker related to breast cancer, and a detection primer and application thereof, wherein the marker is LINC 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 last decades, but about 30% of patients with early breast cancer have relapse, 24% -60% of patients have distant metastasis, and the relapse and metastasis of tumors seriously affect the life quality and survival time of the patients with breast cancer, and also bring great challenges to clinical work. Many studies have shown that tumor recurrence and metastasis are related to their histopathological parameters, including tumor size, lymph node status, histological grade, level of proliferative gene expression, etc. However, the clinical, pathological, and molecular biological characteristics of breast cancer have biological heterogeneity that is not characterized by these traditional histopathological parameters. Therefore, researchers have molecularly typed breast cancer in order to identify and differentiate the clinical pathology and response to treatment of different breast cancer patients, thereby guiding clinical work. The molecular classification of the breast cancer comprises Luminal A type, Luminal B type, HER-2 overexpression type and triple negative breast cancer. Luminal type B breast cancer is divided into two subtypes, 1) HER-2 negative type, namely ER positive and HER-2 negative, and at least one of the following conditions is that Ki-67 is high in expression, PR negative or low in expression and polygene expression analysis indicates high recurrence risk; 2) HER-2 positive type ER positive, HER-2 positive, PR and Ki-67 can be at any level.

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 an application of a reagent for detecting LINC02422 in preparation of a product for diagnosing breast cancer.

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

Further, the product comprises reagents for detecting the expression level of LINC02422 in the sample by sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology. The nucleic acid amplification techniques include 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 agent is selected from: a probe that specifically recognizes LINC 02422; or

And (3) primers for specifically amplifying LINC 02422.

Further, the primer sequence of the specific amplification LINC02422 is shown as SEQ ID No. 1-2.

The invention provides a product for detecting the expression level of LINC02422 in vitro, which comprises a chip, a kit and a nucleic acid membrane strip.

Further, the chip comprises an oligonucleotide probe that specifically recognizes LINC 02422; the kit comprises a primer for specifically amplifying LINC02422, or an oligonucleotide probe for specifically recognizing LINC 02422; the nucleic acid membrane strip includes an oligonucleotide probe that specifically recognizes LINC 02422.

Further, the primer sequence of the specific amplification LINC02422 is shown as SEQ ID No. 1-2.

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 in vitro detection of LINC02422 expression level in preparation of a tool for diagnosing breast cancer.

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

The invention provides application of LINC02422 in construction of a computational model for predicting breast cancer.

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

The invention provides application of LINC02422 in preparation of a pharmaceutical composition for treating breast cancer.

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

Further, the pharmaceutical composition comprises an inhibitor of LINC02422, which inhibitor may reduce the expression level of LINC 02422. The inhibitor is an agent which takes LINC02422 as a target sequence and can inhibit the expression level of LINC02422, and comprises: 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.

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 detection of the expression of LINC02422 gene in breast cancer tissues by QPCR.

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

FIG. 3 is a graph of the effect of LINC02422 on proliferation of breast cancer BT474 cells measured using CCK-8.

FIG. 4 is a graph showing the effect of LINC02422 on the migratory invasion capacity of breast cancer BT474 cells measured using a Transwell chamber.

Detailed Description

According to the invention, the expression of lncRNA in a breast cancer specimen 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 better method are found for diagnosis and targeted therapy of breast cancer. Through screening, the invention discovers the significant upregulation of LINC02422 in the breast cancer for the first time, and further verifies the upregulation of LINC02422 through QPCR, and the difference has statistical significance.

