CN116814793A - Marker for breast cancer diagnosis and/or prognosis diagnosis and application thereof - Google Patents
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The invention relates to the technical field of medical detection, in particular to a marker for breast cancer diagnosis and/or prognosis diagnosis and application thereof. The present study found that hsa_circ_0003963 was significantly down-regulated in breast cancer tissue compared to paracancerous tissue. Analysis of the working characteristics of the subjects showed that hsa_circ_0003963 can better distinguish breast cancer tissue from other tissues, the area under the curve is 0.7183, the sensitivity is 59.65%, and the specificity is 85%. The low expression of hsa_circ_0003963 is associated with positive expression of breast cancer HER-2, larger tumor diameter, more invasive molecular typing. Thus, hsa_circ_0003963 can be used as a marker for breast cancer diagnosis and/or prognosis.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a marker for breast cancer diagnosis and/or prognosis diagnosis and application thereof.
Background
The disclosure of this background section is only intended to increase the 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 already known to those of ordinary skill in the art.
Breast cancer is the most common malignancy, and in order to treat breast cancer in time, early detection and diagnosis of breast cancer are required. Breast ultrasound and molybdenum targets can find early breast cancer, but have the limitations of missed diagnosis, delayed diagnosis and the like. The serum marker examination commonly used in clinic at present has the defect of low specificity and sensitivity. Therefore, it is desirable to provide a minimally invasive tumor marker with both sensitivity and specificity to improve the diagnosis and treatment efficiency of breast cancer.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a marker for breast cancer diagnosis and/or prognosis diagnosis, which has higher specificity and sensitivity for breast cancer diagnosis and prognosis diagnosis. It is another object of the present invention to provide a product containing the marker and its use.
The invention obtains the annular RNA markers which can realize the differentiation research of the breast cancer tissue and the adjacent tissue of the breast cancer with higher specificity, sensitivity and accuracy through the examination and analysis of a large number of annular RNAs of the cancer tissue and the adjacent tissue of the breast cancer, and can diagnose and prognosis the breast cancer by detecting the expression of the annular RNA markers, thereby providing the invention.
Specifically, the invention provides the following technical scheme:
in a first aspect, the invention provides a marker for breast cancer diagnosis and/or prognosis, the marker being a circular RNA which is hsa_circ_0003963. Hsa_circ_0003963 was significantly down-regulated in breast cancer tissue compared to paracancerous tissue. Subject work characteristic curve (ROC) analysis showed that hsa_circ_0003963 can better distinguish breast cancer tissue from its paracancerous tissue, the area under the curve is 0.7183, the sensitivity is 59.65%, and the specificity is 85%. The low expression of hsa_circ_0003963 is associated with positive expression of breast cancer HER-2, larger tumor diameter, more invasive molecular typing. Based on the above studies, it can be inferred that the circRNA is effective in distinguishing breast cancer lesion tissue from normal tissue.
In another aspect, the use of a marker for breast cancer diagnosis and/or prognosis as described above in the manufacture of a product for diagnosing breast cancer.
The product of the invention can detect the expression content of hsa_circ_0003963 in a patient detection sample, thereby confirming whether the patient is a breast cancer patient or not; or the prognosis of the patient is evaluated by detecting the expression level of hsa_circ_0003963 in a breast cancer patient detection sample.
The detection sample of the diagnostic marker is a clinical biological sample of a patient, including but not limited to one or more of serum (plasma), whole blood, secretion, cotton swab, pus, body fluid, tissue, organ, paraffin section, and the like. In one mode of the invention, the test sample is a focal tissue sample or a suspected focal tissue sample of the patient.
According to the research result, in the focus sample of the breast cancer patient, the expression content of hsa_circ_0003963 is lower than that of the tissue beside the cancer, and the reduction of the expression content is positively correlated with the diameter of the breast cancer tumor and the pathological parameters indicating poor prognosis. Thus, in the above applications of each aspect, detecting a reduced hsa_circ_0003963 content in the sample means that there is a greater likelihood that the patient will have breast cancer, or that the breast cancer will have a poor prognosis.
In some embodiments, the product is a drug or a kit.
The types of the reagents in the product are determined according to the detection method of the circRNA, and the detection method is available in the invention, such as a PCR method or Northern Blotting. In some embodiments, the product is for PCR method detection.
In one or more embodiments, the products for detection by the PCR method include primers, sample tissue lysis reagents, PCR reaction systems.
Specifically, the primers include a circRNA primer and a primer of an internal reference marker.
