CN116287258A - Application of novel molecular marker in breast cancer diagnosis or prognosis evaluation - Google Patents
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Abstract
The invention discloses application of a novel molecular marker in breast cancer diagnosis or prognosis evaluation. In particular to application of a molecular marker lncRNA LIVAR and/or a substance for detecting the molecular marker in breast cancer diagnosis, auxiliary diagnosis, screening, prognosis evaluation and risk evaluation, and application of the molecular marker lncRNA LIVAR serving as a target in preventing or treating breast cancer. The expression level of LIVAR is significantly up-regulated in breast cancer patients, and high expression of LIVAR is significantly correlated with poor prognosis of breast cancer (including triple negative breast cancer). Experiments show that inhibiting the expression of LIVAR can obviously inhibit the migration and/or invasion capacity of breast cancer cells and can obviously inhibit the proliferation of the breast cancer cells. The method shows that the LIVAR can be used as a target point of breast cancer treatment, a substance for inhibiting LIVAR expression can be used for preparing a medicament for treating breast cancer, and a substance for detecting LIVAR can be used for diagnosis or prognosis of breast cancer.
Description
Technical Field
The invention belongs to the field of biotechnology and medical diagnosis, relates to application of a novel molecular marker in diagnosis or prognosis evaluation of breast cancer, and in particular relates to a screening method of long non-coding RNA related to breast cancer and application thereof.
Background
Whole genome transcriptome studies have shown that protein coding regions account for only 2% of human genomic sequences, with more than 90% of the sequences transcribed into non-coding RNA (ncRNA) that does not control protein synthesis. Long non-coding RNAs (lncRNAs) are transcripts of over 200 nucleotides in length, are characterized by multiple types, modes of action and numbers, have spatial and temporal specificities, can regulate gene expression at multiple levels, and are involved in multiple biological processes such as proliferation, apoptosis, metastasis, metabolism, drug resistance, and the like. More and more evidences show that the abnormality of the expression quantity of the lncRNA and the genetic polymorphism thereof are closely related to the occurrence and development of tumors, so the lncRNA is hopeful to become a novel tumor diagnosis marker and a treatment target spot, and is used for tumor diagnosis, treatment, recurrence monitoring and the like.
From the data published in 2020 by global cancer statistics report (Global Cancer Observatory), breast cancer has become the most frequently occurring type of cancer. National cancer report issued by the national cancer center 2022, month 2, shows that breast cancer is the fourth leading cancer onset in China, and the annual incidence rate is steadily rising. Breast cancer patients can be divided into 4 subtypes based on the expression of Estrogen Receptor (ER), progestogen Receptor (PR), human epidermal growth factor receptor 2 (HER 2), and KI 67: luminal cell a (lumineal a), luminal cell B (lumineal B), HER2 positive and Triple Negative Breast Cancer (TNBC). Wherein triple negative breast cancers (ER, PR and HER2 are all negative) lack hormone receptors, accounting for about 15% of breast cancer cases, and are insensitive to targeted and endocrine therapies. More than 70% of triple negative breast cancer patients relapse within 3 years after surgery, resulting in poor prognosis. In view of the above, the method for searching and identifying the sensitive and specific novel breast cancer molecular marker and the detection method thereof for diagnosing or assisting in diagnosing breast cancer has important significance for guiding clinical treatment and judging prognosis, and has wide application prospect.
Disclosure of Invention
The invention aims to provide a molecular marker for prognosis evaluation, diagnosis, auxiliary diagnosis, screening, risk evaluation and/or postoperative recurrence or metastasis risk evaluation of breast cancer and/or provide a breast cancer treatment target. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.
