CN112430665B

CN112430665B - Molecular biomarker for diagnosing and treating triple negative breast cancer and application thereof

Info

Publication number: CN112430665B
Application number: CN202011500830.2A
Authority: CN
Inventors: 杨其峰; 宋肖瑾
Original assignee: Qilu Hospital of Shandong University
Current assignee: Qilu Hospital of Shandong University
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2022-06-24
Anticipated expiration: 2040-12-17
Also published as: CN112430665A

Abstract

The invention provides a molecular biomarker for diagnosing and treating triple negative breast cancer and application thereof, belonging to the technical field of crude drug medicines and molecular biology. The invention discovers for the first time that the expression of circEIF3H in a tumor specimen of a triple-negative breast cancer patient is obviously higher than that of breast cancer tissues of other subtypes, meanwhile, the expression quantity of circEIF3H is related to the prognosis of triple-negative breast cancer, and the malignant growth and invasion capacity of breast cancer cells can be controlled by regulating the expression of circEIF 3H. Therefore, the circEIF3H can be used as a molecular biomarker for diagnosing and treating triple negative breast cancer, namely, can be used as a prognostic index and a potential therapeutic target of triple negative breast cancer. The invention provides a more favorable means for the diagnosis and prognosis evaluation analysis of the triple negative breast cancer, and has important significance for the research and treatment of the triple negative breast cancer, thereby having good practical application value.

Description

Molecular biomarker for diagnosing and treating triple negative breast cancer and application thereof

Technical Field

The invention belongs to the technical field of crude drug medicine and molecular biology, and particularly relates to a molecular biomarker for diagnosing and treating triple negative breast cancer and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Breast cancer is one of the most common malignancies worldwide. The latest epidemiological statistical data in 2015 show that 268,600 new breast cancer cases and 69,500 breast cancer death cases exist in China every year, which are one of the countries with the fastest incidence and death rate of breast cancer in the world, and the incidence age and death age of the countries show a trend of gradual younger development. Triple Negative Breast Cancer (TNBC) is a breast cancer subtype, which refers to breast cancer that is negative for both Estrogen Receptor (ER) and Progestogen Receptor (PR) and has no amplification of human epidermal growth factor receptor 2(HER2), and has the characteristics of high invasiveness, high recurrence and metastasis rate, poor patient prognosis and the like. Therefore, the method for searching the biomarkers of breast cancer metastasis and prognosis prediction has important theoretical guidance and clinical significance for finding new treatment targets, improving the curative effect and improving the prognosis of patients.

circular RNAs (circular RNAs) are non-coding RNA molecules with closed circular structures, and have the characteristics of high expression abundance, evolution conservation, high stability and the like. The circular RNA is formed by reversely cutting and cyclizing exons or introns of host genes and does not have a 5 'end cap and a 3' end tail. At present, domestic and foreign researches show that the expression of the circular RNA can be regulated and controlled at each level of genome level, transcription level, post-transcription level, protein translation and the like, and the circular RNA is closely related to the occurrence and development of various human diseases such as tumors and the like. However, the function and action of circRNA is still not completely understood.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a molecular biomarker for diagnosing and treating triple negative breast cancer and application thereof. The invention discovers for the first time that the expression of circEIF3H in a tumor specimen of a triple-negative breast cancer patient is obviously higher than that of breast cancer tissues of other subtypes, meanwhile, the expression quantity of circEIF3H is related to the prognosis of triple-negative breast cancer, and the malignant growth and invasion capacity of breast cancer cells can be controlled by regulating the expression of circEIF 3H. Therefore, the circEIF3H can be used as a molecular biomarker for diagnosing and treating triple negative breast cancer, namely, can be used as a prognostic index and a potential treatment target of triple negative breast cancer, and the invention is completed.

Specifically, the invention relates to the following technical scheme:

in a first aspect of the invention, there is provided the use of a substance for detecting circEIF3H in the manufacture of any one or more of:

(a1) triple negative breast cancer diagnosis or auxiliary diagnosis products;

(a2) triple negative breast cancer prognosis evaluation or auxiliary prognosis evaluation products.

According to the invention, the research shows that the expression level of circEIF3H is abnormally up-regulated in the expression of triple negative breast cancer, meanwhile, the expression of circEIF3H is obviously related to prognosis, and in the total triple negative breast cancer patients, the high expression of circEIF3H indicates poor prognosis. Therefore, the circEIF3H can be used as a new triple negative breast cancer prognostic marker and provides a basis for the evaluation of the postoperative survival period of patients.

