CN109022583A - Hsa_circ_0021977 is preparing the application on Diagnosis of Breast cancer product - Google Patents
Hsa_circ_0021977 is preparing the application on Diagnosis of Breast cancer product Download PDFInfo
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Abstract
The invention discloses a kind of marker for detecting breast cancer, the marker is hsa_circ_0021977, and further confirms that hsa_circ_0021977 is raised in expression in breast.The present invention further discloses a kind of for detecting the diagnostic kit of breast cancer, and the kit includes the primer and specification of specific amplification breast cancer correlation circRNA, and the breast cancer correlation circRNA is hsa_circ_0021977.It can not only quickly and effectively accomplish early detection using hsa_circ_0021977 detection breast cancer, and provide therapy target and important evidence for clinical applications such as gene therapy, drug therapies.
Description
Technical field
The present invention relates to biomedicine fields, are related to diagnostic marker and its application of newborn breast cancer, the specific marker
For hsa_circ_0021977.
Background technique
Breast cancer is the malignant tumour that women is common in global range.Breast cancer incidence constantly rises.It is swollen according to the whole nation
Tumor registration annual report, breast cancer are in China's female malignant first, and in cities such as Beijing, breast cancer incidence is up to
46.6/10 ten thousand, it is 7 times of Midwest city, in the past 20 years, Beijing area breast cancer incidence rises 91%, every year on average
Increase by 4.6%.A large amount of experts and scholars are dedicated to studying breast cancer both at home and abroad, are expected that by the pathogenesis to breast cancer, detection
The research of the various aspects such as means, biomarker, drug therapy improves patient with breast cancer's survival rate and quality of life.
Circular rna is a kind of novel non-coding RNA, and head and the tail form the ring being closed by covalent bond, is shown and line
The RNA different characteristic of property.CircRNA is largely present in eukaryocyte transcript profile, has conservative between species, and expression is stablized
And there is tissue and developing stage specificity.CircRNA is not easy to be degraded by exonuclease, more steady compared with linear rna in body fluid
It is fixed, thus there is the potential using value as clinical diagnosis and prognostic marker.CircRNA is in disease of cardiovascular system, mind
Through playing a significant role in the diseases such as systemic disease, PrPC disease and cancer.However with mRNA, miRNA and lincRNA phase
Than we just start the understanding of circRNA.In terms of the research of breast cancer, circRNA is a completely new research neck
Domain, existing experts and scholars focus on the research field, for example, existing research shows Circular RNA hsa_circ_
0001982 can promote the generation of breast cancer, but there is no a large amount of systematic Studies for circRNA at present, also have no circRNA
Report for breast cancer diagnosis.
Summary of the invention
In order to realize the early detection of breast cancer, early intervention, one of the objects of the present invention is to provide hsa_circ_
0021977 as molecular marked compound is preparing the application on Diagnosis of Breast cancer product.
Further, the hsa_circ_0021977 expresses up-regulation in breast cancer biological sample.
Further, the marker includes the nucleotide sequence as shown in SEQ ID NO:1.
Still further, the product is the product for capableing of quantitative detection hsa_circ_0021977 expression quantity.
Further, the product is the kit that detection individual suffers from breast cancer possibility.
Preferably, the kit includes the primer and specification of the relevant circRNA of specific amplification breast cancer.
Preferably, the relevant circRNA of the breast cancer is hsa_circ_0021977.
Preferably, the primer sequence is as shown in SEQ ID NO:2 and SEQ ID NO:3.
More preferably, the kit further includes 10 × Buffer, dNTP, MgCl2, Taq enzyme and SYBR Green fluorescence
Dyestuff.Preferably, the following contents is indicated in the specification:
When hsa_circ_0021977 expression quantity E1 and normal breast cell in the breast cancer cell or tissue of test object
Or the ratio between hsa_circ_0021977 expression quantity E2 of tissue >=2, then prompt the probability of the test object breast cancer to be higher than common
Crowd.The E1 is the breast cancer cell of test object or the hsa_circ_0021977 expression quantity of tissue；The E2 is
The normal breast cell of normal population or the hsa_circ_0021977 expression quantity of tissue.The normal breast cell or tissue
Including the mammary glandular cell or tissue by cancer.The expression quantity is the relative expression quantity relative to crt gene (such as GAPDH).
Preferably, the kit further includes 10 × Buffer, dNTP, MgCl2, Taq enzyme and SYBR Green fluorescence dye
Material.
