CN115992201A

CN115992201A - Breast cancer marker and application thereof

Info

Publication number: CN115992201A
Application number: CN202211045951.1A
Authority: CN
Inventors: 陈敏; 徐建震; 于浩洋; 张国君
Original assignee: Shenzhen Genebiohealth Co ltd
Current assignee: Shenzhen Genebiohealth Co ltd
Priority date: 2017-05-16
Filing date: 2018-05-10
Publication date: 2023-04-21
Also published as: CN108220433A; CN109415770B; CN108220433B; CN109415770A; WO2018210173A1

Abstract

The present application relates to a circRNA selected from the group consisting of circTADA2A-E6, circTADA2A-E5/E6, circNOL10, circNSUN2, circCSRNP2, circFAM125B, circCDC and circABCC1, as a marker of breast cancer, and uses thereof. The invention also relates to kits, microarrays, medicaments and related methods and uses for breast cancer diagnosis, prognosis and treatment.

Description

Breast cancer marker and application thereof

The application is a divisional application, the application number of the original application is 201880002722.7, the application date is 2018, 5 months and 10 days, and the name of the application is breast cancer marker and application thereof.

Technical Field

The present invention relates to compositions, methods and uses for diagnosis, prognosis and treatment of breast cancer. In particular, the present invention relates to circRNA molecules associated with breast cancer diagnosis, prognosis and treatment, kits and microarrays associated therewith for breast cancer diagnosis, prognosis and treatment, and associated methods and uses.

Background

Breast cancer is a common malignancy, the first of which is in global female cancer incidence. In recent years, the incidence rate and the death rate of breast cancer in China rise year by year, and the health of Chinese females is seriously endangered. Breast cancer is a complex and diverse disease from the point of view of molecular changes associated with clinical medicine, and should be considered as a class of tumors with varying levels of molecules. There are at least 4 molecular subtypes of breast cancer clinically today: luminal A (ER+PR+Her2-), luminal B (ER+PR-Her2-), her2 (ER-PR-and Her2+) and TNBC (ER-PR-and Her2-). Today, detection of biomarkers has also begun to be used for prognosis of breast cancer and adjuvant chemotherapy such as 21-gene signature (Oncotype DX) and European common 70 gene detection Mammaprint (European common 70-gene detection MammaPrint). Thus, it was found that studies to identify and group biomarkers for breast cancer were necessary and important.

Circular RNA (circRNA) is thought to be caused by non-classical splicing of linear pre-mRNA into circular form. The circRNA is evolutionarily conserved, stable in structure and high in abundance in caenorhabditis elegans (C.elegans), zebra fish, drosophila, mice and humans. The abundance of some circrnas is up to 200 times that of its linear counterpart. The circRNA was determined to be a stable structure produced mainly by exons or introns and was produced by reverse splicing (back splicing) or lasso intron (lariat intron) differences (differentially generated), followed by formation of a covalently closed loop structure without either a 5 'cap or 3' tail. The exon circRNA is very stable in cells, compared to an average half-life of 10h for mRNA, with most species of exon circRNA having half-lives exceeding 48h. Both exon and intron circrnas play a role in regulating gene expression. Recent studies have shown that: the circRNA may exert its biological functions by several means, such as actively involved in pre-mRNA splicing, affecting gene expression by binding to proteins, sponges (epigenetic miRNA sponges) that regulate miRNAs by epigenetic regulation, RNA transporters, and promoting transcription of their parent mRNA. To date, over 10,000 human circrnas have been found to be widely distributed and diverse in humans, often with tissue and developmental stage specificity, intimately associated with molecular biology and molecular oncology.

Circular RNAs (circrnas) have been studied as widely distributed and diverse endogenous non-coding RNAs with great regulatory potency, demonstrating that circrnas play an important role in the development of several types of diseases, such as atherosclerosis and neurological disorders. In recent years, the role and function played by circRNA in cancer has become a new focus in the field of cancer research. The unique features of circRNA are constantly being revealed and discovered, which will expand the awareness of cancer, especially in terms of carcinogenesis and malignant progression of cancer. The expression of circRNA is abnormal in human cancers such as esophageal cancer, CRC and gastric cancer. The circITCH shows antitumor function in esophageal squamous cell carcinoma by interacting with mirnas such as miR-7, miR-17 and miR-214. Ahmed et al @ through Ingenuineny IPA tool kit

QIAGEN Redwood City, www.qiagen.com/ingness) enriched RNAs found 312 circrnas that were differentially expressed in breast or ovarian cancer.