Wherein the circEIF3H is formed by circularization of 3-5 exons of a host gene EIF3H, the chromosome position is chr8:117668094-117671219 (database source is rna. sysu. edu. cn/depeBase /), and the nucleotide sequence is shown as SEQ ID No. 1.

In a second aspect of the invention, there is provided the use of a substance for reducing the expression level of circEIF3H in the manufacture of a product.

The research result of the invention shows that circEIF3H can be combined with IGF2BP2 and HuR to form a compound, so that the stability of downstream cancer-promoting gene mRNA is enhanced, and the proliferation and the metastasis of breast cancer are promoted. Therefore, the inhibition of the expression of circEIF3H can effectively reduce the proliferation and metastatic capacity of breast cancer, thereby achieving the effect of treating the breast cancer, and therefore, the product has the functions of any one or more of the following:

(b1) inhibiting growth of triple negative breast cancer cells;

(b2) inhibiting migration of triple negative breast cancer cells;

(b3) inhibiting growth of triple negative breast cancer;

(b4) inhibiting triple negative breast cancer metastasis;

(b5) treating triple negative breast cancer.

In a third aspect of the invention, there is provided the use of a substance for increasing the expression level of circEIF3H in the manufacture of a product.

The function of the product is any one or more of the following:

(c1) promoting breast cancer cell growth;

(c2) promoting migration of triple negative breast cancer cells;

(c3) promoting growth of triple negative breast cancer;

(c4) promoting triple negative breast cancer metastasis;

(c5) and preparing a triple negative breast cancer animal model.

In a fourth aspect of the invention, there is provided a product comprising as an active ingredient a substance for reducing the expression level of circEIF 3H.

The function of the product is any one or more of the following:

(b1) inhibiting growth of triple negative breast cancer cells;

(b2) inhibiting migration of triple negative breast cancer cells;

(b3) inhibiting growth of triple negative breast cancer;

(b4) inhibiting triple negative breast cancer metastasis;

(b5) treating triple negative breast cancer.

In a fifth aspect of the invention, there is provided a product comprising as an active ingredient a substance for increasing the expression level of circEIF 3H.

The function of the product is any one or more of the following:

(c1) promoting the growth of breast cancer cells;

(c2) promoting migration of triple negative breast cancer cells;

(c3) promoting growth of triple negative breast cancer;

(c4) promoting triple negative breast cancer metastasis;

(c5) and preparing a triple negative breast cancer animal model.

In a sixth aspect of the invention there is provided the use of an agent for reducing the expression level of circEIF3H in any one or more of:

(b1) inhibiting growth of triple negative breast cancer cells;

(b2) inhibiting migration of triple negative breast cancer cells;

(b3) inhibiting the growth of triple negative breast cancer;

(b4) inhibiting triple negative breast cancer metastasis;

(b5) treating triple negative breast cancer.

In a seventh aspect of the invention there is provided the use of a substance for increasing the expression level of circEIF3H in any one or more of:

(c1) promoting breast cancer cell growth;

(c2) promoting migration of triple negative breast cancer cells;

(c3) promoting growth of triple negative breast cancer;

(c4) promoting triple negative breast cancer metastasis;

(c5) and preparing a triple negative breast cancer animal model.

The beneficial technical effects of one or more technical schemes are as follows:

the technical scheme proves that the expression of the circEIF3H in a tumor specimen of a triple-negative breast cancer patient is obviously higher than that of breast cancer tissues of other subtypes, the expression quantity of the circEIF3H is related to the prognosis of triple-negative breast cancer, and the malignant growth and invasion capacity of breast cancer cells can be controlled by regulating the expression of the circEIF3H, so that the circEIF3H can be used as an effective medicament for preventing and/or treating the triple-negative breast cancer.

The technical scheme provides a more favorable means for the diagnosis and prognosis evaluation analysis of the triple negative breast cancer, and has important significance for the research and treatment of the triple negative breast cancer. Meanwhile, an experimental basis is laid for developing high-efficiency drugs related to the treatment of the triple negative breast cancer, and a new visual field is provided, so that the method has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a schematic diagram of the structure of circEIF3H according to the present invention.

FIG. 2 shows the linker sequence of circEIF3H as a result of sequencing in the examples of the present invention.