More specifically, the circRNA primers are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
More specifically, the primers of the internal reference markers are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
In particular, the sample tissue lysing reagent includes, but is not limited to, TRIZOL reagent, isopropanol, ethanol, buffer reagent.
In a third aspect, the use of a reagent for detecting a marker for breast cancer diagnosis and/or prognosis as described above in the preparation of a molecular marker formulation for screening breast cancer.
In a fourth aspect, the use of a marker for breast cancer diagnosis and/or prognosis as described above in the manufacture of a medicament for the treatment of breast cancer.
In a fifth aspect, a method for determining prognosis of a breast cancer patient, the method comprising detecting the level of hsa_circ_0003963 in a focal tissue of the patient.
In the above determination method, if the content of hsa_circ_0003963 in the lesion tissue of the patient is lower than that of the control group, it indicates that the breast cancer of the patient has a greater risk of metastasis or exacerbation. The cutoff value of hsa_circ_0003963 is technical content which can be conventionally determined by a person skilled in the art according to factors such as a kit, an expression level calculation method, a sample and the like.
The beneficial effects of the invention are as follows:
the invention provides application of hsa_circ_0003963 as a marker for diagnosis and prognosis of breast cancer, and compared with a beside-cancer tissue, the marker has good detection specificity and has important clinical application value for diagnosis and prognosis of breast cancer.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a graph showing the results of full transcriptome sequencing of breast cancer tissue and paracancerous tissue in accordance with an embodiment of the present invention;
FIG. 2 is a graph showing the results of qRT-PCR detection on breast cancer tissue and paracancestral tissue in the examples of the present invention;
FIG. 3 is a graph showing the results of verification of hsa_circ_0003963 as a marker for diagnosis and prognosis evaluation of breast cancer in the embodiment of the invention, wherein A is the result of qRT-PCR data comparison of hsa_circ_0003963 in breast cancer tissues and beside cancer tissues, B is the result of ROC curve analysis of hsa_circ_0003963, C is the relationship between the expression level of hsa_circ_0003963 and HER-2, D is the result of correlation analysis of the expression level of hsa_circ_0003963 and tumor diameter, E is the relationship between the expression level of hsa_circ_0003963 and breast cancer of different molecular types, F is the relationship between the expression level of hsa_circ_0003963 and breast cancer of Luminal type, and G is the relationship between the expression level of hsa_circ_0003963 and breast cancer of Luminal type, HER-2 positive type and triple negative breast cancer.
Detailed Description
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Examples
(1) Clinical samples
From 5 months 2017 to 7 months 2021, the cancer and paracancerous tissues of breast cancer patients were collected at the zilut hospital at the university of shandong and fresh tissue samples were immediately stored in liquid nitrogen until further analysis. 3 pairs of cancer and paracancerous tissues are used for full transcription sequencing screening; 11, performing qRT-PCR preliminary verification on cancer and tissues beside the cancer; 140 and 57 cases of cancer and other tissues were used for qRT-PCR large sample validation, all breast cancer and other tissues were obtained during surgery. All participants obtained written informed consent. The study was approved by the ethical committee of the Qilu hospital at Shandong university.
(2) RNA extraction (TRIZOL method)
1) 30-50mg of tissue specimens stored in liquid nitrogen were removed and rapidly transferred to EP tubes, 700. Mu.l of RNA-easy Isolation Reagent reagent was added and the tissue was cut thoroughly with tissue shears until it became paste-like (no visible particles were apparent). 280ml of RNase-free ddH was added to the lysate 2 O, mixing the mixture upside down, standing the mixture at room temperature for 5min, and centrifuging the mixture at 12,000rpm (room temperature) for 15min.
2) The centrifuge tube was removed, at which time the solution was separated into an upper aqueous phase (containing RNA) and a dark lower precipitate (containing impurities such as protein, DNA, polysaccharide, etc.), and the upper aqueous phase was carefully aspirated into a new centrifuge tube.
3) Adding equal volume of isopropanol, mixing, standing for 10min.
4) Centrifugation at 12,000rpm (room temperature) for 10min, a white precipitate was usually seen, and the supernatant was carefully discarded.
5) To the EP tube, 1000 μl of 75% ethanol (RNase-free ddH2O formulation) was added, the bottom of the tube was flicked, the pellet was suspended, and turned upside down several times.
6) Centrifugation at 8,000rpm (room temperature) for 3min, discarding the supernatant and air-drying at room temperature. And (3) injection: it is not overly dry, otherwise the RNA sample is difficult to dissolve.
7) Adding an appropriate amount of RNase-free ddH to the EP tube 2 O dissolves the precipitate, vortex for 3min at room temperature, make RNA precipitate fully dissolve.