Further, the present invention also provides a kind of biological agent for assessing breast cancer risk, the biological agent packets
Containing the hsa_circ_0021977 inhibitor effectively measured.
Further, the present invention also provides a kind of pharmaceutical composition for treating breast cancer, described pharmaceutical composition includes
Hsa_circ_0021977 inhibitor further includes pharmaceutically acceptable carrier as active constituent.
Beneficial effects of the present invention are as follows:
The invention discloses a kind of hsa_circ_0021977s relevant to breast cancer, and further confirm the hsa_
Circ_0021977 is raised in expression in breast.The discovery of the RNA can be used for early diagnosing the biomarker of breast cancer
Situations such as object, the therapeutic effect of evaluating breast cancer, relapse and metastasis of assessment breast cancer, provides theoretical foundation.It can further carry out
CircRNA is associated with Journal of Sex Research with miRNA's, to study the pathogenesis of breast cancer.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, reagent used can be commercially available.
Test method without specific conditions in embodiment, usually conventional method in that art, such as according to normal conditions
Such as Sambrook et al., molecular cloning, the condition in laboratory manual (third edition) (Science Press, 2002), or according to
Condition proposed by reagent manufacturing firm.
The present inventor carries out gene expression profile inspection by chip to breast cancer tissue's sample and normal tissue sample
It surveys, genescreen is carried out by bioinformatics method, picks out candidate circRNA hsa_circ_0021977, existing research
In there is no hsa_circ_0021977 and the relevant report of breast cancer, further, inventor has carried out molecular biology method
Verifying, it was confirmed that hsa_circ_0021977 expresses up-regulation in breast cancer cell.
The present invention also uses RT-PCR method to detect above-mentioned circRNA in the expression of breast cancer tissue and normal tissue, and
The circRNA is demonstrated to raise in breast carcinoma.
Terms used herein " expression up-regulation " refer to the sequence corresponding to expressed gene, wherein the measurement card of sequence amount
It is bright, determine that identified in the individual of morbid state different with breast cancer separates from from normal individual or from by stages for breast cancer
Biological sample in same gene compare, the gene from suffer from breast cancer or pass through stages for breast cancer determine breast cancer
Identify that the expression in the biological sample separated in the individual of morbid state increases.According to the present invention, " expression up-regulation "
Refer to the measurement of intensity for hybridization by the method for the invention at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%
Or higher expression increases, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher or is higher than 1
Times, up to 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, 100 times or higher.
Terms used herein " expression " refer to through the measurement of known to those skilled in the art and method described herein
Given nucleic acid or protein can measure.When being related to the corresponding circRNA of molecular marked compound of the present invention, expression can lead to
Hybridization or more is crossed to measure, such as includes green, TaqMan and quantitative real-time RT-PCR using SYBR.
1 circular rna cDNA microarray difference expression gene of embodiment
1, it samples
It is research object that the present invention, which chooses patient 10 to go to a doctor in Cancer Hospital of Chinese Academy of Medical Sciences Breast Surgery,
After the informed consent for obtaining patient, the breast tumor tissues sample and normal galactophore tissue's sample of random collecting some patientss, into
Row detection.
2, Total RNAs extraction is carried out to tissue samples
Using the very fast extraction agent box of total serum IgE, (article No.: 220010) Shanghai Fei Jie Bioisystech Co., Ltd carries out sample
RNA is extracted, and experimental implementation is carried out by product description, and concrete operations are as follows:
1. 300 μ L physiological saline are added in tissue samples, it is lightly ground with electronic tissue grinder, after the completion of grinding
12000rpm is centrifuged 2min, and 100 μ L of supernatant is taken to be transferred in new 1.5mL eppendorf pipe；
2. 500 μ L of RA2 liquid is added, is sufficiently mixed by inversion 5-10 times in the sample tube handled well, 1min is stood；
3. all sucking or pouring into inner sleeve for sample lysate, 12000rpm is centrifuged 1min；
4. taking out inner sleeve, suck and put back to inner sleeve in outer tube after liquid, 500ul washing lotion, 12000rpm centrifugation is added
1min；
5. repeating step 4 to wash again once；
6. taking out inner sleeve, suck and put back to inner sleeve in outer tube after liquid, washing lotion is not added, 12000rpm is centrifuged 1min；
7. inner sleeve is moved into new 1.5mL eppendorf to manage, 1 ‰ DEPC water, 25 μ L is added in film center；
8. after being stored at room temperature 1min, being centrifuged 1min, total serum IgE is obtained；
9. 2 μ L RNA is taken to survey concentration with Nanodrop.With Nanodrop2000 ultraviolet specrophotometer measure RNA purity and
Concentration freezes in -80 DEG C.RNA quality judging standard: the OD of RNA sample260/OD280Value is between 1.7-2.2；Total serum IgE electrophoresis
Map has clearly 28S, 18S band；70 DEG C of water-baths keep the temperature 1 hour after electrophorogram and water-bath keep the temperature before map without obvious
Difference.