FIG. 3 shows that the expression level of circEIF3H in the tumor tissue of the triple negative breast cancer is obviously higher than that of other subtypes.

FIG. 4 shows that the prognosis of the patients with triple-negative breast cancer with high circEIF3H expression is worse than that of the patients with triple-negative breast cancer with low circEIF3H expression.

FIG. 5 shows that NC of the invention is the control group cell, and siRNA1 and siRNA2 are two knockdown group cells for knocking down circEIF 3H. The cells in the knockdown group grew more slowly than the cells in the control group.

FIG. 6 shows that NC of the invention is the control group cell, and siRNA1 and siRNA2 are two knockdown group cells for knocking down circEIF 3H. The cells in the knockdown group were less migratory than those in the control group.

FIG. 7 shows that the RNA-pulldown experiment of the present invention confirmed that circEIF3H binds to IGF2BP2 and HuR.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. The experimental procedures, if specific conditions are not indicated in the following detailed description, are generally in accordance with conventional procedures and conditions of molecular biology within the skill of the art, which are fully explained in the literature. See, e.g., Sambrook et al, "molecular cloning: the techniques and conditions described in the laboratory Manual, or according to the manufacturer's recommendations.

The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.

The skilled artisan will appreciate that the term "expression level" refers to the amount of a gene product present in vivo or in a sample at a particular time point. The expression level can be measured/quantified/detected, for example, by protein or mRNA expressed by the gene. The expression level can be quantified, for example, as follows: normalizing the amount of the gene product of interest present in a sample with the total amount of the gene product of the same type (total protein or mRNA) in the same sample or a reference sample (e.g., a sample obtained from the same individual at the same time or a fraction of the same size (weight, volume) of the same sample), or determining the amount of the gene product of interest/defined sample size (weight, volume, etc.). The expression level can be measured or detected by any method known in the art, such as a method for direct detection and quantification of a gene product of interest (e.g., mass spectrometry), or a method for indirect detection and measurement of a gene product of interest that generally works by binding the gene product of interest to one or more different molecules or detection devices (e.g., primers, probes, antibodies, protein scaffolds) specific for the gene product of interest. Also known to the skilled person is the determination of the level of gene copy, which also includes the determination of the absence or presence of one or more fragments (e.g. by nucleic acid probes or primers, such as quantitative PCR, Multiplex ligation-dependent probe amplification (MLPA) PCR).

The terms "indicator" and "marker" are used interchangeably herein and refer to a sign or signal of a condition or to monitor a condition. Such "disorder" refers to a biological state of a cell, tissue or organ, or to a health and/or disease state of an individual. The indicator may be the presence or absence of molecules including, but not limited to, peptides, proteins, and nucleic acids, or may be a change in the level or pattern of expression of such molecules in a cell, or tissue, organ, or individual. The indicator can be a sign of the occurrence, development or presence of a disease in an individual or of further progression of such a disease. The indicator may also be a sign of the risk of developing a disease in the individual.

The term "up-regulation", "increase" or "increase" of the level of an indicator means that the level of such indicator is reduced in a sample compared to a reference or reference sample.

The term "down-regulation", "reduction" or "decrease" of the level of an indicator refers to a reduction of the level of such indicator in a sample compared to a reference or reference sample.

As mentioned above, triple negative breast cancer is a breast cancer subtype, which refers to breast cancer that is negative for both Estrogen Receptor (ER) and Progesterone Receptor (PR) and has no amplification of human epidermal growth factor receptor 2(HER2), and has the characteristics of high invasiveness, high recurrence and metastasis rate, poor patient prognosis, and the like. Therefore, the method for searching the biomarkers of breast cancer metastasis and prognosis prediction has important theoretical guidance and clinical significance for finding new treatment targets, improving the curative effect and improving the prognosis of patients.

In view of the above, in one embodiment of the present invention, there is provided the use of a substance for detecting circEIF3H in the preparation of any one or more of the following:

(a1) triple negative breast cancer diagnosis or auxiliary diagnosis products;

Wherein the circEIF3H is formed by circularization of 3-5 exons of a host gene EIF3H, the chromosome position is chr8:117668094-117671219 (database source rna. sysu. edu. cn/depeBase /), and the nucleotide sequence is shown as SEQ ID No. 1.

Thus, in the use (a2), the prognostic assessment of triple negative breast cancer comprises a prediction of overall survival of the patient after triple negative breast cancer surgery.