The literature reports that the general features of circrnas are a large and numerous variety, wide distribution, strong evolutionary conservation, tissue specificity, extremely high stability and high abundance expression. The unique advantage of circRNA makes it a novel marker useful for disease diagnosis, prognosis and predictive therapeutic response. First, circRNAs were demonstrated as senescence biomarkers of drosophila. In gastric cancer and hepatocellular carcinoma (HCC), circRNA was found to be a novel cancer biomarker. After analysis of breast cancer circRNA sequencing (circ-Seq) of a large number of samples in the TCGA pool, the recently Kalari study group identified new circrnas with breast cancer specificity, and also classified unique sets of circrnas corresponding to different breast cancer subtypes: triple Negative (TNBC), estrogen receptor positive (er+) and HER2 positive (her2+), from which it can be seen that circRNA is likely a novel marker for breast cancer diagnosis. Furthermore, circRNA is detectable in human body fluids such as plasma, saliva and exosomes (exosomes), suggesting that circRNA is more likely to be clinically useful in cancer diagnosis.

Disclosure of Invention

The inventors studied the expression of circRNA in primary tumor samples of breast cancer patients to identify circRNA that can be used as a marker or target for breast cancer.

According to a first aspect of the present invention there is provided a circRNA for use as a marker (e.g. diagnostic or prognostic marker) or target for breast cancer, the circRNA being selected from one or more of the following: the ingredients include circTADA2A-E6, circTADA2A-E5/E6, circNOL10, circNSUN2, circCSRNP2, circFAM125B, circCDC and circABCC1.

According to a second aspect of the present invention there is provided the use of a reagent and/or microarray for detecting circRNA as described in the first aspect in the preparation of a kit for breast cancer diagnosis or prognosis.

According to a third aspect of the present invention there is provided the use of a specific probe for detecting circRNA as described in the first aspect in the preparation of a microarray for the diagnosis or prognosis of breast cancer.

According to a fourth aspect of the present invention there is provided a kit for diagnosis and/or prognosis of breast cancer comprising reagents or a microarray for measuring the expression level of circRNA according to the first aspect 1.

According to a fifth aspect of the present invention there is provided a method of diagnosing whether a subject has, or is at risk of developing, breast cancer, or prognosticating breast cancer, comprising measuring the expression level of the circRNA of the first aspect in a test sample. The method may comprise:

reverse transcribing RNA from a test sample obtained from the subject to obtain a reverse transcribed product;

detecting the expression level of the circRNA of the first aspect; and

comparing the expression level of the circRNA of the test sample with the expression level of the circRNA of normal breast tissue as a control,

wherein a lower level of expression of the circRNA in the test sample than the control sample is indicative of a subject suffering from or at risk of developing breast cancer, or indicative of a poor prognosis of breast cancer.

According to a sixth aspect of the invention, the use of circTADA2A-E6, an agent that increases the expression level of circTADA2A-E6 and/or a nucleic acid that expresses circTADA2A-E6, in the manufacture of a medicament for inhibiting or treating breast cancer.

According to a seventh aspect of the present invention there is provided a method for identifying an inhibitor of breast cancer, the method comprising identifying an agent that increases the expression level of circRNA in breast cancer cells. The circRNA is preferably circTADA2A-E6 and/or circNOL10.

According to an eighth aspect of the present invention there is provided a breast cancer inhibitor identified by the method of the seventh aspect.

According to a ninth aspect of the present invention there is provided the use of an inhibitor according to the eighth aspect in the manufacture of a medicament for inhibiting or treating breast cancer.

According to a tenth aspect of the present invention there is provided a pharmaceutical composition for use in the inhibition or treatment of breast cancer comprising circRNA, an agent that increases the expression level of circRNA, a nucleic acid that expresses circRNA and/or a breast cancer inhibitor according to the eighth aspect, optionally further comprising an additional agent for use in the inhibition or treatment of breast cancer. The circRNA is preferably circTADA2A-E6 and/or circNOL10.

