CN110592219B - lncRNA diagnosis and treatment marker for breast cancer - Google Patents

Abstract

The invention discloses an lncRNA diagnosis and treatment marker for breast cancer, and particularly relates to an lncRNA marker AC092071.1, wherein the expression of AC092071.1 in a breast cancer patient is found to be up-regulated for the first time by combining a high-throughput sequencing technology and bioinformatics analysis, and AC092071.1 is further verified to be related to the occurrence and development of the breast cancer by QPCR and in vitro cell experiments, so that the AC092071.1 can be used as a biomarker for diagnosis and treatment of the breast cancer.

Description

lncRNA diagnosis and treatment marker for breast cancer

Technical Field

The invention belongs to the field of biological medicines, and relates to an lncRNA diagnosis and treatment marker for breast cancer, in particular to an lncRNA marker AC 092071.1.

Background

The breast cancer originates from mammary epithelial tissue and is a serious disease threatening physical and mental health of women, and 99 percent of breast cancer comes from women. The connection between breast cancer cells is loose, and cancer cells are easy to transfer through blood and lymph nodes after shedding, thereby being threatening to life. The pathogenesis of the breast cancer is not very clear, and through clinical research and epidemiological research, the occurrence of the breast cancer is a process of multi-factor combined action, is generated by various genetic variations, is the result of accumulation of various abnormal oncogenes and cancer suppressor gene actions, and is mainly shown in the following aspects that (a) reproductive factor is researched and shown to be closely related to the secretion of ovarian hormone. Early menstrual onset and late menopause increase estrogen load in women, which is one of the risk factors for breast cancer. Mainly relates to the secretion time of female ovarian hormone. And (II) compared with the general population, the first-class relatives of the breast cancer patients with familial accumulation and genetic factors have 2-3 times higher risk of suffering from breast cancer. And (III) diet factors show that high-fat diet and high-calorie diet increase the incidence rate of breast tumor. It has been found that weight gain is closely related to the incidence of breast cancer, particularly in women after menopause. (IV) history of mammary disorders atypical hyperplasia has a higher relative risk of carcinogenesis as the severity of the hyperplasia increases, and thus the atypical hyperplasia is referred to as precancerous.

With the rapid development of molecular biology (gene chip technology), breast cancer has been found to have different molecular subtypes at the gene level, including molecular subtypes such as Luminal A type, Luminal B type, HER-2 overexpression type, basal cell type and triple negative. The currently internationally recognized breast cancer molecular subtypes mainly comprise 4 types, namely triple negative, HER-2 overexpression type, Luminal A type and Luminal B type. The breast cancer with different molecular types has various characteristics, not only is an important factor for judging the prognosis of a breast cancer patient, but also is an important basis for guiding postoperative adjuvant therapy. Despite the recognized relatively better prognosis for Luminal type A, some patients have poorer prognosis, and further intensive research on Luminal type A breast cancer is necessary.

Long non-coding RNAs (lncRNAs) are a class of nucleotide transcripts lacking protein coding function, comprising 200 to 100000 nucleotides, with highly conserved sequence elements and specific spatial secondary structures, present in the nucleus or cytoplasm. LncRNAs play an important role in the processes of epigenetic regulation, alternative splicing, RNA decay, cell differentiation, cell cycle control, cancer cell metastasis, drug resistance and the like (Lau, E., Non-codingRNA: Zoomin on 1ncRNA functions. nat Rev Genet,2014.15(9): p.574-5.). With the intensive research on lncRNAs, more and more evidence shows that lncRNAs are involved in various biological processes and pathogenesis of diseases, especially tumorigenesis, and that in various human tumors, the lncRNAs are widely differentially expressed (Gao, K., et., Long non-coding RNA ZAS 1 is an unresourceble pathological factor and diabetes cell growth by activation of the Notch signaling pathway biomed Pharmacether, 2017.87: p.555-560.). To date, lncRNAs have made modest advances in tumors, such as HOTAIR and GWS5, which have been identified as oncogenes and cancer suppressor factors, respectively. HOTAIR enables ER protein to be highly expressed, thereby improving the occupancy rate of chromatin and strengthening downstream gene regulation. GAS5 acts as a competitive endogenous RNA to inhibit tumor spread through the action of molecular sponges of miR-21 however, lncRNAs are very large in number, only a few of lncRNAs associated with cancer have been reported, and research on lncRNAs in breast cancer is still in the initial stage. Therefore, the novel lncRNAs crucial to the occurrence, development and typing of breast cancer are still to be discovered and explored by researchers.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides a product for detecting long-chain non-coding RNAAC092071.1, and provides a basis for early diagnosis of breast cancer.

The second objective of the present invention is to provide a method for diagnosing breast cancer, which can diagnose whether a patient has breast cancer by detecting the expression level of lncRNA AC 092071.1.

The third purpose of the invention is to provide a gene closely related to the occurrence and development of breast cancer, and provide a theoretical basis for scientific research of breast cancer, particularly Luminal A-type breast cancer.