To achieve the above object, the present invention provides, first of all, any one of the following uses of a molecular marker and/or a substance detecting the molecular marker:
a1 Use in the diagnosis of breast cancer or in the preparation of a product for the diagnosis of breast cancer;
a2 Use in breast cancer assisted diagnosis or in the manufacture of a product for breast cancer assisted diagnosis;
a3 Use in breast cancer screening or in the preparation of a product for breast cancer screening;
a4 Use in the prognosis evaluation of breast cancer or in the preparation of a product for the prognosis evaluation of breast cancer;
a5 Use in assessing breast cancer risk or in the manufacture of a product for assessing breast cancer risk;
a6 Use in assessing risk of recurrence or metastasis after breast cancer surgery or in the manufacture of a product for assessing risk of recurrence or metastasis after breast cancer surgery;
a7 For the prevention or treatment of breast cancer or for the preparation of a medicament for the prevention or treatment of breast cancer;
a8 The application of the composition in preventing or treating triple negative breast cancer or preparing medicines for preventing or treating triple negative breast cancer;
a9 For inhibiting proliferation of breast cancer cells or for preparing an agent or medicament for inhibiting proliferation of breast cancer cells;
a10 For inhibiting migration, metastasis, diffusion and/or invasion of breast cancer cells or for preparing an agent or a medicament for inhibiting migration, metastasis, diffusion and/or invasion of breast cancer cells;
the molecular marker may be lncRNA (long non-coding RNA), which may be named LIVAR (liver cell viabilityassociated lncRNA), and the lncRNA LIVAR may be any of the following:
b1 Nucleotide sequence is the RNA of SEQ ID No. 1;
b2 A polynucleotide which is of human origin and has more than 80% identity to B1);
preferably, the breast cancer may be a triple negative breast cancer.
Further, the expression level of lncRNA LIVAR is significantly up-regulated in breast cancer patients, and high expression of LIVAR is significantly correlated with poor prognosis of breast cancer. The lncRNA LIVAR can be used as a molecular marker for the fields of diagnosis, auxiliary diagnosis, screening, prognosis evaluation, risk evaluation or postoperative recurrence or metastasis risk evaluation of breast cancer and other molecular diagnosis.
Further, the gene encoding the lncRNA LIVAR is a LIVAR gene, and the nucleotide sequence of the LIVAR gene is shown as SEQ ID No. 4.
Further, the lncRNA LIVAR may be human.
Herein, identity refers to identity of nucleotide sequences. The identity of nucleotide sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of nucleotide sequences, and then the value (%) of identity can be obtained.
Herein, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
In the above application, the substance for detecting the molecular marker may include a reagent and/or an instrument for detecting the molecular marker by reverse transcription-polymerase chain reaction, real-time fluorescent quantitative PCR technique, transcriptome sequencing technique, northern blot, in situ hybridization technique, gene chip technique, nanopore sequencing technique or PacBio sequencing technique.
In the above application, the substance for detecting the molecular marker may be a reagent and/or an instrument for detecting the expression level of the lncRNA LIVAR.
Further, the reagent for detecting the expression level of lncRNA LIVAR comprises a probe which specifically recognizes lncRNA LIVAR and/or a primer which specifically amplifies lncRNA LIVAR.
Further, the reagent for detecting the expression level of the lncRNA LIVAR includes a probe that specifically recognizes the LIVAR gene and/or a primer that specifically amplifies the LIVAR gene.
The probe may be a DNA probe, RNA probe, cDNA probe, cRNA probe or oligonucleotide probe.
The invention also provides kits for breast cancer diagnosis, auxiliary diagnosis, screening, prognostic assessment, risk assessment or post-operative recurrence or metastasis risk assessment, which kits may comprise any of the substances for detecting the molecular markers described herein.
Further, the detection sample of the kit may be a blood sample (e.g., whole blood, plasma, serum), a tissue sample, a saliva sample, a sputum sample, a body fluid sample, etc., but is not limited thereto.
The various reagent components of the kit may be present in separate containers or may be pre-combined in whole or in part into a reagent mixture.
The invention also provides any one of the following applications of the substance for inhibiting the expression of the molecular marker:
c1 For the prevention or treatment of breast cancer or for the preparation of a medicament for the prevention or treatment of breast cancer;
c2 The application of the composition in preventing or treating triple negative breast cancer or preparing medicines for preventing or treating triple negative breast cancer;
c3 For inhibiting proliferation of breast cancer cells or for preparing an agent or medicament for inhibiting proliferation of breast cancer cells;
c4 For inhibiting migration, metastasis, diffusion and/or invasion of breast cancer cells or for preparing an agent or a medicament for inhibiting migration, metastasis, diffusion and/or invasion of breast cancer cells.
Further, the inhibition of the expression of the molecular marker (lncRNA LIVAR) may be achieved by techniques well known to those skilled in the art, such as knockdown of lncRNA by antisense oligonucleotide ASO, knockdown of lncRNA by shRNA or siRNA delivered by plasmid or virus, and inhibition of expression of lncRNA by CRISPRi (gene suppression/silencing) technique, etc.
