CN114921558A

CN114921558A - Application of hsa _ circ _0044235 level in serum as breast cancer diagnosis marker

Info

Publication number: CN114921558A
Application number: CN202210739324.1A
Authority: CN
Inventors: 洪宏海; 李巧玉; 尹萍; 张宗桃; 陈妍; 杨瑾仪; 唐毅
Original assignee: Third Affiliated Hospital of Guangzhou Medical University
Current assignee: Third Affiliated Hospital of Guangzhou Medical University
Priority date: 2022-06-28
Filing date: 2022-06-28
Publication date: 2022-08-19
Anticipated expiration: 2042-06-28
Also published as: CN114921558B

Abstract

The invention discloses application of hsa _ circ _0044235 level in serum as a breast cancer diagnosis marker, and relates to the technical field of biomedicine. The invention claims the application of a reagent for detecting the expression of hsa _ circ _0044235 in preparing a product for diagnosing breast cancer. The present invention also claims a diagnostic kit for breast cancer comprising a reagent for detecting expression of hsa _ circ _ 0044235. The breast cancer diagnostic marker provided by the invention is high in expression in the serum of normal people and low in expression in the serum of breast cancer patients; ROC curve analysis is adopted to prove that the marker has good value for breast cancer diagnosis.

Description

Application of hsa _ circ _0044235 level in serum as breast cancer diagnosis marker

Technical Field

The invention relates to the technical field of biomedicine, in particular to application of a hsa _ circ _0044235 level in serum as a breast cancer diagnosis marker.

Background

The breast cancer is a common tumor, and early discovery and early diagnosis are crucial to reducing the breast cancer death rate and improving the survival rate of patients. In recent years, although the diagnostic techniques for breast cancer have been improved, the early diagnosis of breast cancer still faces a great challenge because of the lack of specific biomarkers and the fact that the diagnosis is often at a middle-late stage.

The existing breast cancer auxiliary detection has the defects that:

firstly, imaging examination: for example, in X-ray examination and B-ultrasonic examination of molybdenum targets, calcific foci are found in the tumor position and size. But the benign and malignant nature of the tumor cannot be determined, the real-time monitoring cannot be realized, and the relative hysteresis is realized; the requirements on doctors and equipment are high.

Pathology examination: the pathological nature 'gold standard' is clear, but there are studies showing: there is "heterogeneity" in tumors, which may miss mutations in specific locations in biopsy procedures that rely on single-site sampling, and also between metastases, even if there are differences between metastases from different locations in the same tumor, with the potential for missed detection. Secondly, pathological examination has strong pain, large wound and poor patient compliance, and dynamic detection of diseases cannot be achieved.

③ the existing protein molecule detection: molecular markers such as CEA, CA125 and CA153 which are commonly used in clinic at present are used for auxiliary detection of breast cancer, but the specificity is poor, the sensitivity is low, joint detection is often needed, and the economic burden of patients is increased invisibly. For example: CA153 lacks sufficient sensitivity and specificity, and has insignificant increase in serum of early breast cancer patients, and is mainly used for prognosis evaluation, postoperative condition monitoring, efficacy evaluation and the like.

Compared with the existing nucleic acid detection technology: the technology used clinically for nucleic acid detection relies primarily on qRT-PCR technology. Although the detection sensitivity is high, the method still has certain limitation when detecting nucleic acid with low copy content or single gene mutation and relies on standard curve quantification.

Digital PCR (digital polymerase chain reaction, ddPCR) is a detection mode with high sensitivity and strong specificity. Based on the principles of poisson distribution and extreme dilution of the sample, the sample is extremely diluted to the single molecule level in a water-in-oil format, i.e. containing 1 or 0 nucleic acid molecule per droplet. The microdroplet form ensures that each nucleic acid molecule is amplified independently and mutually independent. Thermal cycling is then performed, producing an amplification effect. The fluorescence signal only exists in the liquid drop containing the target molecule, and the absolute quantitative detection of the target molecule is finally achieved through the rapid microfluidic analysis of the fluorescence signal of each droplet.

It has been shown that when the target gene is less than 10 ³ At one copy, the real-time fluorescent quantitative pcr (qpcr) detection performance is significantly reduced. But ddPCR is different, the absolute quantification of a target gene can be achieved by amplifying in a microdroplet mode, and compared with qPCR detection, the method is more sensitive and higher in accuracy, is more suitable for low-concentration and low-copy nucleic acid detection, and does not depend on a standard curve.

In recent years, the research of detection based on 'liquid biopsy' is becoming more popular, wherein the detection of circulating tumor cells, non-coding nucleotides and the like plays a great role in early detection of diseases, disease monitoring, evaluation of curative effect and the like. For example: since professor Rajewsky and teams thereof in 2013, 2 months, the circRNACiRS-7(CDR1as) with milestone significance can be used as a molecular sponge of micro RNA-7(miR-7), the circRNA becomes a new hotspot for tumor mechanism research and is also a novel marker for serological detection, and by consulting recent literature, research shows that hsa _ circ _0001017 and hsa _ circ _0061276 can be early gastric cancer prediction factors; however, no report has been made on hsa _ circ _0044235 as a serum detection marker in the field of breast cancer.

Disclosure of Invention

The invention aims to provide application of hsa _ circ _0044235 level in serum as a breast cancer diagnosis marker to solve the problems in the prior art, and the breast cancer diagnosis marker provided by the invention has good value for breast cancer diagnosis.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides application of a reagent for detecting expression of hsa _ circ _0044235 in preparation of a product for diagnosing breast cancer.

Further, the product is a detection reagent or a kit.