In yet another embodiment of the invention, the means for detecting circEIF3H comprises amplification primers for circEIF3H, said amplification primers comprising circEIF3H-F and circEIF 3H-R.

In still another embodiment of the present invention, the circEIF3H-F is any one of the following single-stranded DNAs F1) to F4):

f1) single-stranded DNA shown in a sequence 2 in a sequence table;

f2) single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or 3' -end of f 1);

f3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined by f1) or f 2);

f4) a single-stranded DNA which hybridizes with the single-stranded DNA defined by f1) or f2) under stringent conditions;

in still another embodiment of the present invention, the circEIF3H-R is any one of the following single-stranded DNAs of R1) to R4):

r1) single-stranded DNA shown in sequence 3 in the sequence table;

r2) single-stranded DNA obtained by adding one or more nucleotides to the 5 '-end and/or the 3' -end of r 1);

r3) has 85% or more identity with the single-stranded DNA defined by r1) or r 2);

r4) under stringent conditions with the single-stranded DNA defined by r1) or r 2).

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, or SEQ ID No.4 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In another embodiment of the present invention, the identity of 85% or more may be 85%, 90% or 95% or more.

In one embodiment of the invention, the product comprises a test kit.

In a particular embodiment of the invention there is provided the use of a substance for reducing the expression level of circEIF3H in the manufacture of a product.

(b1) inhibiting growth of triple negative breast cancer cells;

(b2) inhibiting migration of triple negative breast cancer cells;

(b3) inhibiting growth of triple negative breast cancer;

(b4) inhibiting triple negative breast cancer metastasis;

(b5) treating triple negative breast cancer.

Wherein the substance for reducing the expression level of circEIF3H comprises an RNA interference molecule or antisense oligonucleotide against circEIF3H, a small molecule inhibitor, siRNA, and a substance for performing lentiviral infection or gene knockout.

The product may be a medicament.

In one embodiment of the invention, there is provided the use of a substance for increasing the expression level of circEIF3H in the manufacture of a product.

The function of the product is any one or more of the following:

(c1) promoting the proliferation of triple negative breast cancer cells;

(c2) promoting the malignant invasion of triple negative breast cancer cells;

(c3) promoting growth of triple negative breast cancer;

(c4) promoting triple negative breast cancer metastasis;

(c5) and preparing a triple negative breast cancer animal model.

In a specific embodiment of the invention, the substance for improving the expression level of the circEIF3H comprises a substance for up-regulating the expression of the circEIF3H and/or promoting the activity of the circEIF3H based on gene-specific Mimics technology; such as short hairpin RNA (shRNA) artificially synthesized as circEIF3H or a promoter or lentivirus that up-regulates the expression of circEIF 3H; compound accelerators are also included.

The product may be a medicament.

In one embodiment of the invention, there is provided a product whose active ingredient comprises a substance for reducing the expression level of circEIF 3H.

The function of the product is any one or more of the following:

(b1) inhibiting growth of triple negative breast cancer cells;

(b2) inhibiting migration of triple negative breast cancer cells;

(b3) inhibiting growth of triple negative breast cancer;

(b4) inhibiting triple negative breast cancer metastasis;

(b5) treating triple negative breast cancer.

The product may be a medicament.

In one embodiment of the invention, there is provided a product whose active ingredient comprises a substance for increasing the expression level of circEIF 3H.

The function of the product is any one or more of the following:

(c1) promoting the growth of breast cancer cells;

(c2) promoting migration of triple negative breast cancer cells;

(c3) promoting growth of triple negative breast cancer;

(c4) promoting triple negative breast cancer metastasis;

(c5) and preparing a triple negative breast cancer animal model.

The product may be a medicament.

In a particular embodiment of the invention there is provided the use of an agent for reducing the expression level of circEIF3H in any one or more of:

(b1) inhibiting growth of triple negative breast cancer cells;

(b2) inhibiting migration of triple negative breast cancer cells;

(b3) inhibiting growth of triple negative breast cancer;

(b4) inhibiting triple negative breast cancer metastasis;

(b5) treating triple negative breast cancer.

In one embodiment of the invention, there is provided the use of a substance for increasing the expression level of circEIF3H in any one or more of:

(c1) promoting breast cancer cell growth;

(c2) promoting migration of triple negative breast cancer cells;

(c3) promoting growth of triple negative breast cancer;

(c4) promoting triple negative breast cancer metastasis;

(c5) and preparing a triple negative breast cancer animal model.