According to an eleventh aspect of the present invention there is provided a method for inhibiting or treating breast cancer comprising administering to a patient in need thereof an inhibitory therapeutically effective amount of circRNA, an agent that increases the expression level of circRNA, a nucleic acid that expresses circRNA and/or a breast cancer inhibitor as described in the eighth aspect, optionally administering a further agent for inhibiting or treating breast cancer. The circRNA is preferably circTADA2A-E6 and/or circNOL10.

For the uses, microarrays, kits, methods, compositions or inhibitors described above, wherein the breast cancer may be selected from the group consisting of Luminal a and B, HER2+ and triple negative subtype breast cancer, more preferably triple negative subtype breast cancer.

Drawings

FIG. 1. Profile analysis of circular RNA in human normal breast tissue and breast cancer tissue. (a) scatter plot, X-axis: normal breast tissue (standardized), Y-axis: TNBC organization (standardized). The green line represents the fold change. The circRNA above the top green line and below the bottom green line indicated that the circRNA expression level varied by more than 1.5 fold between the two groups of samples. (b) volcanic plot, X axis: log2 (fold change), Y axis: log10 (P value). The vertical green line corresponds to 1.5-fold up and down regulation, and the horizontal green line represents a p value of 0.05. Red dots in the figure represent differentially expressed circrnas of statistical significance. (c) Comparison of circRNA profiles between normal breast tissue and breast cancer tissue (Luminal A and TNBC subtypes). (d) Cluster heatmaps (clustered heatmap) of the differentially expressed circrnas between breast cancer tissue and human normal breast tissue, rows representing the circrnas and columns representing tissue samples. The circrnas were classified according to Pearson correlation. The values represent the average SRPBM converted to log10 for each group. (e) A network of 215 circrnas that are differentially expressed in breast cancer and their target mirnas in breast cancer. The network consists of 212 upregulated circrnas (red nodes), 93 downregulated circrnas (yellow nodes) and their target mirnas (blue nodes). (f) Comparison between microarray data of 8 selected circrnas and qRT-PCR results.

FIG. 2. Expression levels of two circTADA2A and prognostic and predictive value of TNBC by the two circTADA 2A. (a-b) compared to normal breast tissue (n=16), the expression levels of circTADA2A-E6 (a) and circTADA2A-E5/E6 (b) in the following different breast cancer subtypes were verified by qRT-PCR: LA (n=25), LB (n=21), her-2 (n=17) and TNBC (n=115). * P <0.05, P <0.01. (c-d) breast cancer cell lines and immortalized breast cell lines MCF-10A, the expression of circTADA2A-E6 (c) and circTADA2A-E5/E6 (d) was analyzed by qRT-PCR. (E-f) AUC of ROC analysis of cictada 2A-E6 and cictada 2A-E5/E6 in TNBC (n=115), LA (n=25), LB (n=21) and Her-2 (n=17), respectively. Correlation of (g-h) circTADA2A-E6 and circTADA2A-E5/E6 expression with disease-free survival (DFS) (h) and total survival (OS) (g). 8 of the n= 107,115 patients (6.96%) were out of visit after the first surgery.

FIG. 3. Overexpression of circTADA2A-E6 inhibited breast cancer cell proliferation, colony formation, invasion. (a) Construction of lentiviral circTADA2A-E6 expression vector plvx-CMV-circTADA2A-E6-EF 1-ZsGreen-Puro. (b) After transfection with the circTADA2A-E6 vector or control vector in MCF-7 and MDA-MB-231 cells, ectopic circTADA2A-E6 expression was analyzed by qRT-PCR. (c) CCK-8 assay and proliferation rates of MCF-7 and MDA-MB-231 cells with ectopic circTADA2A-E6 expression. (d) Cloning of MCF-7 and MDA-MB-231 cells overexpressing circTADA 2A-E6. Representative plots are shown on the left and quantification of the foci/wells are shown on the right. Error bars represent the mean ± SD of three independent experiments. (e) Representative images of MCF-7 and MDA-MB-231 cell scratch assays overexpressing circTADA 2A-E6. (f) The effect of ectopic circTADA2A-E6 expression on cell invasion was studied using a Boyden cell. Morphological comparisons of cell penetration are shown on the left, while quantification of penetrated cells is shown on the right. Error bars represent the mean ± SD of three independent experiments. * p <0.05, p <0.01. (g) Protein levels of E-cad and vimentin were assessed by Western blotting of MCF-7 and MDA-MB-231 cells over-expressing circTADA 2A-E6. In the figure, "vector control" is the vector control.