3, the quality analysis of RNA sample
RNA extract after agarose gel electrophoresis, from electrophoresis result can with preliminary judgement extract RNA sample it is up-to-standard with
It is no, if to can be used for further transcriptome analysis.And then RNA sample is detected by NanoDrop1000 spectrophotometer
Extract situation, the sample requirement of RNA-seq sequencing: OD260/OD280For 1.8-2.2.
4, expression profiling
Mankind's circular rna chip CapitalBio Technology Human CircRNA Array v24x180K expression
Spectrum chip (CapitalBio Technology, 4 × 180K) detection is starting with the total serum IgE of sample to be tested, is expanded in vitro
Increase and fluorescent marker, amplification and labeling process use Ambion WT Expression kit (Invitrogen, WT
Expression Kit, article No.: 4411973).Reverse transcription synthesizes the first chain cDNA: being originated with total serum IgE, contains T7 promoter sequence
The random primer of column synthesizes the first chain cDNA using First Strand Enzyme Mix.It synthesizes the second chain DNA: using Second
The RNA chain in DNA-RNA heterozygote is converted the second chain cDNA, synthetic dsdna by Strand Enzyme Mix.It is external to turn
Record synthesis cRNA: using the second chain cDNA as template, cRNA is synthesized with T7Enzyme Mix.CRNA purifying: RNA purification column is used
(article No.: 157035437) Qiagen, RNeasy mielute spin column purify cRNA, except salt, the enzyme in dereaction
Equal reagents, and quantitative, Quality Control is carried out to cRNA.Reverse transcription: using cRNA as template, Random Primer is primer,
II enzyme of CbcScript carries out reverse transcription.CDNA that purification and recovery reverse transcription obtains is simultaneously quantitative.Label: it is produced with the cDNA of reverse transcription
Object is template, and Random Primer is primer, with Klenow Fragment enzymatic synthesis cDNA complementary strand and is mixed with fluorescence
The dNTP (Cy3-dCTP) of group purifies simultaneously quantitative mark product.It can be used to chip hybridization with fluorophor DNA.
After mankind's circular rna chip CapitalBio Technology Human CircRNA Array v2 hybrid screens
Tiff format picture data several softwares mentioned using Feature Extraction carry out Preprocessings, then use
GeneSpring GX software calculates gene expression difference and significance,statistical p value.Data handling procedure specifically includes that chip
Scanning tiff figure is handled to obtain raw data file (.txt) with FeatureExtraction software.By raw data file
(.txt) imports GeneSpring software, the parameter informations such as write-in grouping.Carry out data normalization and the QC analysis of each sample.
Cluster analysis and graphical representation are carried out with Cluster3.0 software.According to grouping information, comparison in difference is carried out, difference is obtained
Allogene Circular RNA hsa_circ_0021977, the gene express up-regulation in breast tumor tissues.
Embodiment 2RT-PCR verifies breast cancer tissue and normal galactophore tissue's hsa_circ_0021977 expression
1, material
Choose go to a doctor in Cancer Hospital of Chinese Academy of Medical Sciences Breast Surgery patient be research object, be obtained from 2017
January in June, 2018 receives the breast cancer tumour sample and cancer beside organism's sample of 10 patients of mammary cancer surgery in our hospital.
2, method
2.1 pairs of tissue samples carry out Total RNAs extraction, with the extracting method of embodiment 1.
2.2 reverse transcriptions synthesize cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044)
CDNA reverse transcription is carried out, experimental implementation is carried out by product description, and concrete operations are as follows:
Using Reverse Transcriptase kit, converse record is carried out to l μ g total serum IgE with RT Buffer and synthesizes cDNA.Using 25 μ L
Reaction system, each sample take 1 μ g total serum IgE as template ribonucleic acid.- 20 DEG C are stored in after the cDNA sample of acquisition is diluted 10 times
Refrigerator is spare.
