CN116479131B - Biomarker for diagnosing breast cancer and application thereof - Google Patents
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Abstract
The invention discloses a biomarker for diagnosing breast cancer and application thereof, belonging to the technical field of biomedicine. The biomarker comprises hsa_circ_0000848; the invention also discloses application of hsa_circ_0000848 in preparation of a product for diagnosing breast cancer. The marker hsa_circ_0000848 for diagnosing breast cancer provided by the invention is expressed in normal human serum in a low level, is expressed in serum of a breast cancer patient in a high level, has good value for diagnosing target breast cancer, is proved by experiments to be possibly involved in the occurrence and development of breast cancer, can be used as a biomarker for early diagnosis of breast cancer and a new therapeutic target, and provides a new direction for clinical early diagnosis and long-term prognosis evaluation of breast cancer.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a biomarker for diagnosing breast cancer and application thereof.
Background
Breast cancer is one of the most common malignant tumors in the world and is also the leading cause of tumor death in women. In the past few decades, despite diagnostic and therapeutic improvements, many breast cancer patients still fail to escape the eventual recurrence and metastasis, and the morbidity and mortality of patients diagnosed with advanced breast cancer remain relatively high. It is therefore important to be able to find a biomarker that can be used to diagnose and judge prognosis early.
The existing common auxiliary detection mode for breast cancer comprises the following steps:
(1) imaging examination: in breast cancer examination, imaging examination is an indispensable part, such as ultrasound, molybdenum targets and MR, is an important item of "two cancer screening", and can be used for helping to judge tumor properties, and define tumor positions, sizes and the like. But can be generally found only after forming a tumor or appearing a bright calcification focus, and the benign and malignant tumor quality cannot be clarified, and the relative hysteresis is provided; wherein the ultrasound examination has high requirements for doctors, the molybdenum target examination causes the patients to feel more uncomfortable, and the MR examination has high requirements for equipment.
(2) Pathology examination: "gold standard" for breast cancer diagnosis. Breast cancer is a heterogeneous tumor, i.e., there is a difference or mutation in cell type between the same lesion or primary metastatic lesions, and different cell types direct different treatment regimens. Therefore, the biopsy mode of single point sampling may miss the mutation at a specific part, and there is a possibility of missing the biopsy. Secondly, the puncture pathological examination belongs to an invasive operation, has large wound and high risk, is limited in repeated sampling, and is difficult to achieve ideal dynamic monitoring.
(3) Blood examination: blood examination is a convenient and quick examination mode with small wounds, molecular markers such as CEA, CA125, CA153, CA199 and the like are commonly used for auxiliary detection of breast cancer at present, but even if specificity and sensitivity of combined detection are still low, economic burden of a patient is increased intangibly, and tumor positioning is also undefined.
Liquid biopsy is a monitoring method that uses human body fluid as a sample to diagnose, predict results or monitor disease progression. Compared with the traditional tissue biopsy, the liquid biopsy has the advantages of non-invasive, real-time and accurate. It has proven useful in the management of many human diseases, including cancer, prenatal genetic disease, heart disease, and the like. In many studies, circular RNAs (circRNAs) are involved in the development and progression of various cancers, such as gastric cancer, breast cancer and other types of tumors. In a preliminary study of the expression profile of circRNAs in 348 primary breast cancer tissue specimens, smid et al found that circcnt 2 was a prognostic biomarker for aromatase inhibitor treatment in patients with advanced breast cancer. However, in the study of the breast cancer field, hsa_circ_0000848 has not been reported in the literature as a serum detection marker.
Disclosure of Invention
The invention aims to provide a biomarker for diagnosing breast cancer and application thereof, so as to solve the problems in the prior art, and the hsa_circ_0000848 biomarker provided by the invention can have great clinical value and practical significance for early diagnosis, long-term prognosis and improvement of life quality of breast cancer, can be used as a biomarker for early diagnosis of breast cancer and a new treatment target, and provides a new direction for clinical early diagnosis and long-term prognosis evaluation of breast cancer.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a biomarker for diagnosing breast cancer, which comprises hsa_circ_0000848, wherein the nucleotide sequence of hsa_circ_0000848 is shown in SEQ ID NO: 1.