FIG. 4.CirctaDA2A silencing promotes breast cancer cell proliferation, clonogenic, migration, and invasion. (a) Schematic and target sequence of siRNA that specifically binds to the reverse splice junction of circTADA 2A-E6. (b) qRT-PCR analysis of the expression of the circTADA2A-E6 RNA after transfection of cells with two circTADA2A-E6 siRNA. (c) The effect of the circTADA2A-E6 siRNA on cell proliferation of MCF-7 and MDA-MB-231 cells was evaluated by CCK-8 assay. (d) Colony formation assays on MCF-7 and MDA-MB-231 cells transfected with two circTADA2A-E6 siRNAs. Representative plots are shown on the left and quantification of the foci/wells are shown on the right. Error bars represent mean ± SD. Data represent three independent experiments. (e) The effect of circTADA2A-E6 siRNA expression on cell invasion was studied using a Boyden cell. Morphological comparisons of cell penetration are shown on the left, while quantification of the number of penetrated cells is shown on the right. (f) Wound healing assay of MCF-7 and MDA-MB-231 cells transfected with two circTADA2A-E6 siRNAs. Representative images are shown on the left and quantification of cell migration is shown on the right. (g) Protein levels of E-cad and Vimentin (Vimentin) were assessed by Western blotting after transfection with two types of circTADA2A-E6 siRNA using MCF-7 and MDA-MB-231 cells.

FIG. 5. Expression levels of circNL10, prognostic and predictive value of circNL 10. (a) Real-time fluorescent quantitative PCR analysis of the expression level of circNOL10 in different molecular types of breast cancer tissues and normal breast tissues: LA (n=25), LB (n=21), her-2 (n=17) and TNBC (n=115), normal (Normal) (n=16)..p <0.05, P <0.01. (b) Real-time fluorescent quantitative PCR analysis of expression level of circNOL10 in breast cancer cell line and normal-like breast cell MCF-10A. (c) ROC curve analysis-area under curve of circNOL10 in different molecular types of breast cancer. (d) relationship between expression level of circNOL10 and DFS and OS. The number of cases involved in the analysis was 107 cases (8 out of 115 cases were lost after surgery, the rate of lost visit was 6.96%).

Detailed Description

Before describing the present methods and compositions, it is to be understood that this invention is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The description of the embodiments is intended to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and is not intended to limit the scope of what the inventors regard as their invention nor is it intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for.

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. Some potential and preferred methods and materials are now described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It will be appreciated that in the event of a conflict, the present disclosure will control to any of the publications cited.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features in other several embodiments without departing from the scope or spirit of the present invention. Any recited method may be implemented in the order of recited events or any other order that is logically possible.

As used herein and in the appended claims, unless otherwise indicated, "a" or "an" means "one or more".

When a numerical range is provided, it is to be understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value 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 in or excluded from the range, and each range where either or both of the upper and lower limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded from the range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

A "therapeutic agent" is an atom, molecule, or compound that can be used to treat a disease. Examples of therapeutic agents include antibodies, antibody fragments, peptides, drugs, toxins, enzymes, nucleases, hormones, immunomodulators, circular RNAs, antisense oligonucleotides, small interfering RNAs (sirnas), chelators, boron compounds, photosensitizers, dyes and radioisotopes.

"circular RNA" or "circRNA" means a novel non-coding RNA which exists widely and variously in mammalian cells, and which has endogenous RNA molecules with gene expression regulating action, mainly consisting of exon transcripts and introns which are spliced in a nonlinear reverse direction, and which form closed loops by covalent bonds from end to end, exhibiting characteristics different from those of linear RNA.