Further, the substances that inhibit the expression of the molecular markers include, but are not limited to, one or more of nucleic acid molecules, carbohydrates, small molecule compounds, gene editing vectors, lentiviruses, or adeno-associated viruses.
Further, the nucleic acid molecules include, but are not limited to, antisense oligonucleotides (e.g., antisense RNA), double stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (microRNA), or short hairpin RNA (shRNA).
Further, the antisense oligonucleotide (e.g., antisense RNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (microRNA), or short hairpin RNA (shRNA) is used to inhibit expression of the molecular marker (lncRNA LIVAR).
The antisense oligonucleotide refers to a nucleic acid fragment which can be complementary to a target gene (lncRNA LIVAR or LIVAR gene) and can be bound to the target gene through a base complementary principle so as to inhibit, block or reduce the expression of the lncRNA LIVAR, and the antisense oligonucleotide can be antisense DNA or antisense RNA and can be artificially synthesized or expressed in vivo by a method known to a person skilled in the art; the double-stranded RNA (dsRNA), small interfering RNA (siRNA), micro RNA (miRNA) or short hairpin RNA (shRNA) refers to RNA that inhibits or reduces expression of lncRNALIVAR by RNA interference (RNAi) and can be prepared by methods well known to those skilled in the art.
In the above application, the substance that inhibits expression of the molecular marker includes a substance that silences or knocks out a LIVAR gene, which may be a gene encoding the molecular marker lncRNA LIVAR.
Further, the nucleotide sequence of the LIVAR gene can be shown as SEQ ID No. 4.
In such applications, the agent that silences or knocks out the LIVAR gene may include, but is not limited to siRNA, sgRNA, microRNA, shRNA and/or antisense oligonucleotides.
In the above application, the siRNA may be si-LIVAR, the nucleotide sequence of the sense strand of the si-LIVAR may be SEQ ID No.2, and the nucleotide sequence of the antisense strand may be SEQ ID No.3.
The present invention also provides a pharmaceutical composition, which may comprise any of the substances described herein that inhibit the expression of the molecular marker, which may have at least one of the following uses:
d1 Preventing or treating breast cancer;
d2 Preventing or treating triple negative breast cancer;
d3 Inhibiting breast cancer cell proliferation;
d4 Inhibit breast cancer cell migration, metastasis, spread and/or invasion.
Further, the pharmaceutical composition may also include one or more pharmaceutically acceptable carriers.
The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant, or lubricant.
The present invention also provides a device for breast cancer diagnosis, assisted diagnosis, screening, prognosis evaluation, risk evaluation or postoperative recurrence or metastasis risk evaluation, which device may comprise any one of the substances for detecting the molecular markers described herein and a computer readable storage medium storing a computer program which causes a computer to perform the steps of: and diagnosing breast cancer, performing auxiliary diagnosis, screening, prognosis evaluation, risk evaluation or postoperative recurrence or metastasis risk evaluation according to the expression level of the molecular marker in the sample.
The breast cancer described herein may be triple negative breast cancer.
Herein, the product includes, but is not limited to, reagents, kits, chips, dipsticks, detection cards, high throughput sequencing platforms, or biosensors.
The products described herein may include any of the substances described herein that detect the molecular markers.
The invention also provides methods of diagnosing, aiding in diagnosing or screening for breast cancer, which may include: obtaining a test sample from a subject, and then detecting the expression level of the molecular marker (lncRNA LIVAR) in the test sample using any of the substances described herein that detect the molecular marker, and diagnosing, aiding in diagnosis or screening of breast cancer based on the expression level of the molecular marker.
The invention also provides methods of prognosis, risk assessment, or post-operative recurrence or metastasis risk assessment of breast cancer, which methods may comprise: obtaining a test sample from a subject, and then detecting the expression level of the molecular marker (lncRNA LIVAR) in the test sample using any of the substances described herein that detect the molecular marker, and performing prognosis, risk assessment, or postoperative recurrence or metastasis risk assessment of breast cancer based on the expression level of the molecular marker.
The invention also provides a method of preventing or treating breast cancer, which method may comprise administering to a subject any of the substances described herein that inhibit the expression of the molecular marker (lncRNA LIVAR), or the pharmaceutical composition.
In the above method, the subject may comprise a patient suffering from a breast cancer disease.
In the above method, the breast cancer may comprise triple negative breast cancer, luminal a breast cancer, luminal B breast cancer, or HER2 positive breast cancer.