Further, the method for detecting the expression of hsa _ circ _0044235 specifically comprises the following steps:

(1) extracting total RNA in a human serum sample, and carrying out reverse transcription to obtain cDNA;

(2) the cDNA is taken as a template, a primer pair shown as SEQ ID NO.1-2 and a probe shown as SEQ ID NO.3 are adopted for ddPCR detection.

Further, the reaction system of the ddPCR detection is as follows: 10. mu.L of 2X ddPCR Supermix for probes, 1. mu.L of 20X probe-primer mixture, 8. mu.L of RNase-free water, and 1. mu.L of template cDNA; the preparation system of the 20X probe-primer mixture is as follows: mu.L of 50. mu.M forward primer 36. mu.L, 50. mu.M reverse primer 36. mu.L, 5. mu.L of 100. mu.M probe, 23. mu.L of RNase-free water.

Further, the temperature of the ddPCR detection extension stage is 52.9 ℃.

The invention also provides a primer group for diagnosing breast cancer, which comprises an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO. 2.

The invention also provides a diagnostic kit for breast cancer, which comprises a reagent for detecting the expression of hsa _ circ _ 0044235.

Further, the diagnostic kit comprises the primer set described above.

Further, the diagnostic kit also comprises a probe shown as SEQ ID NO. 3.

The invention discloses the following technical effects:

the marker hsa _ circ _0044235 for diagnosing breast cancer provided by the invention is high in expression in normal human serum and low in expression in serum of a breast cancer patient. The results of the analysis of the clinical diagnostic significance of hsa _ circ _0044235 using the ROC curve indicate that the AUC of the area under the ROC curve is 0.85 (95% CI, 0.798-0.903); sensitivity of 0.913 and specificity of 0.66; the critical value (cut-off value) of 296.5 copies/mu L indicates that the marker has good value for breast cancer diagnosis.

According to the invention, ddPCR is adopted to detect serum hsa _ circ _0044235, and the method has high sensitivity; absolute quantification, independent of standard curve; the influence of nonspecific amplification is small; the sample consumption is less; easily obtained materials, small wound, high patient compliance and the like. In addition, hsa _ circ _0044235 has stable structure; the detection is easy; the specificity is good. The invention combines the advantages of the two methods, and the detection method has good detection value.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings 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 it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a diagram showing the construction of the ddPCR detection method for serum hsa _ circ _0044235, and finding the result of the optimal extension temperature;

FIG. 2 is a diagram showing the result of searching for the optimal RNA concentration of a sample by constructing a ddPCR method for detecting serum hsa _ circ _ 0044235;

FIG. 3 shows the copy number of hsa _ circ _0044235 at different RNA concentrations in samples;

FIG. 4 shows that the content of hsa _ circ _0044235 in serum is detected by ddPCR under the conditions of 52.9 ℃ and the RNA concentration of a sample of 2.5 ng/. mu.L;

FIG. 5 shows the copy number of hsa _ circ _0044235 in the sera of normal and breast cancer patients;

FIG. 6 is a ROC curve plotted against the serum content of hsa _ circ _0044235 in 106 breast cancer patients and 92 normal persons.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description 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 the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated or intervening value in a stated range, and every other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

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 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 herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. 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 present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. It is intended that the specification and examples be considered as exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.

Example 1 measurement of the level of hsa _ circ _0044235 in peripheral blood serum of patients with breast cancer and normal persons

Serum was collected from 106 breast cancer patients and 92 normal persons. And (3) respectively extracting total RNA in the serum, finally detecting the absolute content of hsa _ circ _0044235 in the serum by using a ddPCR probe method, and analyzing the diagnostic value of the serum by using an ROC curve.

hsa _ circ _0044235 (from circBase database):