The invention also provides an application of the hsa_circ_0000848 in preparing a product for diagnosing breast cancer.
Further, the product comprises a reagent or kit for detecting hsa_circ_0000848 expression level.
Further, the reagent or the kit comprises a primer group for detecting the expression level of hsa_circ_0000848, and the nucleotide sequence of the primer group is shown as SEQ ID NO: 2-3.
Further, the product uses serum as a sample to be detected, and the primer group is used for detecting the hsa_circ_0000848 expression level in the sample to be detected so as to diagnose the breast cancer.
The invention also provides a reagent or a kit for diagnosing breast cancer, which comprises a primer group for detecting the expression level of hsa_circ_0000848, and the nucleotide sequence of the primer group is shown as SEQ ID NO: 2-3.
The invention also provides a reagent for interfering hsa_circ_0000848 expression, which comprises siRNA for interfering hsa_circ_0000848 expression, and the nucleotide sequence of the siRNA is shown as SEQ ID NO: 4.
The invention also provides application of the reagent in preparation of medicines for inhibiting breast cancer development.
The invention also provides a breast cancer inhibitor comprising an agent that inhibits hsa_circ_0000848 expression.
Further, the reagent comprises a nucleotide sequence shown as SEQ ID NO:4, and a siRNA as shown in 4.
The invention discloses the following technical effects:
the marker hsa_circ_0000848 for diagnosing breast cancer provided by the invention is expressed low in serum of normal people and expressed high in serum of breast cancer patients. Clinical diagnostic significance of hsa_circ_0000848 was analyzed using the subject work profile, which showed an area under the subject work profile AUC of 0.877 (95% ci, 0.8256-0.928); the maximum value of about dendrin index is 0.571, the sensitivity is 1, the specificity is 0.571, and the marker has good value for diagnosing breast cancer. Further analysis of the diagnostic markers in early patients can be clearly distinguished from normal persons, and may have early diagnostic ability, compared with the Ki-67 low expression group and PR positive group, the high expression of hsa_circ_0000848 in serum of the Ki-67 high expression group and PR negative group suggests that the tumor has stronger invasiveness and poorer prognosis. The results of CCK8 experiments and scratch experiments indicate that interfering with the expression of hsa_circ_0000848 can inhibit proliferation and migration capacity of breast cancer cells.
In conclusion, the hsa_circ_0000848 biomarker provided by the invention can have great clinical value and practical significance for early diagnosis, long-term prognosis and improvement of life quality of breast cancer, and experiments prove that the target possibly participates in the occurrence and development of breast cancer, can be used as a diagnostic biomarker for early stage breast cancer and a novel treatment target, and provides a novel direction for clinical early diagnosis and long-term prognosis evaluation of breast cancer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a comparison of the relative expression levels of hsa_circ_0000848 in serum from normal and breast cancer patients;
FIG. 2 is a graph of the working characteristics of subjects plotted against the hsa_circ_0000848 content in serum of 71 breast cancer patients and 84 normal persons;
FIG. 3 is a comparison of the relative expression levels of RNA in serum from hsa_circ_0000848 in normal versus early breast cancer patients (stage I, II);
FIG. 4 is a comparison of the RNA relative expression levels of hsa_circ_0000848 in the Ki-67 high (+.30%) and low (< 30%) expression groups;
FIG. 5 is a comparison of the RNA relative expression levels of hsa_circ_0000848 in serum in PR positive and negative groups;
FIG. 6 is a comparison of the relative amounts of hsa_circ_0000848 in MDA-MB-231 breast cancer cells after expression of the interfering hsa_circ_0000848;
FIG. 7 is a graph of CCK-8 experiments observing the effect of interfering hsa_circ_0000848 expression on the proliferative capacity of MDA-MB-231 breast cancer cells;
FIG. 8 is a photograph showing cell migration of a scratch test observed under a 10X mirror;
FIG. 9 is a scratch experiment observing the effect of interfering hsa_circ_0000848 expression on the ability of MDA-MB-231 breast cancer cells to migrate.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 detection of hsa_circ_0000848 levels in serum from peripheral blood of patients with breast cancer and normal persons
Serum was collected from 71 breast cancer patients and 84 normal individuals. Breast cancer inclusion criteria: a patient clinically diagnosed with breast cancer; normal person inclusion criteria: the patients with normal blood circulation, blood lipid, blood sugar and blood coagulation in the physical examination patients. Total RNA in serum was extracted separately, and finally the relative content of hsa_circ_0000848 in serum was detected using qPCR and its diagnostic value was analyzed using the subject working profile.