2.3Real-Time PCR
2.3.1 instrument and analysis method
With StepOne Plus real-time PCR (ABI7300), using 2-ΔΔCtMethod carries out data relative quantitative assay.
2.3.2 design of primers
Using online primer-design software, gene order such as SEQ ID NO.1, interior participation in the election GAPDH, by reviving after design of primers
The synthesis of the biotech inc Zhou Hongxun.Specific primer sequence is as shown in table 1:
1 primer sequence of table
Operating process is as shown in table 2:
2 SYBR Green of table, I PCR reaction system
| Component | Additional amount |
| 2×Talent qPCR PreMix | 10μL |
| Upstream primer (10 μM) | 0.6μL |
| Downstream primer (10 μM) | 0.6μL |
| RNA template | 1μL |
| 50×ROX Reference Dye | 2μL |
| RNase-free ddH2O | Polishing is to 20 μ L |
With SuperReal PreMix Plus (SYBR Green) (TIANGEN, article No.: FP205-02) respectively to purpose
Gene primer and reference gene primer are expanded.Experimental implementation is carried out by product description.Amplification program such as table 3:
3 PCR condition of table
After reaction, the PCR product of 5 μ l is taken to carry out 2% agarose electrophoresis, with quick plastic recovery kit
The segment that size is 103bp is carried out gel extraction and is sequenced by (invitrogen company), is as a result carried out with blast software homologous
Property analysis.
3, experimental result
Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；Initial data is exported from quantitation software, is obtained
CT value is obtained, passes through and calculates Δ CT (Δ CT=target gene CT average value-reference gene CT average value), Δ Δ CT (Δ Δ CT=
Sample to be tested Δ CT- standard Δ CT), 2-ΔΔCTValue, obtains sample gene relative expression quantity (RQ, Relative quantity).
Specific data are as shown in table 4 and table 5.RQ is more than or equal to 1, and illustration purpose gene is higher than normal cream in the expression of breast cancer tissue
Glandular tissue, for RQ less than 1, expression of the illustration purpose gene in breast cancer tissue will be lower than normal galactophore tissue.
Data pass through statistical calculations, Circular RNA hsa_circ_0021977 phase in breast tumor tissues sample
There is statistics to Circular RNA hsa_circ_0021977 relative expression quantity difference in expression quantity and normal galactophore tissue's sample
It learns meaning (P=0.002).
Hsa_circ_0021977 relative expression quantity in 4 breast tumor tissues of table
| Sample (breast tumor tissues sample) | Hsa_circ_0021977 relative expression quantity |
| BCA0010-T | 1.0000 |
| BCA0011-T | 2.5109 |
| BCA0006-T | 2.4857 |
| BCA0009-T | 2.4208 |
| BCA0007-T | 1.5859 |
Hsa_circ_0021977 relative expression quantity in 5 normal tissue of table
| Sample (normal galactophore tissue's sample) | Hsa_circ_0021977 relative expression quantity |
| PBCA0106-P | 1.0000 |
| PBCA0121-P | 0.0000 |
| PBCA0184-P | 0.0731 |
| PBCA0219-P | 0.0247 |
| PBCA0007-P | 0.4419 |
Result above demonstrates the result that hsa_circ_0021977 in embodiment 1 expresses up-regulation in patient with breast cancer；
It after RT-PCR product is recovered, is sequenced on the full-automatic sequenator of ABI3730, the nucleotide sequence of the segment of the 103bp such as SEQ
Shown in ID NO.1.With 10 software of Vector NTI advance (Invitrogen company) by the sequence and hsa_circ_
0021977 entire RNA sequence is compared, and comparison result is shown, the nucleotides sequence as shown in SEQ ID NO:1 is classified as hsa_
A part of sequence of circ_0021977 gene, coincidence rate 100%.
3 breast cancer detection kit of embodiment
Based on the primer sets that embodiment 2 obtains, the kit of the present invention for breast cancer, the kit are assembled
The primer pair of cDNA including specific amplified hsa_circ_0021977 is as shown in SEQ ID NO:2 and SEQ ID NO:3, and spy
The primer pair of different amplification reference gene (GAPDH) is as shown in SEQ ID NO:4 and SEQ ID NO:5；It further include SYBR Green
Polymerase chain reaction system, such as PCR buffer, SYBR Green fluorescent dye, dNTPs.The ingredient of the PCR buffer is
25mM KCL, 2.5mM MgCL2, 200mM (NH4)2SO4。
By the optimization to primer concentration and annealing temperature, determine that optimal reaction system is as shown in table 6:
6 PCR reaction system of table
| Component | Additional amount |
| SYBR Green polymerase chain reaction system | 12.5μL |
| Upstream primer (10 μM) | 0.5μL |
| Downstream primer (10 μM) | 0.5μL |
| Template cDNA | 2.0μL |
| Sterile purified water is added | To 25 μ L |
Optimum reaction condition are as follows:
95 DEG C of initial denaturation 5min, (95 DEG C of denaturation 15sec, 60 DEG C of annealing 45sec, 72 DEG C of extension 35sec) × 40 circulations,