The invention provides the following technical scheme:

use of circRNA as a marker for breast cancer, wherein the circRNA is selected from one or more of the following: the circlTADA 2A-E6, circlTADA 2A-E5/E6, circlNOL 10, circlNSUN 2, circlCSRNP 2, circlFAM 125B, circCDC and circlABCC 1 are preferably selected from one or more of the following: the circTADA2A-E6, the circTADA2A-E5/E6 and the circNOL10.

2. Use of a reagent and/or microarray for detecting circRNA as described in 1 above, preferably comprising a specific probe, for the preparation of a kit for breast cancer diagnosis.

3. Use of a specific probe for detecting circRNA as described in 1 above for the preparation of a microarray for breast cancer diagnosis.

4. A kit for diagnosing breast cancer, comprising a reagent or microarray for measuring the level of circRNA according to 1 above, the reagent preferably comprising a specific probe.

5. A method of diagnosing whether a subject has or is at risk of developing breast cancer comprising measuring the level of circRNA according to 1 above in a test sample, the method preferably comprising:

(1) Reverse transcribing RNA from a test sample obtained from the subject to obtain a reverse transcribed product;

(2) Measuring the level of circRNA according to 1 above; and

(3) Comparing the level of the circRNA of the test sample with the level of the circRNA of normal breast tissue as a control,

wherein a lower level of the circRNA in the test sample than the control sample is indicative of the subject suffering from or at risk of developing breast cancer.

6. The method of item 5 above, wherein step (2) comprises hybridizing the reverse transcription product to the circRNA-specific probe or microarray for detecting the circRNA.

7. The method of 5 above, comprising measuring the level of the circRNA by qRT-PCR.

8. The use of the circRNA according to 1 above as a prognostic marker for breast cancer.

9. Use of a reagent or microarray for measuring the level of circTADA2A-E6 and/or circNOL10, preferably comprising a specific probe, in the preparation of a kit for prognosis of breast cancer.

Use of a specific probe of circtada2a-E6 and/or circNOL10 for the preparation of a microarray for prognosis of breast cancer.

11. A kit for breast cancer prognosis comprising reagents and/or microarrays for detecting circTADA2A-E6 and/or circNOL10, said reagents preferably comprising specific probes.

12. A method for prognosis of a breast cancer patient comprising measuring the level of circTADA2A-E6 and/or circNOL10 in a test sample, said method preferably comprising:

(2) Detecting the level of circTADA2A-E6 and/or circNOL 10; and

(3) Comparing the level of the circTADA2A-E6 and/or circNOL10 of the test sample with the level of the circTADA2A-E6 and/or circNOL10 of normal breast tissue as a control,

wherein a lower level of the circTADA2A-E6 and/or circNOL10 in the test sample than the level of the circTADA2A-E6 and/or circNOL10 in the control sample is indicative of a poorer prognosis of the breast cancer.

13. The method according to above 12, wherein step (2) comprises hybridizing the reverse transcription product with a specific probe of the circTADA2A-E6 and/or circNOL10 or a microarray for detecting the circTADA2A-E6 and/or circNOL10.

14. The method according to 12 above, comprising measuring the level of the circTADA2A-E6 and/or circNOL10 by qRT-PCR.

Use of circTADA2A-E6 and/or circNOL10, an agent which increases the level of circTADA2A-E6 and/or circNOL10 and/or a nucleic acid which expresses circTADA2A-E6 and/or circNOL10 in the manufacture of a medicament for the inhibition or treatment of breast cancer.

16. A method for identifying a breast cancer inhibitor, the method comprising identifying an agent that increases the level of circTADA2A-E6 and/or circNOL10 in a breast cancer cell.

17. A breast cancer inhibitor identified by the method according to 16 above.

18. Use of an inhibitor according to 17 above in the manufacture of a medicament for inhibiting or treating breast cancer, wherein the medicament optionally further comprises an additional agent for inhibiting or treating breast cancer.

19. A pharmaceutical composition for inhibiting or treating breast cancer comprising circTADA2A-E6, an agent that increases the level of circTADA2A-E6, a nucleic acid that expresses circTADA2A-E6, and/or a breast cancer inhibitor according to 17 above, optionally further comprising an additional agent for inhibiting or treating breast cancer.