The LINC02422 gene is located on chromosome 12 with gene ID 105369723, and includes the LINC02422 gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed LINC02422, as well as any form of LINC02422 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of LINC 02422. The term encompasses, for example, the LINC02422 gene, the gene sequence of human LINC02422 (NR _135029.1), and 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 the difference in the level of expression of the RNA of one or more biomarkers of the invention and/or one or more splice variants of said biomarker LncRNA in one sample as determined by the amount or level of LncRNA compared to the level of expression of the same one or more biomarkers of the invention in a second sample. Differential expression can be determined as described herein and 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 as measured by the amount of RNA compared to the measurable expression level of the given biomarker in a second sample. The term "differential expression" or "change in expression level" may also mean an increase or decrease in the measurable expression level of a given biomarker in a sample population as compared to the measurable expression level of the biomarker in a second sample population. As used herein, "differential expression" can be determined as the ratio of the expression level of a given biomarker relative to the average expression level of the given biomarker in a control, wherein the ratio is not equal to 1.0. Differential expression can also be measured using p-values. When using a p-value, biomarkers are identified as differentially expressed between the first and second populations when the p-value is less than 0.1. More preferably, the p-value is less than 0.05. Even more preferably, the p-value is less than 0.01. Still more preferably, the p-value is less than 0.005. Most preferably, the p value is less than 0.001. When differential expression is determined based on the ratio, the RNA is differentially expressed if the ratio of the expression levels in the first and second samples is greater than or less than 1.0. For example, a ratio of greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or a ratio less than 1, such as 0.8, 0.6, 0.4, 0.2, 0.1, 0.05. In another embodiment of the invention, the nucleic acid transcript is differentially expressed if the ratio of the average expression level of the first population to the average expression level of the second population is greater than or less than 1.0. For example, a ratio of greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or a ratio less than 1, such as 0.8, 0.6, 0.4, 0.2, 0.1, 0.05. In another embodiment of the invention, a nucleic acid transcript is differentially expressed if the ratio of the expression level in the first sample to the average expression level in the second population is greater than or less than 1.0, for example including ratios greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or ratios less than 1, for example 0.8, 0.6, 0.4, 0.2, 0.1, 0.05.

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.

The invention can amplify nucleic acids (e.g., ncRNA) prior to or simultaneously with detection. 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 (LCR), 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 into 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 referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies; ligase chain reaction of LCR 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.beta.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 products, including but not limited to chips, nucleic acid membrane strips, or kits, for detecting the expression level of LINC02422 gene in a sample. Wherein the chip includes: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to a part or all of the sequence shown in LINC 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 aims at LINC 02422; 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.

The invention provides a kit which can be used for detecting the expression of LINC 02422. 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 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 invention provides application of LINC02422 in preparing a calculation model for predicting breast cancer. As the skilled artisan will appreciate, the measurement of two or more markers may be used to improve the diagnostic question in the survey. The biochemical markers may be determined individually, or in one embodiment of the invention, they may be determined simultaneously, for example using a chip or bead-based array technology. The concentration of the biomarkers is then interpreted independently, for example using individual retention of each marker, or a combination thereof.

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.

The logarithmic function used to correlate marker combinations with disease preferably employs algorithms developed and obtained by applying statistical methods. For example, suitable statistical methods are Discriminant Analysis (DA) (i.e., linear, quadratic, regular DA), Kernel methods (i.e., SVM), nonparametric methods (i.e., k-nearest neighbor classifiers), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest methods, boosting/bagging methods), generalized linear models (i.e., logistic regression), principal component-based methods (i.e., SIMCA), generalized additive models, fuzzy logic-based methods, neural network-and genetic algorithm-based methods. The skilled person will not have problems in selecting a suitable statistical method to evaluate the marker combinations of the invention and thereby obtain a suitable mathematical algorithm. In one embodiment, the statistical method used to obtain the mathematical algorithm used in assessing breast cancer is selected from DA (i.e., linear, quadratic, regular discriminant analysis), Kernel method (i.e., SVM), non-parametric method (i.e., k-nearest neighbor classifier), PLS (partial least squares), tree-based method (i.e., logistic regression, CART, random forest method, boosting method), or generalized linear model (i.e., logarithmic regression).

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 type B 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

Extraction of tissue total RNA Using TRIZOL 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 the expression of LINC02422 is remarkably up-regulated in breast cancer patients, and suggests that LINC02422 can be used as a possible detection target for early diagnosis of breast cancer.