The fourth purpose of the invention is to provide a molecular target for the precise treatment of breast cancer.

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

the invention provides application of long-chain non-coding RNA AC092071.1 in preparation of a product for diagnosing breast cancer. Among them, AC092071.1 is located on human chromosome 19, and includes AC092071.1 gene and its homologues, mutations, and isoforms. The term encompasses full-length, unprocessed AC092071.1, as well as any form of AC092071.1 that results from processing in the cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of AC 092071.1. The term encompasses gene sequences such as the AC092071.1 gene, human AC092071.1, and from any other vertebrate source. A representative AC092071.1 sequence is shown in ENST00000597260.1, and those skilled in the art will understand that when the sequencing result is subjected to bioinformatics analysis, the sequencing result is usually aligned with a known genome, and the expression of the gene can be regarded as long as the sequencing fragment can be aligned with the relevant gene, so that different transcripts of AC092071.1 are also included in the present invention.

Further, AC092071.1 was upregulated in luminel type a breast cancer patients, and when the expression level of AC092071.1 in the subject sample was significantly upregulated, it indicated that the subject had, or was at risk for, breast cancer.

Further, the product comprises: the expression level of the AC092071.1 gene is detected by methods of sequencing technology, nucleic acid hybridization technology and nucleic acid amplification technology.

Illustrative, non-limiting examples of such 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.

Such 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.

The nucleic acid amplification technique is selected from the group consisting of 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). Among them, PCR requires reverse transcription of RNA into DNA before amplification (RT-PCR), TMA and NASBA to directly amplify RNA.

The invention provides a product for detecting the expression level of lncRNA AC092071.1 in a sample in vitro, and the product comprises a preparation, a chip or a kit. The "sample" includes cells, tissues or body fluids. 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. Preferably, the sample is tissue or blood. In a specific embodiment of the invention, the selected sample is tissue.

Further, the formulation, chip or kit comprises a specific primer pair or probe for AC 092071.1.

Further, the specific primer pair is used for detecting SYBR Green, Taqman probes, molecular beacons, double-hybrid probes and composite probes.

In the present invention, a "probe" refers to a molecule that is capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

The probe has a base sequence complementary to a specific base sequence of a target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA or RNA, and may be polynucleotides obtained by replacing nucleotides in a part or all of them with artificial Nucleic acids such as PNA (polypeptide Nucleic Acid), LNA (registered trademark, locked Nucleic Acid, bridge Nucleic Acid, crosslinked Nucleic Acid), ENA (registered trademark, 2 '-O, 4' -C-Ethylene-Bridged Nucleic acids), GNA (glyceronucleic Acid), and TNA (Threose Nucleic Acid).

The term "hybridization" in the context of the present invention is used to refer to the pairing of complementary nucleic acids. Hybridization and hybridization strength (i.e., strength of association between nucleic acids) are affected by factors such as: the degree of complementarity between nucleic acids, the stringency of the conditions involved, the Tm of the hybrids formed, and the ratio of G: C within the nucleic acids. A single molecule that contains within its structure a pair of complementary nucleic acids is said to be "self-hybridizing".

Furthermore, the sequences of the specific primer pair are shown as SEQ ID NO.1 and SEQ ID NO. 2.

The invention provides application of AC092071.1 in construction of 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 invention provides application of AC092071.1 in preparing a medicament for treating breast cancer.

Further, the medicament comprises an inhibitor of AC 092071.1. The inhibitor comprises an agent which takes AC092071.1 as a target sequence and can inhibit the expression level of AC092071.1, 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.

Preferably, the inhibitor is a small interfering RNA (siRNA).

The invention provides a medicament for treating breast cancer, which comprises an inhibitor of AC092071.1 and/or a pharmaceutically acceptable carrier. Wherein the inhibitor comprises an agent which takes AC092071.1 as a target sequence and can inhibit the expression level of AC092071.1, 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.

Preferably, the nucleic acid inhibitor is an siRNA.

In the specific embodiment of the invention, 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.

The gene detection kit or the gene chip can be used for detecting the expression levels of a plurality of genes (for example, a plurality of genes related to breast cancer) including the AC092071.1 gene, and can be used for simultaneously detecting a plurality of markers of the breast cancer, so that the accuracy of breast cancer diagnosis can be greatly improved.

The term "diagnosis" in the present invention means to identify a disease by the signs and symptoms of the disease or genetic analysis, pathological analysis, histological analysis, and the like.

The term "treating" in the present invention means reducing the number of cancer cells; reducing the size of the primary tumor; inhibit (i.e., slow to some extent, preferably prevent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent, preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or to alleviate one or more symptoms associated with the condition to some extent.

The invention has the advantages and beneficial effects that:

the invention discloses a molecular marker for diagnosing breast cancer, which can judge early breast cancer by detecting the expression level of a marker AC092071.1, thereby carrying out early intervention and improving the survival rate of patients.

The invention provides a molecular target for treating breast cancer, which changes the expression level of AC092071.1 by targeting a target AC092071.1, thereby playing a role in treating breast cancer.