20. A method for inhibiting or treating breast cancer comprising administering to a patient in need thereof an inhibiting therapeutically effective amount of cictada 2A-E6, an agent that increases the level of cictada 2A-E6, a nucleic acid that expresses cictada 2A-E6, and/or a breast cancer inhibitor according to 17 above, optionally with other agents for inhibiting or treating breast cancer.

21. The use, microarray, kit, method, composition or inhibitor according to any one of the preceding claims 1-20, wherein the breast cancer is selected from the group consisting of luminel a and B, HER2+ and a triple negative subtype breast cancer, more preferably a triple negative subtype breast cancer.

22. The use, composition or method according to any one of the preceding claims 15 and 19-21, wherein the nucleic acid is a vector for expressing circTADA2A-E6 and/or circNOL10.

Examples

The invention will be further described with reference to the accompanying drawings and the following specific examples. It should be understood that the invention is not limited to these specific embodiments.

Materials and methods

Clinical population and sample collection

From breast cancer surgical specimens between 2009 6 and 2015 of the oncology hospital affiliated to the university of shan university of china, a total of 121 breast cancer tissues (LA, n=25, lbn=21, her-2, n=17, tnbc, n=58) and 16 normal breast tissues were collected. Another 57 TNBC were collected from tumor hospitals and Yi people's hospitals, respectively, in Zhejiang province, china. The medical ethics committee of the university of Shandong university medical institute, zhejiang province tumor hospital and Linyi people hospital approves the proposal, and participants signed written informed consent prior to surgery.

All fresh tumor tissue samples were immediately preserved

RNA stabilizing solution (++>

RNA Stabilization Solution, invitrogen, beijin, china). To allow the solution to penetrate the tissue sufficiently, the tissue is kept at 4 ℃ overnight, then the supernatant is removed and transferred to-80 ℃ for long term storage. Corresponding adjacent non-tumor tissue was taken 5cm from the margin of the cancer and evaluated by an experienced pathologist to be free of significant tumor cells. Tumors were staged according to the tumor-lymph node-metastasis (TNM) staging system of international union for cancer (International Union Against Cancer). According to the United states national Integrated cancer network (National Comprehensive Cancer Networ)k (NCCN)) clinical practice guidelines for oncology (v.1.2011), the histological grading is assessed. H for normal mammary tissue&E staining and verification by pathologists.

circRNA microarray hybridization

Sample preparation and microarray hybridization were performed based on the Arraystar standard protocol (Arraystar Inc.). Briefly, total RNA was digested with rnase (Epicentre, inc.), linear RNA was removed and circular RNA was enriched. The enriched circular RNA was then amplified and transcribed into fluorescent cRNA by random priming (Arraystar Super RNA Labeling Kit; arraystar). The labeled cRNA was purified with the RNeasy Mini Kit (Qiagen). The concentration and specific activity of labeled cRNA (pmol Cy 3/. Mu.g cRNA) was determined using a NanoDrop ND-1000. The labeled cRNA was hybridized to Arraystar Human circRNA Array (8×15k, arraystar). After washing the slide, the array was scanned with Agilent Scanner G2505C.

circRNA microarray data analysis

Data was extracted using Agilent Feature Extraction software (version 11.0.1.1). A series of data processing, including quantile normalization (quantile normalization), is performed with the R software package. Differentially expressed circrnas were selected based on fold change cutoff (FC > =1.5) and statistically significant P-values (P-value < =0.05). The miRNA target of the circRNA and the circRNA-miRNA interaction were predicted using the TargetScan42 and miRanda43 based Arraystar homemade computer program. To concentrate the targeted miRNA profile, the efficacy of the predicted miRNA targets are scored and ranked (rank) using the miRNA support vector regression (mirSVR) algorithm. Accordingly, for each circRNA, the 5 mirnas with highest mirSVR scores were identified to build a "top 5" circRNA-miRNA network (1 circRNA linked to 5 mirnas).

Verification of candidate circRNA Using qRT-PCR

Total tissue RNA was extracted from breast cancer tissue and normal breast tissue using Trizol reagent (Life Technologies, carlsbad, USA) according to the manufacturer's instructions. The purity and concentration of the RNA samples were determined using a NanoDrop ND-1000 (Thermo Fisher Scientific, wilmington, DE). RNA integrity was assessed by electrophoresis on denaturing agarose gels. RNA was prepared and stored at-80℃for validation experiments.