genomic sequence

>hsa_circ_0044235|NM_001114091|CDC27

GCTGCTATATGGCAAGCACTAAACCACTATGCTTACCGAGATGCGGTTTTCCTCGCAGAACGCCTTTATGCAGAAGGTTTGAAATCTATTCATTGCTGTAGAATGATAAATGAATAACATATATGAAATTCCATAACTTTTTATCACTGTAACAAGATTCTAGCTATCATCATCTTGTTGTTTTTCTGATTGACACTAGTTCATAAAATCTTTACTTTTTTTCTGTGTAGTTCCCTTACATACTACTTTTAATAACTATGTAGAATTGATTCAGGTTTATATGCTATGATTTACTTAATCATTTTTCAATCTTTAGGTTTTTTTTTCCCCCAGGGTTTTTGTTGTTTTGGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAAACAGGGTCTCACTCTGTCACCCATGTTGGAGTACAGTGGGACCATCTCAGCTCACTGCAACCTCTACCTCCCAGGCTCAAGCGATCCTCCCACATCAGCCTCCCAAGTAGCTGGGACCGCAGGCACGGACCACCACGCCTGGCTAATTTTTTTTTTTTTTTTGGTAGAGATAGGGTTTTGCTATGTTGCCCAGGCTGGTCTCAAATTCCTGAGCTTAGGTGATCCACCTGCCTTGGCCTCCCAAAGTGCTGGGATTATACTCATGGGCCACCATGCCATGTATTTTTTTTTTTTTTTTTTTTTTTTAAGAAAAGAGTCTGTATTCTATGGTGAAGAACATGGATTTAGACTCTATAGCCTTGGAACAAATCCTAACCTGTCACTTAAATGTACAGTTTTATAACCTTGGACGAGTTACTTTAATATCTGAAAATTCAGATATTTCATCTATAAAATAATCATTGCCCCTTAGAATTAAAAATTCAATGAGATGGTTTATTTAAAGCCCTTAGCACAGCCATCTGGCTTGGTAATCAATGCTTAATAAACACCACTGATAATGTATGTTTTGTTTCTGTCATTATTATAATTCCCTTTCTAAGATGCTGATATAGATCATTGTGTACAGCTTTTTTTTTTTTTTGAGTCATTTCCTTGGGTTATGTACTTTTAAATGGAATTTACAAGTCAAACAGTATGGGTTTAGTAGCTGAGAGTTGTGAGAACAAGGCCTAAACCTCAGGGGCAGGAAATTTCACAAAAGGACAAAGAGCAATGCTTGGAAGTAGTTTTGAGAAGTTAAGTGGAAACCAGCCTTACCTTGACTCTGAGATAGTGGGATGTAAGGTGATTACTGTCCATGTGAGAGAGCTGTCTCCTCGGTGGAGAATGGGAATTCAGTGAGAAAAGTGAGGAAGATCTGGGCAGCAGGAGTTTAAGGTCCCAATGAAAAGATTAGAAAGGTGAGAAGGAATGGAAGGCTTAGAAAGAAGATTATACAATAGATTAATCTACTCCATTCACTTATGCAAAAGTGATGTTTATTGTGAATAATCTATTTAAAAATATTTTCATTGATACCTAGGCAGGCCCCATATTTTGCTGTATTTTTAAAAATTTTTGTTGAGTCAGAATCTCACTCTGTCGCCCAGGCTAGAGTACAGTGGCACAATCATAGCTCACTGCAGCCTTGAACTCCTGGACTGAAGTGATCCTCCTGCCTCAGCCTCCCAAAGAGCTGGAGTTACAGATGTGAGCCACTGTACTCGGCCACTGTCTTCTATTTCTAGTATTTCCTTTTTGTTTTTGTTTATTATTAAAGAATAGGCAGTTTCTAGTTTATTATAGTTAGGCTCTTAAAAATTGTAAATTCATGGTTTAGAATTCAATTAGATTTTCCCATAGAATTGGTGGCAAGATTCCCAGATTAGTCCACATAAAGCTGTTTAATCCACGTTATTTATGTACCTGATACAATGCTTGGCACTTGGAAAGTTGCTCAGTAAATGTTAGTTTCTTTCAATATAGAACCTCACCATTCGGTTTAACCATATTTATATGAGGAAAGATTTTAGAATTAAGTGTAGTAAGAGCAGATACTATCCTGGCTCAGTAATAATAACAATAATTCTATCACTTATTGATTTCTTACTGTATACTAGGTACTCTAAGTATTTAATATTAGTTACTAATTTAATCTTCAATAATCCTATCAAATAGACATTGCTATTGCCATTTTCTGGATGAAGAACTGAGACTAATGTCCACAGGCGGTAACTAGTAATCTGTCTGATTCCGCTTTCCCAAGAGTAGCAATTACCACTTCTCTGATTTGCAACCATAGTAGGCATCACTAATCCATTACAGTACTCATTCCCACTGAACCAACCTCAGAATTCTTGGCAACAGTTTTCTTGGTGGACAATATCAGTTGATATGCTAGATGTAACCTGTTTGCTCAGGGGCCTCCTATGTCACTAGTAGCAGTTTCTATATTTGGAGAAAGACATCCAACAAGGCATGAGGCTAAAGCAGTTGATCAGTAGGATCTTCTGTAAGTCAAATTCTTGAAATAATTTCTCAGTTTCACTAGTTCTTTGTCGCCCATTTGTCTTTCTCTCGAGATTTATAGTCGGCCTGAGACTGTGGGATCAGAAAGAAGGAACAGCAGAATGATATAAGAGAGAATCTGGGAGGCTGAGGTGGGCAGATCACCTGAGGTTGGGAATTTGAGACCAGCCTGACCAACATGGAGAAACCCCATCTCTACTAAAAAATACAAAATTAGCCGGTGTAGTGGCACATGCCTGTAATCCCAGCCACCCAGGAGGCTGAGGCAGGAGAATTGCTTGAACCCGGGAGGCGGAGGTTGCGGTGAGCCGAGATTACACCACTGCACTCCAGCCTGGGCAACAGAGCGAGACTCCGTCTCAAAAAAAAAACGGAAAAAGGAAAGAGAATCTGAATTCAGATACTCATTTGCCATTTGATCTTAAGCCAAAAGTCATTCTGAGCCTTATTTTCCTTATCTTTAAAATAGAAGAGAAATATCTGTTTAACAAGATAGTTTTAAGGACTAAATACAGTAAAATATGTAAAGAGTCTGCTGTAGTTTCTGGCACATAGAAAGTCCTTTACAGGTTCCCTCTCCCCTAACATATCCTAATACAGTGACTTTTCAGGGTTTTTTTTTTTTTTTCTTATTTTTATTTATTTATTTATTTTTGAGATAGCCTGTCACCCAGGTTGGAGTGCAGTCATGTGATCTTTTCTCACTTCAGCCTTGACCTCCCAGGCCCAAACAATCCTCCCACCTCAACCTCCCTACTAGATGAGACTACAGGCACATGCTACCATGCCCAGCTAATTTTTGTATTTTTGAGAACACGAGATCTCACAATGTTGCCCAGGCCAGTCTTAAACTCCTGGTCTCAAGCAGTCCTCCCACCTCCGTCTCCCAAAGTGCTGGGATTACAGACGTGAGCCACTGTGGCCTGGCCAGATTTTTTTTACCCTTATTAGATATGATACACTCTAATATTTTCCATTGAGTGGTTTTGTTGTTGTTGTTTTAACGTAGGCTGATAACCACTAAATTGACATCACAGTCCACTAGTGAGTTGAGATTTGCTGTTTTGGAAAAACTGTCCTAATTTTACCATCTCCCTCCAATAAATTCCCTTCCCCCATTGTAAGCATGTATTTTTCTTTCCCACCACCAATACCTGATCCCCTTGACCAAGGTTTTTCTCTTTTGTTAATTAACTGTTAAGAATGAATAGAAATAAGTGCTATACAACAAATAGAAATAGTGTTAGACTATGAGGAAAAAAATACATTAATGCAACTACTATGATAGCTCTTTGGAGATATGTGTGTGTGTGTGTGTGTGTGTACACACACATATAAAATAAGAGTTATGTTCAGAAAATCAACCAGACATAAGTTTTGTGTGCATTTTTTTCATAAGTAATTGCTACCTATGGAACTCTATGAGACTAAGAGGATTGTAACTAGTGAAAGGAAAAGAAAGAGAAACAGCTGAAAACCTTGTGTCAACATGTATTCCTAGGTTATTTTGAGGGTCCTAGCCCAGAGTTCAAAATTGCTTAAGGTCTACTGTTTTATCTTAAATTAATGTGGGTTTTACATCCCACACTGCAAAATGTTTTTGTTTGTTTGTTTCAGATAGGGTCTCCCTCTGTCACCCAGGCTGCAGTGCAGTGGCCCAATCTCGGCTGACTGCCTCCCAGGTTCAGGCAATTCTCCTGCATCAGCCTCCTGAGTAGCTGGGATTATAAGCGCCTGCGGCCACGCCCACCTAATTTTTGTATTTTTAGTAGAGATGGAGTTTCACCATGTTGGCTAAGCTGGACTCAAACTCCTAGCCTCAAGTGATCTGTCCACCTCGGCCTCCCAAAGTGCTGGGAATACAGGCGGGAGCCACTGTATCCGGTCTTTATTTTTTAATATTAGTACTTTTTAAACATTTTTTTTTAAGTCAAGATCTCCCTATGTTGCCCATGCTGGCCTCAAACACTTGGGCTCAAGCGATCCTCCCAGGTAGCTAGGAATACAGGCATGTGCTGCTGCGCCCTACTTGAATGCATTTTAAATGACCGTAAAATCGTTCACCAATTTTCTACCTGTCTTAGTGATTAGTGGGTAAGATGTATAGATTGCATCGGGTGTATCAATTTTAGTAAATTAGGCTCAGTGGGAGAGAACTGAACCAGAGTAAAACGTTCAGCATCTGTTTCTTTTCTATTAACCAGGATACTGAGAGCCCAAACTCACAGAAATATATGCGTACAAATGGCAACATATAAAAAAAAACTTATTTACCAGATTGTGTTAAGATCAGTATTAAAGAATATCCAGAATTCTACTAAAGATGTGGAATAATGTATCATTTTGAGGCCTTTATTGATCCAAGACTCACCTATTTTCATTGAATTAACTTGGATGTTTCACAGACACATCAAACTCAATATATTCAAATCTAACCTAAGATACCACTAACTGAATTTTCCTTATCTCCCGTTCCCTGCCTGTTCCTTTTCCCCTCCCAAATTTATGTTCCTTCTTAAAACCCTTCAGAAGTATCTAATTGTTGTAAGGATCTGTTCCTAAATCTTTACACAGTTTACAAAGCCCTGCACAATTTGGCCATCTTTTTTCTCCTCATATATTGCCATTTTCTGCCTCAAACACTAAGGAGCCTTTTAAAACTTCTTCCAGCCCTCTTTTATTTGTGGTCTTTTTACTCTAAGCGTGGTTTTAAAATACTTAAGAGCACTATTAACCCCATCATTAACAACAGGTATAAAACCAGTTGGTATTGAAATGCAGGATGTAAATAAATGTAGAAAAGATAAACATTGAGAATAGTATTCATATGCTAAATTTGCATATGTAGATGAAATGTTCAACAGTAGAAATGACTCTGAACAACAGAATGAATAATGAAATATAAGAAAAGTATAGCCTCCATACAATTGAAAACAGAAAAAAAAAAAGTTGATGAAATAGAATTACTTGAGCATGTGGCTCAGGGACACTTATGTACCCCCTGCATTAATATCAAATATCTATTCAGGTATTGTAAATTTAAGAGAAGGCCAATAATTAAAATAATAGCACATACAGCCTTCACTATGTGTCAGATACTATCCTAATTGCTGTGCATGTATTAAGTCATTTAATCCTAACAGCAACCTTTAAAGGTAGGTATCATAGTCCTTACCGGATGAAAAAAAAATGAGGGTCAGAGAAGTTAAGTAACTCTGCCCTAAGTCATAGCTAGTAGGTTGAAGTGCCAGAATCTGAACCCAAGCAGTCTAGCTTCAGAGTCGGTACTTTAATCCACATGTCATACTACTTATAGTTGATTAAAAAACTTAACAGATAATCGATTTGTTTTATTTTTTTTCTTTTGAGACAGAGTCTCGCTCTGTTGCCCAGGCTGGAGTGCAGTGGCGTGATCTCAGCTTACTGCAACCTCTGCCTCCCAGGTTCAAGCGATTCTTGTACCTCTACCTCCCAAGAAGCTGGAATTACAGACATGCACCACTATGCCTGATTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCGCCATGTTGGCCAGGCTGGTCTCCAGCTCGTGGACTCAAGTGATCTGCCCACCTTGGCCTCCCAGAGTGCTGAGATTACAGGCATGAGCCACTGTGCCTGGCCCAGATAATCAATTTGAAACATGTGTCACAAATCATAAAAAGTTCCATAGGTTCTATGCCCTTAAATTCACACAACATCAGAGTTCATGACAGGGTAGCCACAGAGTACTCTAACCTCTGAGGGCTTCTCTGATATTTGTATAGTTTATCAAGGCTAGGTAAAGAACAGTCACCTCAACAAATTTTAAATTTGTCAAAATTGGTCCATTTTAGAAAGGCTGCTTATTTAGATTATAAATCAGCGTGAAGAAGAGGCGTCTATCTGGTTTAAAAGCTTTGAAAGACTAATTTACCTTTCTGTTTGATGTAAATGTATCTGGAGGATGCAAGGTTAAACCCCTCTTAATTATTTTGGACTTACACTCAAAAGACCTTTCCAATTTTGAAGCCAAACCTGTTTGTAAGAAGAGGAACTTCAGTAGTGCTGCTACAACTTACTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTCTCTCTCTCTTTCTCTCTTTCTTTCTTTCTCTCTCTCCTTTCCTTTCCTGTCCTGTCCTGTCCTGTCCTGTTTCGATAGTCCTGTTTCGACCGAGTTTCGCTGTTGCCCAGGCTGGAGTGCAGTGGCATGATCTCGGCTCACTGCAACCACTGCCTCCCGGGTTTAAGTGATTGTTCTGCCTCAGCCTCCCGAGTAGCTGGGATCACAGGTGCACGCCACCACACCCGGCTGATTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGCCTTGAACTCTTGACCTCAGGTGATCCACCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCTTAATTTGTTTGTTTTTTCACTTTAGCAGTACATTCTGGAAATCTTTCTATGTCTGTATTTTTCATCACATGGACATAGCATAATTTAACTAGTACACGATTGATTTCTCAATATTACTTTAAGAGTGACTTAATGGAGTCAATGCTAACTTCATTCATGATTATGCAATATATTATGGACCTTATAATTCTAATCCCATATTTTAATTAAGTCTGTAAATAAAGGATGGTAAATAAGTGACATGCAAACATCTATAACTGTTTCTGGATCCTGTTATTTTGATCACTACTCTTGGTAGCTAGGCCATGATCAGCCTCAAGAAGTTTTCTGAACATGGCATTTCAGACAGCCATTATCCGTAAGTTGGAGTTACTGTTCTAGATGAAATCTTTGTCAATCCTATCCAGTAAACATATTCTGTGCTTTTTAGATTTATGGCTGCTTACCACTGCTTCTTTAGTTGTGTTTGGGGTACTGTTATCTTTTTCTGCTGCCAAGTTACTTGTGAGGAGCCTAAGAAACCACCCGTGTCACAACACTTCAGATTGCTCTGGATTTGCTTCCTTATCTGACAGTTTCATTATATTTATTTATTTATTTAAACAGAATCTCACTCTGTCACCCAGGCTGGAATGCGGTGGCACGATCTCGGCTCACTGCAGTCTCTGCCTCCTGGGTCCAGGCGATTCTCCTGCCTCAGCCTCCGGAGTAGCTGGGATTACAGGCTTGCGCCACCGCGCCTGGCTAATTTTTGTATTTTTAGTAGAAGACGGGGTTTCGCCATGTTGGCCAGGCTGGGCTTGAATTCCTGACCTCAGGTGATCTGCCTGCCTCAGCCTCCCAAAGTGCTAGGATTACAGGCATGAGCCACCACGCCTGGCCAGTTTCATTATTTTTAAGTTTTGCCTAAATCACCAATCCGTATTCCCATTTGAACATATAATTTTTAAGTTACTATGCTTCAAAAAGCTAATTGCAGTATTACACTAAAATTACACACGCACATGTACACACAAAAAGAATAGTTGGTGTCTTTATTAGGCAATTTTATTGCAGTGAAAGTGGACCTACACATAATCAAATATTTGTAAACAATTGAGACAGCATTACTGTGACTGGGAGTTAAATTAGTTTGTGGGAGCCATTGTTTATTGGTATAATCAATAGTTTTTAAAAACTTATAAGGTGAAAAGAAACCAGACCATTGTAAAATCTAGAGGAAATCAAACTTTCTAACTTATGTAAATTAGAAGTTGTAGCTCAGTAAAATTAATAGTTTATTCCTACTGCAGTGAGCGCCCCATTCTTTTTTTTTTTTTGTAGCTGCAGCACTAATACTACTATACATCTCATTAAAACACATCTGCCAAAATTTCTTATCATTTACTATTTATTATTTTTTACACTGTGGTTTCATAAAAAACCATAAGATGGACCAGAGAAGAAGGGTTCTGTTGGTTTTTGCTTGCACATATCTGTGACAAAAAACTTAGAAATAAAATTTCAAATTACTTTTTTATCCAAACTCTTAGAATGATGTTAACTATTTAAAAAGATGTGGCAAGTTTGTCTTGTACTTTACAACAGTGGTTCTCAAACTTGAGAATGCCACAGAATCACTTGCAGAGTTTGTTAACCACGTAGATTACTGGTTCCTACTCCCAGAATTACTAATTTAGTGGGTATGAGATGGGACACAAAAATTTTCATTTCTAGCAAGTTTCCAAGTTATGCTGATACTGGTACAAGGACCACACTTTGAGAACCACTGGCTTAGAAGATCCTTCAGCTCACTGGTATTATTGCATAACCCATTTTTAGAAGCACAGTTTTGTGGTTTTCCCTCTCTTTCCATCCTCTTTGTTGTTGCCTCTGTAGACCCTGGATTGTGGGGATAGAAGTGAAGTGATGATCAGTTTAGTAGAGACATAGACTAAATAAGGGTGAAAAATGCCACTTGCATTAAGAAGGCAGGGGGCATTTCCTAATGTAAAAAGGAGGGACAGATTGATAGATAACTCAGCTCTTGCTATTGTAATGTAATTTAATTATTGTTACTATAGTCTGTCAACTGAATGTTAAAATGGTGTGAGCATTCACAATTTATAATTTATCTTAAATATAATTTAATGTATATTTGATGTTAAAGCAAAACAAGTGGATTTTTTTCAATCTGGTCTTTTTAAAATATAGAAATTATTTCATATTGGTAGTTAGATTTATTTACACTCAGGTTTCTCTTGCTAATGAAATTTGACTTCTATCCTCCCGAAAGATTTATTAGAAAAATATTACCTAAAAACAAGATCCTTTACTGGTAGTCTGTTTAAAGGCAGGGGTGTTTCTGTGTGTGTATGCTTTTCTTAAATATTTACAGAGAATACCATGATTGTCTTCATTGTATTATTTCCTTGGTTTCTTGTAGAGAAAAGTAATCAAGATCTAAGGAACTGACTTTGCTATAAGCAAACCGTTTCTCCATAATTCAAATTTGAATTAATAAGTGATTGTTTTGTGTTAAAATAGCAAGCCTAATTTTTTTTTTTGCTAAACTTGTTTATTGCATGATAAACTGATTATCATTCAATATACTGAACAATAGCCTTTTTAAAAAATGTCTGTTTTTAGTACACTCAGAAGAAGCCTTGTTTTTACTGGCAACCTGTTATTACCGCTCAGGAAAGGCATATAAAGCATATAGACTCTTGAAAGGACACAGTTGTACTACACCGCAATGCAAATACCTGCTTGCAAAATGTTGTGTTGATCTCAGCAAGTAAGTTTTAAAATATTTTCTAGCTATTTTGTTTTACTTACACTGTTGAATTTTGGATAAAATTCCTTTCAGATTAAACTCTGATTCCCTTTCTTCCCTGTTTTCCATCTATTTCAGTAACATTCGAAATGTTTACACTGTTGCCTATGGCATTATTTATATTATGGAGATTTCATTGTTATGGTAATAAATAATAGAGTTTAACTCTGGAGAAACTTAAGTTATAACCGATTCTTATTGGTTTCTTAAATGATCACAAATATTTCGTTTTCTTTGCTTTCAGTGTTAGACCAATTTGAGTAAGCCTGAAAACCCACCAGCTCACTTGAGATTAAGGCTCCCTTGTGTTTAGGTGATTGTATAGTTTCCTTTCAAGATCATTTTATTGAGTACTTCTTTTGCACATTACAGTTAAACCAAGTCAGCTGTCCCTATACCAAATTTGGAGGCATTTAGAGATACAGATAGTTAGGTTCTATTCAATTCTTTTCTGTACTGTAAGAGATGGAATAATGGTAAAAGGGACTAAACGGTCACATGTGTAAACGGTGTTAGATATTTGTAAAAGCCTGTATGTATAATTGGCTTGTAGGTTTACAAGGGGAAGATACAAGTAAGTCTCACCATAGACTGATCAATAACTATATTATCTGTCCAGTGACTAATTGATGGCTTTTTAATTAAAGCTAGCTAAAATATCTGAAATTTCTGAGACATTTGTTGTTGAATTACTTTTAAATTTCAAAGATCCTAATGTGGAATTCTTAAAAATCTTTCTTATGTAATTTGCCAGTTCCTTTTTCTTTTTTCCTTTTTTTAGAGAAAGGGTCCTGCTGTGTTGCTCAGGCTGGAGTGCAGTGGGGTGATGATGGCTCACTGCAGTCCCCATCTGGGCCTAAGCGATCCTCCCATCTCAGCCTCCCACGTAGCTGGAGCTGCAGACACGTGTCACTACACCTGGCTAATTTTCTTATTTTTTGTATAGATGGGGTCTCACTATGTTGCCCAGGCTGGTTTTGAACTCTTGGGTTCAAGCTGTCCTCCCACCTTGGCCTCCCACAATGCTGGGGTTATAGGCTTGAGCCACTGTGCCGGGCATAAGTATGCAATTTTTAATTAAACTATGTTAGTCTTGTTTAATTTGCATATACATTTCTATTTTAAGGAAGTAAAAAGGACCACCATGTTTGTTAATGAAGAACCATTTATATTTTCTCCCCCAGTATAGTAATCATCTTCTGTTCCTTCTGACATGAGTAATGAATATGTTGCTCAGACTGGATTTGTACATACAGAGACTAAATGTAATTGATTCTGTTTTTAAAGGTAAAAGTGAAATTTTAAGATCATAAATTTCCAGCCTCTCAAATGATTCTAAATAAGTGTTTGCCACTATTGTAATTAAAAAAAATTTTTTTTACAAAAAACAAATTGGCCAGGTGCAGTGTCTCATGCTTGTGATCCCATGAGATGAGAGGATTGCATGAGCCCAGGAGTTTGAAACCAGCCTGGTCAACATAGTGAGACCCTGTCTCTACAAAAAAATTAAAAATTAGCTGGGTGTGGTGGTGTGCACCTGTAGTCCCAGCTACTCACAAGGCTGATGCAAGAGGCTCACCTGAGTCCAGGAATTTGAGGCCATGTGAACTATGATTGTACTACTGCACTCTAGCCTGGGTAACAAAGCAAGACCCTATCTCTTTAAAGAAAGGAAGACAGAGAGAGGGAGGGATGAAGGAAGGAAGGAAAAGGAAAGAGAGAGAGAGAAAAAATTTGTGTCTCCATAGTTAGATGCACTTTGGTAATGAAGAATATGTTTACATGAACTGCTTTTTATATTGGTTTTTAAAAATCTTTAAATTTGTATTTTAAAGGCTTGCAGAAGGGGAACAAATCTTATCTGGTGGAGTGTTTAATAAGCAGAAAAGCCATGATGATATTGTTACTGAGTTTGGTGATTCAGCTTGCTTTACTCTTTCATTGTTGGGACATGTATATTG。