The nucleotide sequence of hsa_circ_0000848 is shown below (5 '-3'):
GGAGGCTTAATTAACGTTTGTAAATCCCTCTTGAGATCAAAGCCACTGCAGAAGGGCAGAGCACCATTTCACAAAAATGCTTGTGGTTGCCTGTGTGGAGCTGGCTTACCTTTGATCTGAGATGGGCTGACACCCTGAATGTGGTCTCAGCTCTGCTCTGAGATGCCCGGCTTTTCCTTTGAAACACACCTTGCCCCTGGGATTCTGCCAGCCAAAGGTGGCGGGGAGTGGCTGTGGATAAGTCATTATGTTTAATGTGGGGGT(SEQ ID NO：1)。
1. experimental method
(1) Serum RNA extraction
Taking 250 mu L of serum of a breast cancer patient or normal human to 1.5mLRNase-free centrifuge tubes, respectively adding 750 mu L of Trizol reagent, manually shaking and mixing for 15s, standing for 5 min at room temperature, adding 0.2mL of chloroform, covering a cover, manually shaking for 15s, and standing for 3 min at room temperature. The homogenate was found to separate significantly into three layers by centrifugation at 12000rcf for 15 minutes at 4 ℃. A new 1.5mL centrifuge tube was taken, the upper colorless clear solution was carefully transferred to the new centrifuge tube, an equal volume of 500. Mu.L isopropyl alcohol was added, and the mixture was stirred and mixed by hand, and left at room temperature for 10 minutes. Again centrifuged at 12000rcf for 10 min at 4 ℃. The liquid was aspirated, and 1mL of 70% alcohol was added to each tube to wash the precipitate. After centrifugation at 7500rcf for 5 min at 4℃the liquid phase was aspirated, dried at room temperature for 5 min and the pellet was dissolved in 30. Mu. LRNase-free water to give total RNA.
(2) Reverse transcription
1) RNA concentration was determined using an ultra-micro nucleic acid protein determinator (DanoDrop 2000).
2) The reverse transcription was performed strictly according to the instructions of Takara reverse transcription kit using Takara reverse transcription kit.
3) The prepared cDNA sample is stored at-20 deg.C for later use.
(3) qPCR detection
Primer design: the specific primer sequence Forward primer (5 '-3') was designed using PrimerExpress3.0.1 software: TGAGATCAAAGCCACTGCAGAA (SEQ ID NO: 2); reverseprimer (5 '-3'): ACACAGGCAACCACAAGCATT (SEQ ID NO: 3).
The reaction system is shown in Table 1:
TABLE 1
Note that: an internal reference gene is required to be arranged in each sample, and the actin gene is adopted as the internal reference gene;
the experiment is provided with blank control and plate control at the same time, wherein the blank control is that no sample is added in the system, RNase-free water is used for supplementing corresponding content, and the plate control is that a group of system containing the same cDNA is added between two plates of PCR for comparison between the plates; the experimental reaction system configuration was all operated on ice.
The reaction procedure is shown in Table 2:
TABLE 2
Detecting and calculating: and detecting a sample CT value by using the LightCycler480 software, adjusting the background value to be 4-13, calculating the obtained CT value by using a delta CT method, and finally carrying out data analysis and drawing by using SPSSStatics 26 and GraphPadprism9 software.
2. Subject work characteristic analysis
The experimental process comprises the following steps: experimental data were calculated using SPSSStatistics26 and subject work characteristics were plotted, and the area under the curve (AUC) was calculated to assess the diagnostic ability of hsa_circ_0000848 in breast cancer serum. The closer the AUC is to 1, the more diagnostic is considered to be, and the less diagnostic is considered to be equal to 0.5. The sensitivity and specificity of the optimal threshold value are obtained after further calculation of the about log index.