The molecular markers (lncRNA LIVAR) are also within the scope of the invention.
Any of the substances described herein for detecting the molecular markers are also within the scope of the present invention.
Any of the substances described herein that inhibit the expression of the molecular markers are also within the scope of the present invention.
The invention also provides a substance inhibiting the expression of the molecular marker as described in any one of the text, which is used for preventing or treating breast cancer.
The purpose of the above-described applications and methods may be for disease diagnosis purposes, disease prognosis purposes and/or disease treatment purposes, as well as for non-disease diagnosis purposes, non-disease prognosis purposes and non-disease treatment purposes; their direct purpose may be information of intermediate results of obtaining disease diagnosis results, disease prognosis results and/or disease treatment results, and their direct purpose may be non-disease diagnosis purpose, non-disease prognosis purpose and/or non-disease treatment purpose.
The invention aims at overcoming the defects of the prior art and providing a novel molecular marker related to breast cancer, and a kit and a device which can be used for the diagnosis method. The molecular marker developed by the invention can be a molecular target LIVAR (liver cell viability associated lncRNA) related to breast cancer diagnosis and prognosis, is a reliable molecular marker, can be applied to the preparation of a kit for detecting and evaluating breast cancer prognosis, can be used for diagnosing and monitoring breast cancer patients, and can be used for determining a further treatment method and improving the curative effect.
Through extensive and intensive studies, the inventors discovered that the expression level of long non-coding RNA LIVAR in breast cancer patients is significantly up-regulated by mining data in a public database, and that high expression of LIVAR is significantly correlated with poor prognosis of breast cancer (including triple negative breast cancer), and developed molecular markers based on the same, which can be used in the fields of diagnosis, auxiliary diagnosis, screening, prognosis evaluation, risk evaluation or postoperative recurrence or metastasis risk evaluation of breast cancer. From the Kaplan-Meier survival analysis results, it can be seen that high expression of LIVAR correlates with poor prognosis of breast cancer. Phenotypic experiments such as cell migration and proliferation further demonstrate that LIVAR correlates with proliferation and migration of breast cancer cells. In summary, the present invention identifies a breast cancer diagnostic and prognostic predictor LIVAR that can be used to assess the likelihood of a patient having breast cancer or developing the disease over a later period of time.
The invention also designs siRNA targeting LIVAR gene as a substance for inhibiting the expression of the molecular marker (lncRNALIVAR) to knock down the expression of the LIVAR gene, and the result further verifies that the knock down LIVAR gene can inhibit the expression of the LIVAR gene, can obviously inhibit the migration and/or invasion capacity of breast cancer cells, and can obviously inhibit the proliferation of the breast cancer cells. The LIVAR can influence proliferation of breast cancer cells, can be used as a drug target point for breast cancer treatment, can be used for preparing a drug for treating breast cancer, and further verifies that the LIVAR-detecting substance can be applied to diagnosis, auxiliary diagnosis or prognosis of breast cancer.
In summary, the development of the molecular marker lncRNA LIVAR for breast cancer and the substance for inhibiting the expression of the molecular marker for diagnosis, auxiliary diagnosis or prognosis evaluation has important clinical application value for diagnosis, prognosis and risk prediction of breast cancer.
Drawings
FIG. 1 is a graph showing LIVAR expression level and survival analysis of breast cancer tissues in example 1 of the present invention. In fig. 1, a is analysis of difference in expression level of LIVAR in breast cancer tissue and normal breast tissue, B in fig. 1 is analysis result of Kaplan-Meier survival of breast cancer patient, and C in fig. 1 is analysis result of Kaplan-Meier survival of triple negative breast cancer patient.
FIG. 2 shows the experimental results of the effect of LIVAR on the migration ability of cells in example 2 of the present invention, and the cells used in the experiment were triple negative breast cancer cell line MDA-MB-231. Where si-LIVAR is the experimental group with LIVAR knocked down in the cells and si-NC is the corresponding negative control group.
FIG. 3 is a graph showing the effect of LIVAR on cell proliferation activity after knocking down LIVAR gene in MDA-MB-231 cells according to the experimental result of LIVAR on cell proliferation activity in example 3 of the present invention. si-LIVAR is the knockdown experimental group, and si-NC is the negative control group.