8) The concentration and purity of RNA were measured. RNase-free ddH was used first 2 And (3) zeroing the O, taking 1 μl of dissolved RNA, detecting the concentration of the RNA on a micro-spectrophotometer, and recording, wherein the OD260/OD280 ratio is 1.8-2.2, which indicates that the purity is higher. RNA was stored in-80℃refrigerator when not in use.
(3) Whole transcriptome sequencing
rRNAs in total RNA were removed using Ribo-Zero rRNA Removal Kits (Illumina, USA). The RNA was pre-treated using TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA) to construct a sequencing library. Library quality control and quantification was performed using a BioAnalyzer 2100 instrument (Agilent Technologies, usa). According to Illumina sequencing instructions, 10pM library was denatured into single stranded DNA molecules, captured on Illumina flowcell, amplified in situ into clusters (clusters), and 150cycle sequenced using double ended mode (PE mode) on Illumina HiSeq sequencer. rRNAs in total RNA were removed using Ribo-Zero rRNA Removal Kits (Illumina, USA). The RNA was pre-treated using TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA) to construct a sequencing library. Library quality control and quantification was performed using a BioAnalyzer 2100 instrument (Agilent Technologies, usa). According to Illumina sequencing instructions, 10pM library was denatured into single stranded DNA molecules, captured on Illumina flowcell, amplified in situ into clusters (clusters), and 150cycle sequenced using double ended mode (PE mode) on Illumina HiSeq sequencer. Double-ended reads were harvested by sequencing with an Illumina HiSeq4000 sequencer. Quality control was performed using Q30, and cutadapts software (v1.9.3) was used to remove linkers, to remove low quality reads, and to obtain high quality reads. High quality reads were aligned to the reference genome/transcriptome using STAR software (v2.5.1b) and circular RNA detection and identification using DCC software (v0.4.4). Data normalization and differential expression circRNA screening was performed using the edge software (v3.16.5).
(4) Reverse transcription
1) Denaturation of RNA
After 5 minutes at 65℃the RNA was immediately cooled on ice.
2) Preparing a reaction solution
3) Reverse transcription reaction
The reverse transcription reaction was carried out at 37℃for 15 minutes.
↓
The enzyme deactivation was carried out at 98℃for 5 minutes.
↓
After the reaction, the mixture was stored at-20 ℃.
(5) Real-time quantitative polymerase chain reaction (qRT-PCR)
1) The solutions required for the various PCR reactions were melted and mixed and placed on ice.
2) Preparing a PCR reaction system
The solution was mixed at the bottom of the flick tube and centrifuged briefly at 5000 rpm.
3) Sample adding: 9ul of the mixture was added to each well corresponding to the PCR plate. The corresponding 1. Mu.lc DNA was then added. Carefully stick the Sealing Film and briefly mix by centrifugation. The prepared PCR plate was placed on ice before setting up the PCR program.
4) The PCR plate was placed on a realtem PCR apparatus to perform PCR reaction. All indexes are carried out according to the following procedures: 95 ℃ for 30s;40 PCR cycles (95 ℃,5s;55 ℃,15s;72 ℃,15s (fluorescence is collected)).
5) Melting curve analysis, GAPDH as an internal reference normalized the expression of circRNA, and 2 was used -△Ct And (5) calculating by a method.
TABLE 1 primer sequences involved in PCR reactions
(6) Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 (San Diego, CA) and SPSS25.0 (SPSS, chicago, IL, USA). Student t-test and chi-square test were used to analyze the differences between the two groups. Subject work characteristic curve (ROC) analysis was used to determine the diagnostic value of circRNA. P <0.05 is considered statistically significant.
3. Experimental results
3.1 Breast cancer expression of hsa_circ_0003963 was significantly down-regulated compared to its paracancerous tissues
Whole transcriptome sequencing was first performed on breast cancer and paracancerous tissues at 3, and circRNAs were screened for fold differences >2 and P-values < 0.05. Sequencing results showed that circRNA hsa_circ_0003963 was significantly down-regulated in breast cancer tissue compared to paracancerous tissue (fig. 1). Subsequently, qRT-PCR was performed on breast cancer and paracancerous tissues at 11 in this example, showing that hsa_circ_0003963 was significantly down-regulated in breast cancer patient tissues compared to paracancerous tissues (fig. 2, p=0.0026).
Continuing to verify hsa_circ_0003963 expression in 140 breast cancer tissues and 57 paracancerous tissues, hsa_circ_0003963 was found to be significantly down-regulated in breast cancer tissues (FIG. 3A, P < 0.0001).