By using

II First Strand cDNASynthesis Kit (Genesed, china), cDNA was prepared from total RNA. Primers used in qRT-PCR were designed as convergent primers (convergentprimer) to detect loop junctions (circular junction) and span reverse splice junctions. From a database "circinter"https:// The sequence of 3 kinds of circRNA was obtained by circinter. Delta Ct for dataAnd (5) analyzing by a method. All primers were synthesized by general Biotech. All results are expressed as mean ± SD of three independent experiments.

Annotation and functional prediction of validated candidate circTADA2A-E6

The MiRanda is combined according to TargetScan (http:// www.targetscan.org /)http:// wwwMicrora. Org /) analysis, the verified candidate circrnas were used as seeds to enrich the circRNA-miRNA-gene network. The Cytoscape (http:// www.cytoscape.org /) was used to build a circRNA-miRNA-mRNA interaction network for circTADA 2A. Predicted gene function in the network is annotated with GO and KEGG path analysis. The light is transmitted through Database for Annotation, visualization and Integrated Discovery (DAVID;http:// www.david.abcc.ncifcrf.gov/) Biological pathways defined by Kyoto Encyclopedia of Genes and Genomes (KEGG), biocarta and reactiomer (http:// www.genome.jp/KEGG /) were identified.

Detection of putative miRNA binding sites

FASTA files in the circRNA that putative miRNA binding sites were obtained from miRBase release 20.0 (http:// www.mirbase.org /). Only mature mirnas were considered for seed analysis. The miRNA was aligned with circRNA. The putative target site of a miRNA is a 6 nucleotide long sequence in the genome representing the reverse complement of nucleotides 2-7 of the mature miRNA sequence.

cDNA Synthesis and qRT-PCR for mRNA and circRNA

Using

II First Strand cDNA Synthesis Kit (Geneseed, china) cDNA was synthesized from total RNA according to the supplied protocol. By base->

The ABI Prism 7500 sequence detection System (Applied Biosystems, foster City, calif., USA) of Green method measures the relative gene expression of circular RNAs. qPCR Ct values were automatically calculated using the manufacturer's software. Human-ACTB was used as an internal control. Specifically, the abundance of the circRNA was determined using divergent primers (primers) annealed to the distal end of the circRNA. The PCR products were directly Sanger sequenced using sequencing primers.

cDNA synthesis and qRT-PCR for miRNA

For quantitative determination of the amount of mature miRNA, mir-X was used ^TM mature miRNA was reverse transcribed by the miRNA First-Strand Synthesis (Takara, china) and PCR amplified using Mir-X miRNA qRT-PCR SYBR Kit on the ABI Prism 7500 sequence detection system (Applied Biosystems, foster City, calif., USA) with small core U6B (RNU 6B) RNA as an internal standard control. With 2 ^-ΔΔCt The method was used for relative quantification.

construction of the circTADA2A vector

To outline the circRNA, the (genomic region) mature sequence of circTADA2A-E6 was synthesized and cloned into pLCDH-ciR-copGGFP-pur (Geneseed, guangzhou, china). And verified by sequencing.

circTADA2A siRNA interference

siRNA to the circTADA2A sequence was designed and synthesized by Ribobio (Guangzhou, china) and targeted to the junction region (junction region) of the circTADA2A sequence. The siRNA target sequence is as follows:

1：5’-CCATTTCACTGCAGGATGT-3’；

2：5’-CACTGCAGGATGTAGCCAA-3’；

3：5’-TTCCATTTCACTGCAGGAT-3’。

oligonucleotide transfection

miRNA mimics and inhibitors are synthesized by Ribobio (Guangzhou, china). Lipofectamine for cells ^TM 3000(invitrogen,USA)And (5) transfection.

Western blot analysis

Protein extraction and western blotting were performed as described previously.