Heliced sequences

>hsa_circ_0044235|NM_001114091|CDC27

GCTGCTATATGGCAAGCACTAAACCACTATGCTTACCGAGATGCGGTTTTCCTCGCAGAACGCCTTTATGCAGAAGTACACTCAGAAGAAGCCTTGTTTTTACTGGCAACCTGTTATTACCGCTCAGGAAAGGCATATAAAGCATATAGACTCTTGAAAGGACACAGTTGTACTACACCGCAATGCAAATACCTGCTTGCAAAATGTTGTGTTGATCTCAGCAAGCTTGCAGAAGGGGAACAAATCTTATCTGGTGGAGTGTTTAATAAGCAGAAAAGCCATGATGATATTGTTACTGAGTTTGGTGATTCAGCTTGCTTTACTCTTTCATTGTTGGGACATGTATATTG。

1. Experimental methods

(1) Serum RNA extraction

Taking 250 mu L of serum of a breast cancer patient or a normal person to a centrifuge tube of 1.5mL of RNase-free, respectively adding 750 mu L of Trizol reagent, manually shaking and mixing uniformly for 15s violently, standing for 5 minutes at room temperature, adding 0.2mL of chloroform, covering a cover, manually shaking violently for 15s, and standing for 3 minutes at room temperature. Centrifugation at 12000rcf for 15 min at 4 ℃ revealed a distinct separation of the homogenate into three layers. A new 1.5mL centrifuge tube was taken, 500. mu.L of the upper colorless transparent liquid phase was carefully transferred to the new tube, an equal volume of 500. mu.L of isopropanol was added, mixed by hand shaking, and left at room temperature for 10 minutes. Centrifuge again at 12000rcf for 10 min at 4 ℃. The liquid was aspirated, and 1mL of 70% ethanol was added to each tube to wash the precipitate. After centrifugation at 7500rcf for 5 minutes at 4 ℃, the liquid phase was aspirated, dried at room temperature for 5 minutes, and the precipitate was dissolved with 30. mu. LRNase-free water to obtain total RNA.

(2) Reverse transcription

RNA concentration was measured using a ultramicro nucleic acid protein measuring instrument (Dano Drop 2000).

② the reverse transcription kit of Takara company is used, and the strict execution is carried out according to the reverse transcription kit instruction of Takara company.

③ storing the successfully prepared sample cDNA at-20 ℃ for later use.

(3) Construction of ddPCR detection method

Designing a probe primer: primer Express 3.0.1 software was used to design primers and probe sequences specific to it, Forward Primer (5 '-3'): TTGGGACATGTATATTGGCTGCTAT (SEQ ID NO. 1); reverse primer (5 '-3'): AACCGCATCTCGGTAAGCAT (SEQ ID NO. 2); probe (5 '-3'): FAM-TGGCAAGCACTAAAC-MGB (SEQ ID NO. 3).

ddPCR reaction system (20. mu.L reaction system in Table 1):

TABLE 1

Reagent composition	μ L/reaction well (20 μ L)	Final concentration
			2X ddPCR Supermix for probes (no dUTP)	10	1X
20X Probe-primer mixture	1	Primer 900 nM/probe 250nM
			RNase-free water	Variable	—
Sample cDNA	Variable	The total amount is less than or equal to 300ng

Remarking:

firstly, according to the previous experiment, when the final concentration of a sample is 2.5 ng/mu L in a 20 mu L reaction system, obvious fluorescence value distinction can be seen, and the sample dosage is less. The later experiments were all used in the experiments with a sample final concentration of 2.5 ng/. mu.L. The amount of RNase-free water was adjusted accordingly to the initial concentration of the sample.

And setting blank control in the experiment, i.e. adding no sample in the system and using RNase-free water to supplement the corresponding content.

And thirdly, the experimental system configuration is operated on ice.

And fourthly, ensuring that the final concentration of the primer is 900nM and the final concentration of the probe is 250nM in a 20 microliter system. The 20 Xprobe-primer mixture is configured as in Table 2;

TABLE 2

ddPCR operation steps:

firstly, 20 mu L of reaction system is configured, and can be proportionally configured to reduce sample adding loss and ensure enough reaction system.

② transfer 20. mu.L of the reaction system to the well position of the microdroplet generation card.

And thirdly, adding 70 mu L of microdroplet generation oil to a corresponding hole of the generation card, covering a rubber mat, putting the generation card into a microdroplet generation instrument, and absorbing 40 mu L of microdroplets to transfer to a 96-well plate.

Fourthly, after the membrane is sealed by a Bio-Rad PX1 PCR heat sealing instrument, the membrane is placed in a Bio-Rad T100PCR gradient PCR instrument for amplification at 52.9 ℃.

Fifthly, transferring the 96-well plate to a ddPCR analyzer, and selecting absolute quantitation (ABS) for data analysis.