3. Analysis of the relationship between serum hsa_circ_0000848 and clinical and pathological conditions
(1) Detection of hsa_circ_0000848 content in serum of early stage patients (I, II stage breast cancer patients) and normal people
The experimental process comprises the following steps: according to clinical pathology data of breast cancer patients, 47 early patients with a clinical stage of I, II are screened from 71 patients and compared with 84 normal patients, and the data are statistically analyzed by Mann-WhitneyU test.
(2) Detection of hsa_circ_0000848 content in serum of Ki-67 low expression group and Ki-67 high expression group
The experimental process comprises the following steps: according to clinical pathology data of breast cancer patients, 31 Ki-67 ∈ 30% of patients (high expression group) are compared with 40 Ki-67<30% of patients (low expression group), and data are statistically analyzed by Mann-WhitneyU test.
(3) Detection of hsa_circ_0000848 content in serum of PR-positive and PR-negative groups
The experimental process comprises the following steps: according to clinical pathology data of breast cancer patients, 14 PR expression negative and 49 PR expression negative patients are compared, and statistical analysis is carried out on the data by using Mann-WhitneyU test.
4. Experimental results:
(1) qPCR detection of relative serum hsa_circ_0000848 content
To investigate whether hsa_circ_0000848 is differentially expressed in normal human and patient serum. The invention determines the relative content of hsa_circ_0000848 in serum of 71 patients and 84 normal persons. The results show that: hsa_circ_0000848 was expressed low in normal human serum and high in serum from breast cancer patients (FIG. 1).
(2) Analysis of subject working characteristics serum hsa_circ_0000848 value in breast cancer diagnosis
Secondly, in order to explore the diagnostic value of hsa_circ_0000848 as a serum marker of breast cancer, the invention adopts a subject working characteristic curve to analyze the clinical diagnostic significance, and the area under the subject working characteristic curve AUC is 0.877 (95% CI, 0.8256-0.928); the maximum value of about dendrin index is 0.571, the sensitivity is 1, the specificity is 0.571 (figure 2), and the serum marker has good value for diagnosing breast cancer.
(3) Analysis of the relationship between serum hsa_circ_0000848 and clinical and pathological conditions
Further analysis, hsa_circ_0000848 was highly expressed in serum of 35 early patients (I, II stage breast cancer patients) (FIG. 3), and was clearly distinguishable from normal persons, with early diagnostic ability; compared to the Ki-67 low-expression group and the PR-positive group, hsa_circ_0000848 was highly expressed in serum in the Ki-67 high-expression group and the PR-negative group (fig. 4, 5), indicating that high expression of hsa_circ_0000848 may suggest that the tumor is more invasive and worse prognosis.
Example 2 interference of hsa_circ_0000848 expression and exploration of functional Effect in breast cancer
(1) Cell culture
Breast cancer cells (MDA-MB-231) were cultured using DMEM medium containing 10% FBS and 0.5mL of diabody (penicillin + streptomycin) was added. All cells were cultured in 5% CO 2 Is placed in a 37℃cell incubator.
(2) siRNA transfected cells
1) siRNA sequence: si-hsa_circ_0000848 (5 '-3'): AAUGUGGGGGUGGAGGCUUTT (SEQ ID NO: 4); si-NC (5 '-3'): UUCUCCGAACGUGUCACGUTT (SEQ ID NO: 5).
2) 2mL of DMEM medium containing 10% FBS without double antibodies is added after plating in a 6-hole plate, the culture is carried out in an incubator overnight, when the cells are attached and the density is 50%, the medium without double antibodies is sucked out, after the cells are washed 2 times by PBS, 1mL of Opti-MEM medium is added to each hole, and the culture is put into the incubator for standby.
3) Setting an experimental group and a negative control group, and preparing transfection solution according to a proportion in each hole: negative control group: 500 mu LOpti-MEM medium +5 mu LLipofectamine RNAiMAX, experimental group: 500. Mu.LOpti-MEM medium+5. Mu.Lsi-hsa_circ_ 0000848 (20. Mu.M), control: 500. Mu.LOpti-MEM medium+5. Mu.Lsi-NC (20. Mu.M) was placed in each of the two tubes, allowed to stand for 3 minutes, mixed, and incubated at room temperature for 20 minutes.