Drawings

FIG. 1 is a graph showing the detection of the expression of AC092071.1 gene in breast cancer tissue by QPCR.

FIG. 2 is a graph of the detection of siRNA silencing AC092071.1 using QPCR.

FIG. 3 is a graph showing the effect of AC092071.1 on the proliferation of MCF-7 cells measured by CCK.

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 were not subjected to chemotherapy, radiotherapy and endocrine treatment before operation, 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 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 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 AC092071.1 is significantly up-regulated in breast cancer patients, and suggests that AC092071.1 may be applied to early diagnosis of breast cancer as a detection target.

TABLE 2 sequencing data

Example 2 QPCR sequencing verification of differential expression of AC092071.1 Gene

1. Large sample QPCR validation of differential expression of the AC092071.1 gene 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 AC092071.1 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, and AC092071.1 was upregulated in breast cancer tissues compared to normal tissues, with the difference being statistically significant (P <0.05), consistent with the high throughput sequencing results, wherein AC092071.1 was significantly upregulated in 26 samples, with no significant change in 24 samples, 23 of 26 upregulated samples were breast cancer tissues, and 3 were normal tissues, as shown in table 5. Suggesting that whether the subject has the breast cancer can be judged by detecting the level of AC092071.1, when the level of AC092071.1 is obviously increased, the subject has the breast cancer or has the risk of having the breast cancer, and siRNA or shRNA for reducing the AC092071.1 level can be designed to treat the breast cancer through the relationship between AC092071.1 and the breast cancer.

TABLE 5 Positive in disease Table of AC092071.1

Example 3 expression of AC092071.1 in a Breast cancer cell line

1. Cell culture

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

2. siRNA sequences

siRNA-NC and siRNA-AC092071.1 used in the present application were purchased from Shanghai Ji code pharmaceutical technology, Inc., and the sequences of silencing AC092071.1 are shown in Table 6.

TABLE 6 siRNA sequences

3. Transfection

Lipofectamin from Invitrogen was used^TM2000 reagents were used for cell transfection and the experiments were divided into three groups: a control group (MCF-7), a negative control group (siRNA-NC) and an experimental group (siRNA1, siRNA2), wherein the siRNA of the negative control group has no homology with the sequence of the AC092071.1 gene.

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

4. QPCR detection of the expression level of AC092071.1

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 siRNA1-2 small molecule has better silencing effect on AC092071.1, wherein the silencing effect of siRNA1 is the most significant (P <0.05) (FIG. 2), so that siRNA1 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 assay for the Effect of the AC092071.1 Gene on Breast cancer cell proliferation

The breast cancer cells transfected with siRNA1 were used as experimental groups and siRNA-NC transfected cells were used as control groups, and the cells were added to a 96-well plate at 3000 cells per well, with 5 duplicate wells per group. The method is used for detecting the detection time points of 24h, 48h, 72h and 96h respectively. Adding 10 mul of CCK-8 detection solution into the cell wells every 24h, continuously putting the 96-well plate into a cell culture box for incubation for about 4h, detecting the absorbance value of each well at the wavelength of 450nm by using a microplate reader, recording data, and continuously measuring for 96 h. And drawing a growth curve according to the average value of the detected OD values.

The growth curve results show that the proliferation capacity of the cells after transfection of siRNA1 in the experimental group is obviously lower than that of the control group (FIG. 3), which indicates that AC092071.1 influences the proliferation of breast cancer cells, and the proliferation capacity of the breast cancer cells can be changed by changing the expression level of AC 092071.1.

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

Sequence listing

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Claims (11)

1. The application of the reagent for detecting the long-chain non-coding RNA AC092071.1 in preparing a product for diagnosing breast cancer.

2. Use according to claim 1, characterized in that the product comprises: the expression level of the AC092071.1 gene is detected by a sequencing technology, a nucleic acid hybridization technology, a nucleic acid amplification technology or an immunoassay method.

3. Use according to claim 2, wherein the nucleic acid amplification technique is selected from the group consisting of polymerase chain reaction, reverse transcription polymerase chain reaction, transcription mediated amplification, ligase chain reaction, strand displacement amplification.

4. The use according to claim 1, characterized in that the product comprises a formulation, a chip, or a kit.

5. The use of claim 4, wherein the formulation, chip or kit comprises a primer pair or probe specific for AC 092071.1.

6. The use according to claim 5, wherein the specific primer pair is used for detection of SYBR Green, Taqman probes, molecular beacons, double-hybrid probes, composite probes.

7. The use according to claim 6, wherein the sequence of the specific primer pair is shown as SEQ ID No.1 and SEQ ID No. 2.

Use of AC092071.1 in the construction of a computational model for predicting breast cancer.

Use of an inhibitor of AC092071.1 in the manufacture of a medicament for the treatment of breast cancer, wherein the inhibitor reduces the expression level of AC 092071.1.

10. The use of claim 9, wherein the inhibitor is an siRNA.

11. A medicament for the treatment of breast cancer, said medicament comprising an inhibitor of AC 092071.1.

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