After droplets are formed in the ddPCR system constructed by the method, a sample is uniformly dispersed into the droplets, and amplification reactions in the droplets do not influence each other; all microdroplets contain or do not contain target genes, and the 5 'end of the probe in the microdroplets is marked with FAM fluorescent group, and the 3' end of the probe in the microdroplets is marked with MGB fluorescent quencher; normally, the fluorescence of FAM is quenched by MGB fluorescence to present a basic fluorescence value, and during amplification, a fluorescent group and a quenching group are hydrolyzed by exonuclease to present a remarkable amplification fluorescence value; and finally, counting the number of the micro-drops with the amplified fluorescence value by using a ddPCR (polymerase chain reaction) analysis instrument to achieve absolute quantification.

The optimal extension temperature of the probe primer and the optimal final concentration of the sample RNA are searched.

And performing ddPCR detection by using the optimal sample RNA concentration, transferring a 20-mu L system to a middle hole of a microdroplet generation card, adding 70-mu L microdroplet generation oil into a lower row of holes, putting the microdroplet generation oil into an instrument, sucking 40-mu L microdroplet system from an upper row of holes after microdroplets are formed, and transferring the microdroplet system to a corresponding hole of a 96-well plate. PCR amplification is performed using the found optimal extension temperature as the extension temperature.

And placing the amplified 96-well plate in a ddPCR analyzer, selecting a corresponding reagent and absolute quantitative parameters, waiting for the reading result of the analyzer, and finally performing data analysis and drawing by using GraphPad Prism 9 software.

(3) The diagnostic value of serum hsa _ circ _0044235 levels in breast cancer was analyzed using the ROC curve (SPSS Statistics 25 software).

2. Results of the experiment

(1) Constructing a method for detecting serum hsa _ circ _0044235 by ddPCR, and searching for the optimal extension temperature

As shown in FIG. 1, the extension temperature of the ddPCR detection serum hsa _ circ _0044235 has a good fluorescence differentiation effect at 52.9 ℃, and is the optimal extension temperature.

And (3) amplification procedure:

TABLE 3

(2) Constructing a method for detecting serum hsa _ circ _0044235 by ddPCR, and searching the optimal sample RNA concentration

As shown in FIG. 2 and FIG. 3, for the normal human serum test, good fluorescence discrimination effect can be obtained by hsa _ circ _0044235 test when the RNA concentration of the sample is 2.5 ng/. mu.L, and the sample dosage is less; the copy number of hsa _ circ _0044235 also increased with increasing RNA concentration.

An amplification system: since the concentration of cDNA extracted from this sample was 50 ng/. mu.L. The following system was therefore configured: 2XddPCR Supermix for probes (no dUTP) 10. mu.L, 20 XProbe-primer mix 1. mu.L; 8 mu L of RNase-free water; mu.L of template cDNA was obtained.

(3) ddPCR detection of serum hsa _ circ _0044235 content

As shown in FIG. 4, in the ddPCR assay at 52.9 ℃ and with the normal human serum sample as the template and the sample RNA concentration of 2.5 ng/. mu.L, it can be seen that the fluorescence values of hsa _ circ _0044235 are significantly differentiated and can be well detected (FIG. 4), which indicates that we successfully construct the ddPCR assay for the content of hsa _ circ _0044235 in the serum.

(4) ROC curve analysis value of serum hsa _ circ _0044235 in breast cancer diagnosis

To investigate whether hsa _ circ _0044235 was differentially expressed in the sera of normal and patients. We determined the absolute content of hsa _ circ _0044235 in serum from 106 patients and 96 normal persons. The results show that: hsa _ circ _0044235 was highly expressed in normal human serum and low expressed in serum of breast cancer patients (FIG. 5).

Secondly, in order to explore the diagnostic value of hsa _ circ _0044235 as a breast cancer serum marker, the ROC curve is adopted to analyze the clinical diagnostic significance of the hsa _ circ _0044235, and the AUC (AUC) of the area under the ROC curve is 0.85 (95% CI, 0.798-0.903); sensitivity of 0.913, specificity of 0.66; the cut-off value (296.5 copies/. mu.L) (FIG. 6) indicates that the serum marker has good value for breast cancer diagnosis.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> Guangzhou university of medical sciences attached to the third Hospital (Guangzhou intensive pregnant and lying-in woman treatment center, Guangzhou soft hospital)

<120> use of hsa _ circ _0044235 level in serum as a diagnostic marker for breast cancer

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttgggacatg tatattggct gctat 25

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aaccgcatct cggtaagcat 20

<210> 3

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tggcaagcac taaac 15

Claims

1. The application of the reagent for detecting the expression of hsa _ circ _0044235 in preparing a product for diagnosing breast cancer.

2. The use according to claim 1, wherein the product is a detection reagent or kit.

3. The use of claim 1, wherein the method of detecting the expression of hsa _ circ _0044235 specifically comprises:

4. The use of claim 3, wherein the reaction system for ddPCR detection is: 10. mu.L of 2X ddPCR Supermix for probes, 1. mu.L of 20X probe-primer mixture, 8. mu.L of RNase-free water, 1. mu.L of template cDNA; the preparation system of the 20X probe-primer mixture is as follows: mu.L of 50. mu.M forward primer 36. mu.L, 50. mu.M reverse primer 36. mu.L, 5. mu.L of 100. mu.M probe, 23. mu.L of RNase-free water.

5. The use of claim 3, wherein the temperature of the ddPCR detection extension stage is 52.9 ℃.

6. A primer group for diagnosing breast cancer is characterized by comprising an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO. 2.

7. A diagnostic kit for breast cancer comprising a reagent for detecting the expression of hsa _ circ _ 0044235.

8. The diagnostic kit of claim 7, wherein the diagnostic kit comprises the primer set of claim 6.

9. The diagnostic kit of claim 8, further comprising a probe as set forth in SEQ ID No. 3.