According to the present invention, the concept of "treatment" means any measure suitable for the treatment of a disease associated with triple negative breast cancer, or the prophylactic treatment of such manifested disease or manifested symptoms, or the avoidance of recurrence of such disease, e.g. recurrence after the end of a treatment period or treatment of symptoms of an already established disease, or the prevention or suppression or reduction of the occurrence of such disease or symptoms with prior intervention.

According to the invention, the above-mentioned medicament also comprises at least one pharmaceutically inactive ingredient.

The pharmaceutically inactive ingredients may be carriers, excipients, diluents and the like which are generally used in pharmacy. Further, the composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

Such pharmaceutically inactive ingredients, which may include carriers, excipients and diluents, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

In still another embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In yet another embodiment of the present invention, the medicament of the present invention may be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In still another embodiment of the present invention, the subject to which the medicament is administered may be a human or non-human mammal, such as a mouse, rat, guinea pig, rabbit, dog, monkey, orangutan, or the like.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1

1. Sample collection

Tumor tissues of patients undergoing breast cancer surgery during the period of 2015-2019 at the eastern university Qilu hospital and 100 pairs of normal control tissues were collected and signed for written informed consent on all patients participating in the study.

2. circRNA quantitative PCR experiment:

2.1 extraction of tissue Total RNA

Total RNA is extracted from the tissues of breast cancer patients by using Trizol reagent, and the concentration and purity of the RNA are detected by using corresponding equipment.

2.2 reverse transcription of Total RNA

Reverse transcription was performed using a reverse transcription kit from Takara, starting with 1. mu.l of RNA, and reverse transcription of RNA was performed according to the provided instructions.

The system is shown in the following table:

setting of specific reaction programs in the PCR instrument:

2.3circRNA quantitative PCR

The expression level of the circRNA in the tumor tissue and the paracancer control tissue was detected by a real-time quantitative PCR instrument (Roche LightCycler 480).

The specific reaction system is as follows:

setting of specific reaction programs in the PCR instrument:

2.4 quantitative PCR primers

The circRNA provided in this example is differentially expressed circRNA selected from triple negative subtype breast cancer tissue and paracancer control tissue by bioinformatics method, the structural pattern diagram of the circRNA is shown in fig. 1, the linear sequence thereof is shown in SEQ ID No.1, and β -actin is used as reference gene for its quantification. The primer sequences are shown in the following table:

example 2

Meanwhile, according to the invention, by carrying out statistics on the expression quantity of the circEIF3H of the sampled triple-negative breast cancer patient and carrying out comprehensive statistical analysis by combining the treatment effect, as shown in figure 4, the prognosis of the triple-negative breast cancer patient with high-expression circEIF3H is found to be worse than that of the triple-negative breast cancer patient with low-expression circEIF 3H.

Example 3

Influence on growth rate and migration ability of cancer cells.

Effect of circEIF3H on growth Rate of Breast cancer cells

1.1 transfection of cells

Take 6cm petri dish as an example:

(1) appropriate cell culture dishes were selected according to experimental requirements, cells were counted the day before transfection and plated as required for transfection.

(2) The cell culture medium is replaced with an appropriate amount of antibiotic-free medium before transfection, e.g. 2ml of medium is added to a 6cm dish.

(3) The EP tubes were removed and labeled with the names of lipo or transfected siRNA, respectively.

siRNA sequence:

si-circEIF3H-1：CAGCAGUCCAAUAUCAGAU(SEQ ID No.6)；

si-circEIF3H-2：CAGCCUUGCCAGCAGUCCA(SEQ ID No.7)。

(4) mu.l of opti-MEM was dissolved in 10. mu.l of liposome and 10. mu.l of 20mM siRNA in a 6cm dish according to the ratio recommended by Lipofectamine2000 instructions, and the mixture was gently mixed and allowed to stand at room temperature for 5 min.

(5) Mixing the liposome solution and siRNA solution, and standing at room temperature for 20 min.

(6) The final transfection mixture was then added dropwise to the cell culture medium uniformly.

(7) Placing the culture dish into an incubator, culturing for 6-8 hours, and then changing to a normal culture medium.

(8) Functional cell experiments can be performed 24-48 hours after transfection.