Fig. 4 is a graph of the work of subjects with LIVAR as a molecular marker for breast cancer diagnosis.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The following examples used GraphPad Prism 9 statistical software to treat the data, the experimental results were expressed as mean ± standard deviation, using the unpaired t assay test method, P < 0.05 (x) indicated a statistical difference, P < 0.01 (x) indicated a significant statistical difference, and P < 0.001 (x) indicated a significant statistical difference.
MDA-MB-231 cells in the examples below were derived from American type culture Collection (AmericanType Culture Collection, ATCC) Cat#CRM-HTB-26.
The partial sequences involved in the examples below are shown in Table 1.
TABLE 1 lncRNALIVAR related sequences
EXAMPLE 1 LIVAR expression level in breast cancer tissue and survival analysis
The invention has been extensively studied and analyzed to determine a new prognosis-related long non-coding RNA (lncRNA) between tumor and non-cancerous samples in breast cancer data, designated LIVAR (liver cellviability associated lncRNA). LIVAR has a high expression trend in breast cancer samples and leads to low survival in patients, up-regulation of LIVAR also being associated with a poor prognosis in triple negative breast cancer subtypes. As a novel molecular marker, LIVAR has not been reported for use in the prognosis of cancer survival. Therefore, we propose that LIVAR can be used as an effective treatment target in breast cancer, and is a promising new strategy, so that the treatment effect of patients is improved.
The nucleotide sequence of LIVAR is shown in SEQ ID No. 1:
the gene encoding the lncRNA LIVAR is a LIVAR gene, and the nucleotide sequence of the LIVAR gene is shown as SEQ ID No. 4:
the present inventors downloaded RNA-Seq and clinical data of breast cancer using a cancer genomic map (The Cancer Genome Atlas, TCGA) website, and analyzed the expression level of LIVAR in 1109 tumor tissues and 113 normal breast tissues. Then, a breast cancer tumor sample is reserved, and according to the expression quantity and survival condition of LIVAR, a about dengue index of 3.5 can be calculated and used as an optimal critical value. And taking the critical value as a bifurcation point, dividing the samples into 2 groups of high expression and low expression, and performing Kaplan-Meier survival analysis. Then, subtype of tumor samples is separated according to clinical data, the reduction index is also calculated for the samples of the triple negative type, and the optimal critical value of 4.5 is calculated and is also used for carrying out survival analysis after distinguishing high and low expression groups.
The analysis results are shown in FIG. 1, and the expression level of LIVAR in tumor tissues of breast cancer is significantly higher than that of LIVAR in normal tissues in FIG. 1. From the Kaplan-Meier curves of B and C in fig. 1, it can be seen that the LIVAR high expression group had poor overall survival in breast cancer samples (B in fig. 1), and also had poor prognosis in triple negative typing (C in fig. 1). The result shows that LIVAR can be used as a molecular marker for diagnosis and prognosis evaluation of breast cancer and can be used as an effective treatment target of breast cancer.
Example 2 influence of LIVAR on cell migration ability
After knocking down the LIVAR gene in breast cancer cells, a cell migration experiment is carried out, and the steps are as follows:
(1) Transfection of si-LIVAR in MDA-MB-231 cells reduced expression of LIVAR gene, si-NC being the corresponding negative control group;
the siRNA (i.e., si-LIVAR) sequences used to knock down LIVAR gene expression were as follows:
si-LIVAR sense strand: 5'-GGUUGGGACAGAUCUAUUUTT-3' (SEQ ID No. 2),
si-LIVAR antisense strand: 5'-AAAUAGAUCUGUCCCAACCTT-3' (SEQ ID No. 3).
The nucleotide sequence of the negative control si-NC is: 5'-UUCUCCGAACGUGUCACGUTT-3'.
Cells were seeded in six well plates and after cell attachment, a transfection complex prepared from siRNA and transfection reagent was added, and serum-free medium was used during transfection. After the culture medium is placed in an incubator for about 6 hours, the culture medium is changed into a complete culture medium, the culture is continued for 48 hours, and the subsequent operation is carried out.
(2) Cell suspensions were prepared, the number of cells therein was counted using a cell counter, diluted to an appropriate concentration with DMEM medium, and added to a 24-well plate with an upper chamber. Each upper chamber is added with a liquid containing 1X 10 4 -5×10 4 Cell number 400. Mu.L cell suspension, and 600. Mu.L complete medium containing DMEM+FBS (10X) +pen/strep (100X) was supplemented to the lower chamber, 3-4 wells per group, at 37℃with 5% CO 2 Is cultured at constant temperature for 24-48 hours.