TABLE 2 sequencing data

Example 2 QPCR sequencing validation of differential expression of LINC02422 Gene

1. Large sample QPCR validation of differential LINC02422 gene expression was performed on 25 cancer and normal tissue samples of luminal B breast cancer patients collected as described in 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

Add 5 XgDNA Eraser B. mu.ffer 2.0. mu.l, gDNA Eraser 1.0. mu.l, total RNA 1. mu.g, and RNase Free ddH into the tube₂O 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 withBuffer 2 4.0μl，RT Enzyme Mix I 1.0μl，RT Primer 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

Designing primers according to the gene sequences of LINC02422 and GADPH, wherein the specific primer sequences are as follows:

LINC02422 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 usingPremix Ex Taq^TMII (Takara Code: DRR081) kit is configured with a PCR reaction system in a Thermal CyclerPCR 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 (2X) 12.5. mu.l, forward (reverse) primers 1. mu.l each, DNA template 2. mu.l, and sterile distilled water 8.5. mu.l.

Reaction conditions are as follows: 30s at 95 ℃ (5 s at 95 ℃, 30s at 60 ℃) multiplied by 40

5. Results

The QPCR result is shown in figure 1, compared with the normal tissue, the expression of LINC02422 in the breast cancer tissue is up-regulated, the difference is statistically significant (P <0.05), and the result is consistent with the high-throughput sequencing result, which indicates that LINC02422 can be used as a biomarker for diagnosis and treatment of the breast cancer.

Among them, LINC02422 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 3 expression of LINC02422 in a breast cancer cell line

1. Cell culture

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

2. Transfection

General siRNA-NC, siRNA-LINC02422 used herein was purchased from Shanghai Ji code pharmaceutical technology, Inc., and the siRNA1-3 sequence silencing LINC02422 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)

Lipofectamin from Invitrogen was used^TM2000 kit, transfecting LINC02422 siRNA to breast cancer BT474 cells in logarithmic phase of growth, preparing cells planted in a 6-well plate in an incubator in advance before cell transfection, and changing the liquid of the cells in the 6-well plate and continuing culturing 24h after transfection. 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 the expression level of LINC02422

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 LINC02422 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 LINC02422 compared to the control group and the negative control group, which had statistical significance (P <0.05), wherein the effect of siRNA2 was the most significant, and therefore siRNA2 was selected for the subsequent experiments.

Example 4 CCK-8 assay to examine the Effect of LINC02422 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 significantly lower than that of the control group (FIG. 3), indicating that LINC02422 affects the proliferation of breast cancer cells, and that the proliferation capacity of breast cancer cells can be changed by changing the expression level of LINC 02422.

Example 5 Transwell Chamber examination of the Effect of LINC02422 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 adding amount of the upper chamber is 1 multiplied by 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

The results of Transwell experiments are shown in fig. 4, the cell migration and invasion number of the experimental group transfected with siRNA are remarkably reduced compared with that of the control group (P <0.05), which indicates that the expression level of LINC02422 is related to the migration and invasion of breast cancer cells, and LINC02422 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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Claims (10)

1. Application of a reagent for detecting LINC02422 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 LINC02422 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 that specifically recognizes LINC 02422; or

A primer for specifically amplifying LINC 02422;

preferably, the primer sequence for specifically amplifying LINC02422 is shown as SEQ ID No. 1-2.

4. A product for detecting the expression level of LINC02422 in vitro is characterized by comprising a chip, a kit and a nucleic acid membrane strip.

5. The product of claim 4, wherein the chip comprises oligonucleotide probes that specifically recognize LINC 02422; the kit comprises a primer for specifically amplifying LINC02422, or an oligonucleotide probe for specifically recognizing LINC 02422; the nucleic acid membrane strip comprises an oligonucleotide probe that specifically recognizes LINC 02422; preferably, the primer sequence for specifically amplifying LINC02422 is shown as SEQ ID No. 1-2.

6. The product of claim 5, 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.

7. Use of a product according to any one of claims 4 to 6 in the manufacture of a means for diagnosing breast cancer.

Use of LINC02422 in the construction of a computational model for predicting breast cancer.

Application of LINC02422 in preparing medicine for treating breast cancer is provided.

10. The use of claim 9, wherein the medicament comprises an inhibitor of LINC 02422; preferably, the inhibitor is siRNA.