1.2 MTT assay

(1) And (3) carrying out conventional digestion and centrifugation on transfected cells, suspending a proper amount of culture medium, counting the cells, adding the cells into a 96-well plate, adding 1500 cells per 100 mu l of each hole according to experiment requirements, cell characteristics and the like, and setting 3-5 multiple holes.

(2) The 96-well plates were placed in a cell incubator and cultured for 1 to 5 days, one 96-well plate was removed every day at a fixed time, 20. mu.l of MTT (5mg/ml) was added to each well, and then the plate was placed in the cell incubator again and cultured, taking care to keep out light.

(3) After 4-6 hours, the 96-well plate is taken out, the liquid in the hole is slowly sucked out, and a 1ml empty needle can be used for replacing a gun head to avoid sucking purple precipitates as much as possible. Then, 10. mu.l DMSO was added to the wells to dissolve the precipitate, the mixture was shaken at room temperature for 10min, and the absorbance in the wells was measured with a microplate reader.

(4) Plotting cell proliferation curves: the relative absorbance values measured by the machine and calculated relative to the values of the first day are plotted on the ordinate and the time (days or hours) on the abscissa, and the results of each day are statistically analyzed.

1.3 the results show that: in contrast to tumor cells normally expressing EIF3H, the assay showed that two knockdown cells with circEIF3H knocked down grew more slowly than the control cells. As shown in fig. 5.

Effect of circEIF3H on the migratory Capacity of Breast cancer cells

2.1 transfection of cells

2.2 Transwell cell migration assay

(1) And (4) performing conventional digestion and centrifugation on the treated cells, adding a proper amount of FBS-free culture medium for resuspension, and counting the cells.

(2) Add 700. mu.l of medium containing 20% FBS to the lower chamber (24-well plate), then place the chamber in the lower chamber and take care to ensure that there are no air bubbles between the lower part of the chamber membrane and the medium, then add 200. mu.l of cell suspension to the upper chamber, containing 1 x 10⁵And (4) cells. The position of the cell was adjusted so that the cell was located in the center of the 24 wells. The 24-well plate was placed in a cell incubator for further culture.

(3) Depending on the migration ability of the cells, the cells are cultured for a specific period of time, generally 24 hours.

(4) The transwell chamber was removed, washed 3 times with PBS, and the cells on the top of the chamber membrane were gently wiped off with a cotton swab.

(5) The transwell cell was placed in methanol for 15min for immobilization.

(6) The methanol remaining on the upper part of the cell membrane and the side wall of the cell was gently wiped off with a cotton swab.

(7) The transwell cell was placed in 0.2% crystal violet and stained for 20 min.

(8) And cleaning the small chamber with clear water, wiping water on the inner side of the small chamber with a cotton swab, and drying.

(9) And (3) placing the cell under a microscope for observation, adjusting light intensity and exposure time, taking a picture under a 10-time lens, randomly selecting 5 visual fields for counting, and mapping and performing statistical analysis.

2.3 the results show: compared with tumor cells normally expressing EIF3H, two knockdown cells with circEIF3H knocked down were less migratory than the control cells. As shown in fig. 6.

Based on the above experimental results we have reason to believe that circEIF3H may be a novel target for the treatment of triple negative breast cancer patients.

Example 4

The embodiment simultaneously designs a detection kit for prognosis prediction of triple negative breast cancer, and a detection system of the kit comprises a reverse transcription reaction system, a qPCR reaction system and conventional reagents for configuring the reverse transcription system and the qPCR reaction system.

1. The tissue diagnosis kit for triple negative breast cancer consists of the following components (50 reactions):

100ml of isopropanol, 100ml of trichloromethane, 50ml of Trizol, 10ml of DEPC water or non-enzyme water, 10ml of double distilled water, 1ml of 5 Xreverse transcription buffer, 1ml of dNTP mixed solution, 500. mu.l of RNase protein inhibitor, 50. mu.l of reverse transcriptase solution, 2 XSYBR Premix 2ml,

10 μ M circEIF3H specific PCR primers 200 μ l,

the forward primer is 5'-GTCCTAATGTGGGAACTT-3' (SEQ ID No.2)

The reverse primer is 5'-ATGCTGGTAACTAAACTGA-3' (SEQ ID No.3)

10 mu M beta-actin specific PCR primer 200 mu l,

the forward primer is 5'-CACCATTGGCAATGAGCGGTTC-3' (SEQ ID No.4)