(3) Cells in the lower chamber were fixed with methanol, stained with 0.1% or 0.5% crystal violet, observed under a microscope and photographed, and the number of migrated cells (cells/chamber) was counted.
The experimental results are shown in fig. 2, which shows that after the LIVAR gene is knocked down, the migration number of breast cancer cells is obviously reduced, migration and/or invasion capacity is obviously inhibited, and the migration capacity of LIVAR controllable cells can be used as a drug target point for breast cancer treatment, and a substance for inhibiting LIVAR expression can be used for preparing drugs for treating breast cancer. The substance for detecting LIVAR can be applied to diagnosis, auxiliary diagnosis or prognosis of breast cancer.
EXAMPLE 3 Effect of LIVAR on cell proliferation Activity
Proliferation experiments were performed using cells knocked down with the LIVAR gene as follows:
(1) Preparation of cell suspensions from MDA-MB-231 cells transfected with si-LIVAR and si-NC control, plating in 96-well plates, 5 duplicate wells per group, 200. Mu.L of each well containing 2X 10 3 The cells were cultured in 5 plates at 37℃in 5% CO 2 A constant temperature incubator.
The siRNA (i.e., si-LIVAR) sequences used to knock down LIVAR gene expression were as follows:
si-LIVAR sense strand: 5'-GGUUGGGACAGAUCUAUUUTT-3' (SEQ ID No. 2),
si-LIVAR antisense strand: 5'-AAAUAGAUCUGUCCCAACCTT-3' (SEQ ID No. 3).
The nucleotide sequence of the negative control si-NC is: 5'-UUCUCCGAACGUGUCACGUTT-3'.
(2) After the cells are completely adhered, taking one 96-well plate in the step (1), adding 10 mu L of CCK-8 per 100 mu L of culture medium to prepare reagent, adding 200 mu L of reagent into the holes with inoculated cells in a liquid exchange mode, carrying out light-shielding treatment during operation, and putting the cells back into an incubator after the addition. After 1 hour, the absorbance at 450nm was measured with a microplate reader and recorded.
(3) After 24, 48, 72 and 96 hours, a 96-well plate of step (1) was taken, the medium of each multiplex well was replaced with 200. Mu.L of medium+CCK-8 reagent, and absorbance was measured after incubation in an incubator for 1 hour. And counting the light absorption values measured for 5 consecutive days, and drawing a curve by taking time as an abscissa and the light absorption value as an ordinate.
The experimental results are shown in FIG. 3, which shows that the proliferation activity of breast cancer cells is reduced after the LIVAR gene is knocked down, and the proliferation capacity of the LIVAR regulatable cells is demonstrated. It is demonstrated that lowering the expression level of the LIVAR gene can significantly inhibit the growth of breast cancer cells, i.e., inhibit the proliferation capacity of breast cancer cells. Further shows that LIVAR can influence proliferation of breast cancer cells, can be used as a drug target point for breast cancer treatment, and a substance for inhibiting LIVAR expression can be used for preparing a drug for treating breast cancer. The substance for detecting LIVAR can be applied to diagnosis, auxiliary diagnosis or prognosis of breast cancer.
Example 4 Performance of LIVAR as molecular marker for breast cancer diagnosis
Based on information of Estrogen Receptor (ER), progestogen Receptor (PR), and human epidermal growth factor receptor 2 (HER 2) in the breast cancer samples and clinical data downloaded by TCGA, the samples of ER, PR, HER2 were all negative were determined as triple negative. And then taking the results of the survival situation, the age and the TNM stage of the subtype patient, and carrying out multi-index joint prediction ROC curve analysis.