72 DEG C of extension 15min.
A small amount of mammary glandular cell of 30 breast cancer patients to be detected is taken, the artificial Chinese Academy of Medical Sciences of breast cancer disease to be checked is swollen
Tumor hospital Breast Surgery is gone to a doctor patient with breast cancer.All clinical samples of this research know to patient and inform and through this
Hospital Ethical Committee passes through.RNA is extracted from mammary glandular cell using conventional method (or using specific kit), uses examination
Reagent in agent box carries out PCR according to optimal reaction system and condition and reacts, use in kit normal galactophore tissue cDNA as
Control cDNA in Real-Time PCR quantitative detection detects the hsa_circ_0021977 of tissue samples with respect to normal breast
The variation of tissue expression amount, analysis detection are examined as a result, comparing between sample and control using t, and P < 0.05 is significant difference, is judged to examine
This positive of test sample.
By 30 patients with breast cancer to be detected and cancer in sample, 24 are groups by cancer in expression of tumor tissue level
2-100 times in knitting, in addition 6 expression quantity do not find differences, and clinical detection result and kit prepared by the present invention detection are tied
Fruit is almost the same.Infer accordingly, the diagnostic kit of this breast cancer can clearly distinguish patient with breast cancer, and as clinic
Diagnostic clue is provided.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Cancer Hospital of Chinese Academy of Medical Sciences
<120>hsa_circ_0021977 is preparing the application on Diagnosis of Breast cancer product
<130> P18042
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 103
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<213> Homo sapiens
<400> 1
gatggcttcc agcccaacac ccaagttaag gtaattgcag ccacaaacag ggtggacatc 60
ctggaccccg ccctcctccg ctcgggccgc cttgaccgca aga 103
<210> 2
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<212> DNA
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gatggcttcc agcccaacac 20
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tcttgcggtc aaggcggccc 20
Claims (10)
1.hsa_circ_0021977 preparing the application on Diagnosis of Breast cancer product as molecular marked compound.
2. application as described in claim 1, which is characterized in that the hsa_circ_0021977 is in breast cancer biological sample
Middle expression up-regulation.
3. application as described in claim 1, which is characterized in that the marker includes the nucleosides as shown in SEQ ID NO:1
Acid sequence.
4. application as described in claim 1, which is characterized in that the product is being capable of quantitative detection hsa_circ_0021977
The product of expression quantity.
5. application as claimed in claim 4, which is characterized in that the product is the examination that detection individual suffers from breast cancer possibility
Agent box.
6. application as claimed in claim 5, which is characterized in that the kit includes that specific amplification breast cancer is relevant
The primer and specification of circRNA.
7. application as claimed in claim 6, which is characterized in that the primer sequence such as SEQ ID NO:2 and SEQ ID NO:3
It is shown.
8. application as claimed in claim, which is characterized in that the kit further includes 10 × Buffer, dNTP, MgCl2、
Taq enzyme and SYBR Green fluorescent dye.
9. a kind of biological agent for assessing breast cancer risk, which is characterized in that the biological agent includes effectively to measure
Hsa_circ_0021977 inhibitor.
10. a kind of pharmaceutical composition for treating breast cancer, which is characterized in that described pharmaceutical composition includes hsa_circ_
0021977 inhibitor further includes pharmaceutically acceptable carrier as active constituent.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057764A (en) * | 2019-12-25 | 2020-04-24 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of CircRNA PVT1 and peptide fragment in tumor growth prediction, metastasis prediction, prognosis evaluation and treatment |
| CN111647598A (en) * | 2020-02-11 | 2020-09-11 | 昆明医科大学 | siRNA for inhibiting expression of hsa _ circ _0027477 and application thereof |
| CN113913524A (en) * | 2021-11-09 | 2022-01-11 | 上海市第十人民医院 | Early breast cancer diagnosis model and diagnosis system |
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