The reverse primer is 5'-AGGTCTTTGCGGATGTCCACGT-3' (SEQ ID No.5)

2. Extraction of tissue total RNA

2.1 reverse transcription of Total RNA, the system is shown in the following table:

setting of specific reaction programs in the PCR instrument:

2.2circRNA quantitative PCR

The specific reaction system is as follows:

setting of specific reaction programs in the PCR instrument:

2.3 determination of DCT index: DCT refers to the difference between the average Ct values of circEIF3H to be detected and the beta-actin in the same sample. Namely, circEIF3H DCT is determined by obtaining a relative quantitative Δ CT value as the difference between the Mean CT value of circEIF3H and β -actin in this example, circEIF3H Mean CT-control Mean CT.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Qilu Hospital of Shandong university

<120> molecular biomarker for diagnosing and treating triple negative breast cancer and application thereof

<130>

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 418

<212> DNA

<213> circEIF3H

<400> 1

tccaatatca gatggaaatg atgcggagcc ttcgccatgt aaacattgat catcttcacg 60

tgggctggta tcagtccaca tactatggct cattcgttac ccgggcactc ctggactctc 120

agtttagtta ccagcatgcc attgaagaat ctgtcgttct catttatgat cccataaaaa 180

ctgcccaagg atctctctca ctaaaggcat acagactgac tcctaaactg atggaagttt 240

gtaaagaaaa ggatttttcc cctgaagcat tgaaaaaagc aaatatcacc tttgagtaca 300

tgtttgaaga agtgccgatt gtaattaaaa attcacatct gatcaatgtc ctaatgtggg 360

aacttgaaaa gaagtcagct gttgcagata aacatgaatt gctcagcctt gccagcag 418

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence

<400> 2

gtcctaatgt gggaactt 18

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence

<400> 3

atgctggtaa ctaaactga 19

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence

<400> 4

caccattggc aatgagcggt tc 22

<210> 5

<211> 22

<212> DNA

<213> Artificial sequence

<400> 5

aggtctttgc ggatgtccac gt 22

<210> 6

<211> 19

<212> RNA

<213> si-circEIF3H-1

<400> 6

cagcagucca auaucagau 19

<210> 7

<211> 19

<212> RNA

<213> si-circEIF3H-2

<400> 7

cagccuugcc agcagucca 19

Claims

1. Use of a substance for detecting circEIF3H in the preparation of any one or more of:

(a1) diagnosing triple negative breast cancer;

(a2) triple negative breast cancer prognosis evaluation;

wherein the nucleotide sequence of the circEIF3H is shown as SEQ ID No. 1.

2. Use of the substance for detecting circEIF3H according to claim 1 in the preparation of any one or more of the following products, wherein the diagnosis of triple negative breast cancer further comprises an auxiliary diagnostic product; the negative breast cancer prognosis evaluation also comprises an auxiliary prognosis evaluation product.

3. The use of claim 1, wherein in said use (a2), the prognostic assessment of triple negative breast cancer comprises prediction of the overall post-operative survival of the patient with triple negative breast cancer.

4. Use according to claim 1 or 2, wherein the means for detecting circEIF3H comprises amplification primers for circEIF3H, said amplification primers comprising circEIF3H-F and circEIF 3H-R;

the circEIF3H-F is a single-stranded DNA shown in a sequence 2 in a sequence table;

the circEIF3H-R is single-stranded DNA shown in a sequence 3 in a sequence table.

5. The use of claim 1 or 2, wherein the product comprises a test kit.

6. Use of a substance for reducing the expression level of circEIF3H in the manufacture of a product; the function of the product is any one or more of the following:

(b1) inhibiting growth of triple negative breast cancer cells;

(b2) inhibiting metastasis of triple negative breast cancer cells.

7. The application of a substance for improving the expression level of circEIF3H in preparing products; the function of the product is any one or more of the following:

(c1) promoting the proliferation of triple negative breast cancer cells;

(c2) promoting the malignant invasion of triple negative breast cancer cells;

(c3) promoting the growth of triple negative breast cancer cells;

(c4) promoting metastasis of triple negative breast cancer cells;

(c5) and preparing a triple negative breast cancer animal model.

8. Application of a substance for improving the expression level of circEIF3H in preparing a triple negative breast cancer animal model.

9. The use of claim 4 or 6, wherein the product is a medicament.