As shown in figure 4, the area under the ROC curve of LIVAR serving as a molecular marker for diagnosing triple negative breast cancer is 0.829, which shows that the LIVAR is used for diagnosing breast cancer, especially triple negative breast cancer, and has good specificity and high sensitivity, and the LIVAR serving as a novel molecular marker for breast cancer has great potential value and can provide a powerful molecular biological tool for screening, diagnosing or assisting diagnosis of breast cancer.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. Use of a molecular marker and/or a substance detecting said molecular marker for any of the following:
a1 Use in the diagnosis of breast cancer or in the preparation of a product for the diagnosis of breast cancer;
a2 Use in breast cancer assisted diagnosis or in the manufacture of a product for breast cancer assisted diagnosis;
a3 Use in breast cancer screening or in the preparation of a product for breast cancer screening;
a4 Use in the prognosis evaluation of breast cancer or in the preparation of a product for the prognosis evaluation of breast cancer;
a5 Use in assessing breast cancer risk or in the manufacture of a product for assessing breast cancer risk;
a6 Use in assessing risk of recurrence or metastasis after breast cancer surgery or in the manufacture of a product for assessing risk of recurrence or metastasis after breast cancer surgery;
a7 For the prevention or treatment of breast cancer or for the preparation of a medicament for the prevention or treatment of breast cancer;
a8 The application of the composition in preventing or treating triple negative breast cancer or preparing medicines for preventing or treating triple negative breast cancer;
a9 For inhibiting proliferation of breast cancer cells or for preparing an agent or medicament for inhibiting proliferation of breast cancer cells;
a10 For inhibiting migration, metastasis, diffusion and/or invasion of breast cancer cells or for preparing an agent or a medicament for inhibiting migration, metastasis, diffusion and/or invasion of breast cancer cells;
the molecular marker is lncRNA, and the lncRNA is any one of the following:
b1 Nucleotide sequence is the RNA of SEQ ID No. 1;
b2 A polynucleotide which is of human origin and has more than 80% identity to B1);
preferably, the breast cancer is a triple negative breast cancer.
2. The use according to claim 1, wherein the substance for detecting the molecular marker comprises reagents and/or instruments for detecting the molecular marker by reverse transcription-polymerase chain reaction, real-time fluorescent quantitative PCR technique, transcriptome sequencing technique, northern blot, in situ hybridization technique, gene chip technique, nanopore sequencing technique or PacBio sequencing technique.
3. The use according to claim 1 or 2, wherein the substance for detecting the molecular marker is a reagent and/or an instrument for detecting the expression level of lncRNA as claimed in claim 1.
4. A kit for breast cancer diagnosis, auxiliary diagnosis, screening, prognosis evaluation, risk evaluation or postoperative recurrence or metastasis risk evaluation, characterized in that the kit comprises a substance for detecting the molecular marker according to any one of claims 1-3.
5. Use of any one of the following substances which inhibit the expression of a molecular marker as defined in claim 1:
c1 For the prevention or treatment of breast cancer or for the preparation of a medicament for the prevention or treatment of breast cancer;
c2 The application of the composition in preventing or treating triple negative breast cancer or preparing medicines for preventing or treating triple negative breast cancer;
c3 For inhibiting proliferation of breast cancer cells or for preparing an agent or medicament for inhibiting proliferation of breast cancer cells;
c4 For inhibiting migration, metastasis, diffusion and/or invasion of breast cancer cells or for preparing an agent or a medicament for inhibiting migration, metastasis, diffusion and/or invasion of breast cancer cells.
6. The use according to claim 5, wherein the substance inhibiting the expression of the molecular marker of claim 1 comprises a substance silencing or knocking out the LIVAR gene, which is a gene encoding the molecular marker of claim 1.
7. The use according to claim 6, wherein the agent that silences or knocks out the LIVAR gene comprises, but is not limited to siRNA, sgRNA, microRNA, shRNA and/or antisense oligonucleotides.
8. The use according to claim 7, wherein the siRNA is si-LIVAR, the sense strand of which has the nucleotide sequence of SEQ ID No.2 and the antisense strand has the nucleotide sequence of SEQ ID No.3.
9. A pharmaceutical composition comprising a substance according to any one of claims 5-8 that inhibits the expression of a molecular marker according to claim 1, said pharmaceutical composition having at least one of the following uses:
d1 Preventing or treating breast cancer;
d2 Preventing or treating triple negative breast cancer;
d3 Inhibiting breast cancer cell proliferation;
d4 Inhibit breast cancer cell migration, metastasis, spread and/or invasion.
10. A device for breast cancer diagnosis, assisted diagnosis, screening, prognosis evaluation, risk evaluation or postoperative recurrence or metastasis risk evaluation, characterized in that it comprises a substance for detecting said molecular marker according to any one of claims 1-3 and a computer readable storage medium storing a computer program for causing a computer to execute the steps of: and diagnosing breast cancer, performing auxiliary diagnosis, screening, prognosis evaluation, risk evaluation or postoperative recurrence or metastasis risk evaluation according to the expression level of the molecular marker in the sample.
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