3.2 diagnostic value of differentially expressed hsa_circ_0003963
The diagnostic properties of differentially expressed circrnas were tested in 140 breast cancer tissues and 57 paracancerous tissues using ROC curve analysis and calculation of the area under the subject's working characteristics curve (AUC). The results showed that the AUC of hsa_circ_0003963 was 0.7183 (fig. 3b, p < 0.0001), indicating that hsa_circ_0003963 can distinguish breast cancer well from paracancerous tissue, with sensitivity and specificity of hsa_circ_0003963 being 59.65% and 85%, respectively.
3.3hsa_circ_0003963 expression was correlated with patient clinical pathology parameters suggesting that it is likely to predict patient prognosis.
Analysis of hsa_circ_0003963 expression levels in 140 breast cancer tissues and 57 paracancerous tissues in a patient with breast cancer showed that hsa_circ_0003963 expression was closely related to various clinical pathological parameters. Expression of hsa_circ_0003963 was up-regulated in breast cancer tissue in patients with negative HER-2 expression compared to patients with positive HER-2 expression (fig. 3c, p=0.0057). In terms of tumor diameter, the results of the correlation analysis showed that the expression level of hsa_circ_0003963 was inversely correlated with tumor diameter (fig. 3D,Spearman correlation r = -0.2142, p=0.0110). Furthermore, hsa_circ_0003963 also showed differential expression in different molecular genotypes of breast cancer (fig. 3e, p=0.0443). Wherein hsa_circ_0003963 was expressed at a higher level in the Luminal type B breast cancer patients than in the HER-2 positive breast cancer patients (fig. 3e, luminal type B vs HER-2 positive, p=0.0074), and hsa_circ_0003963 was expressed at a higher level in the Luminal type breast cancer patients than in the non-Luminal type breast cancer patients (fig. 3f, luminal type vs non-Luminal type, p=0.0090). hsa_circ_0003963 was differentially expressed in the Luminal type, HER-2 positive and triple negative breast cancer patients (FIG. 3G, P=0.0187), wherein hsa_circ_0003963 was expressed in the Luminal type breast cancer patients more than in the HER-2 positive and triple negative breast cancer patients (FIG. 3G, luminal type vs HER-2 positive, P=0.0059). Whereas chi-square test analysis of hsa_circ_0003963 expression levels were independent of lymph node metastasis, estrogen Receptor (ER), progestin Receptor (PR), ki-67 expression levels, tumor stage, histological classification (Table 2).
TABLE 2 correlation of hsa_circ_0003963 expression with clinical pathological characteristics of breast cancer patients
*：P<0.05
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A marker for breast cancer diagnosis and/or prognosis, characterized in that the marker is a circular RNA which is hsa_circ_0003963.
2. Use of a marker for breast cancer diagnosis and/or prognosis as claimed in claim 1 in the manufacture of a product for diagnosing breast cancer.
3. The use according to claim 2, wherein the product is a medicament or a kit.
4. The use according to claim 2, wherein the product is for detection by PCR methods.
5. The method according to claim 4, wherein the products for PCR detection comprise primers, sample tissue lysis reagents, and PCR reaction systems.
6. The use of claim 5, wherein the primers comprise a circRNA primer and a primer for an internal reference marker.
7. The use according to claim 6, wherein the circRNA primer is shown in SEQ ID NO.1 and SEQ ID NO.2, respectively;
or the primers of the internal reference markers are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
8. The use of claim 5, wherein the sample tissue lysing agent includes, but is not limited to, TRIZOL agent, isopropyl alcohol, ethanol, buffer agent.
9. Use of a reagent for detecting the marker for breast cancer diagnosis and/or prognosis according to claim 1 in the preparation of a preparation for screening a molecular marker for breast cancer.
10. Use of a marker for breast cancer diagnosis and/or prognosis as claimed in claim 1 in the manufacture of a medicament for the treatment of breast cancer.
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| CN202310924284.2A CN116814793A (en) | 2023-07-26 | 2023-07-26 | Marker for breast cancer diagnosis and/or prognosis diagnosis and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117604107A (en) * | 2024-01-23 | 2024-02-27 | 杭州华得森生物技术有限公司 | Use of FOXO6 and SNX4 in combination for diagnosing breast cancer |
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2023
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117604107A (en) * | 2024-01-23 | 2024-02-27 | 杭州华得森生物技术有限公司 | Use of FOXO6 and SNX4 in combination for diagnosing breast cancer |
| CN117604107B (en) * | 2024-01-23 | 2024-04-12 | 杭州华得森生物技术有限公司 | Use of FOXO6 and SNX4 in combination for diagnosing breast cancer |
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