4) Sucking out the Opti-MEM culture medium in the 6-hole plate, adding 1mL of the mixed solution prepared in the step 3), shaking gently, and then placing into an incubator for culture. And after 6 hours, the culture medium is replaced by a double-antibody-free culture medium to be continuously cultured for 48 hours.
5) The transfection efficiency was determined by RNA extraction, reverse transcription and PCR after denaturing the cells by adding 1 mLTrilzol reagent per well.
(3)CCK8
1) After 24h of cell transfection, cells were collected and resuspended after digestion with pancreatin in 6-well plates, 100 μl of cell suspension (about 2000 cells) was added to 96-well plates per well, 6 duplicate wells and 3 blank wells (medium only, no cells) were set up, and PBS solution filled the edge wells. Placed in an incubator for 24 hours.
2) 10. Mu.LCCK-8 solution was added to each well and incubated in an incubator for 4h.
3) Absorbance values at 450nm were measured for 24h, 48h, 72h, 96h with a microplate reader.
(4) Scratch test (also known as wound healing test)
1) After 6-well plates 5 lines were drawn, with a spacing of at least 0.5cm between the lines.
2) Cells were transfected for 48h and scored with a 100 μl gun head perpendicular to the bottom cross line.
3) After 3 washes with PBS, 2mL of double-antibody-free medium/well was added and placed in an incubator.
4) Photographing and recording under microscope at 0h, 12h and 24h respectively
5) The scratch area was calculated by Photoshop and ImageJ software.
2. Experimental results
Investigation of the influence of hsa_circ_0000848 on the function of breast cancer cells
To further explore the effect of hsa_circ_0000848 on breast cancer cell function, the present invention uses siRNA to knock down the interference to hsa_circ_0000848 and verifies the knock down efficiency by qPCR (fig. 6). The results of CCK8 experiments (FIG. 7) and scratch experiments (FIGS. 8 and 9) show that the knockdown of hsa_circ_0000848 can inhibit proliferation and migration capacity of breast cancer cells, and the hsa_circ_0000848 possibly participates in the occurrence and development of breast cancer and can be used as a new treatment target.
In conclusion, the hsa_circ_0000848 biomarker provided by the invention can have great clinical value and practical significance for early diagnosis, long-term prognosis and improvement of life quality of breast cancer, and experiments prove that the target possibly participates in the occurrence and development of breast cancer, can be used as a diagnostic biomarker for early stage breast cancer and a novel treatment target, and provides a novel direction for clinical early diagnosis and long-term prognosis evaluation of breast cancer.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (5)
1. Use of hsa_circ_0000848 for the preparation of a product for diagnosing breast cancer, wherein the hsa_circ_0000848 has the nucleotide sequence as set forth in SEQ ID NO: 1.
2. The use according to claim 1, wherein the product comprises a reagent or kit for detecting hsa_circ_0000848 expression levels.
3. The use according to claim 2, wherein the reagent or kit comprises a primer set for detecting hsa_circ_0000848 expression level, the nucleotide sequence of which is set forth in SEQ ID NO: 2-3.
4. The use according to claim 1, wherein the product uses serum as a test sample and the primer set according to claim 3 is used to detect hsa_circ_0000848 expression level in the test sample for diagnosing breast cancer.
5. Use of an agent that interferes with hsa_circ_0000848 expression in the manufacture of a medicament for inhibiting the progression of breast cancer, the agent comprising an siRNA that interferes with hsa_circ_0000848 expression, the nucleotide sequence of which is set forth in SEQ ID NO: 4.
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| Circular RNA hsa_circ_0000848 Promotes Trophoblast Cell Migration and Invasion and Inhibits Cell Apoptosis by Sponging hsa-miR-6768-5p;Wang H等;《Frontiers in Cell and Developmental Biology》;第8卷;第3页左栏第4段 * |
| Circular RNA hsa_circ_0000848 Regulates Cardiomyocyte Proliferation and Apoptosis Under Hypoxia via Recruiting ELAVL1 and Stabilizing SMAD7 mRNA;Shuai Cao等;《Anatol J Cardiol》;第26卷(第3期);189–197 * |
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