Proliferation, cell cycle, apoptosis and cell invasion assays, in vitro tumorigenesis assays

Proliferation assay (CCK-8) proliferation of MCF-7 and MDA-MB-231 cells was tested using the CCK-8 kit (Doindo, japan). About 1x10 to be transfected ³ Cells/well were incubated in triplicate in 96-well plates. 24. At 48, 72, 96 hours, CCK-8 reagent was added to each well and incubated for 1.5 hours at 37 ℃. The optical density at 450nm was measured with an automatic microtiter plate reader (Synergy 4; bioTek, winioski, VT, USA).

Cell migration invasion assay (Transwell invasion assay) invasion assay was performed with cell culture insert (8. Mu.M pore size; BD) and matrigel invasion cell (BD), respectively. The transfected cells were serum starved for 24h, 1X10 in serum-free medium ⁴ MDA-MB-231 cells or 2X 10 ⁴ MCF-7 cells were seeded into the upper chamber. Cells were fixed with 4% paraformaldehyde at 20 hours (MDA-MB-231) or 48 hours (MCF-7), respectively, for invasive assays, followed by staining with 0.1% crystal violet. Each assay was performed in triplicate. The number of cells in 3 fields of view in each well was counted by two investigators.

For the colony formation assay, 100 MDA-MB-231 cells or 200 MCF-7 cells were plated in triplicate in 12-well plates and allowed to grow until visible colonies appear. Colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet and counted.

Statistical analysis

All statistical data were analyzed with Statistical Product and Service Solutions SPSS software 16.0.16.0 (SPSS, chicago, IL), graphPad Prism 5.0 (GraphPad Software, la Jolla, calif.) and SigmaPlat 10.0 (SigmaPlot Software, la Jolla, calif.). The difference in the level of circRNA between breast cancer tissue and normal breast tissue was determined using t-test pair-wise data. The correlation between the circRNA levels and the clinical pathology factors was further analyzed by one-way analysis of variance (ANOVA). A subject operating characteristic (ROC) curve is established to evaluate the diagnostic value thereof. The cutoff value of the circRNA was analyzed with SigmaPlot 12.3. Experimental reproducibility was determined by Pearson correlation test (Pearson correlation test). All experiments were performed in triplicate. Data are presented as mean ± SEM (unless otherwise indicated), and were statistically analyzed using a double sided Student t test. P <0.05 is considered statistically significant.

Example 1 screening and validation of differentially expressed circRNA in breast cancer

High throughput sequencing of the circRNA chip helps to identify the differentially expressed circRNA between breast cancer and normal breast tissue. Differential expression of circrnas from 8 breast cancer tissues (TNBC, n=4, luminal a, n=4) and three normal breast tissues was analyzed by chip technology, resulting in differential expression profiles of breast cancer and normal tissues. The scatter diagram shows that the difference expression of the circular RNAs between the two groups is more than 1.5 times; volcanic images show differentially expressed circular RNAs of statistical significance between the two groups. In total 140 up-regulated and 95 down-regulated circular transcripts were found in breast cancer compared to normal breast tissue (fig. 1 c), and this expression profile data was consistent with other studies, indicating that the circrnas were mostly from exons and introns of the gene-containing region. Notably, the circrnas showed different expression clusters for primary tumor and normal breast tissue (fig. 1 d). The interactions between these circrnas and their target mirnas can be predicted theoretically by software from complementary miRNA matching sequences. A total of 465 mirnas were predicted to bind to circRNA. The complete circRNA-miRNA interaction network was mapped by Cytoscape software (fig. 1 e). All of these data indicate that: there are a wide variety of differentially expressed circrnas in breast cancer that may be useful in disease screening and diagnosis.

The circRNA differential expressed in breast cancer TNBC tissues is screened by a circRNA microarray, and 8 kinds of circRNA with the down-regulated expression differential multiple more than 1.5 times are screened. The results are shown in Table 1.

TABLE 1 screening of circRNA differentially expressed in breast cancer TNBC tissue by circRNA microarray

Fluorescent quantitative PCR confirmed differentially expressed circRNA in triple negative breast cancer tissue

The 8 down-regulated circrnas described above (see table 1) were selected and these differentially expressed circrnas were further confirmed in 178 breast tissues by real-time fluorescent quantitative PCR (qRT-PCR). The length of 8 circrnas ranges from 158nt to 843nt, all of these 8 circrnas are exon spliced, and primers (outward-circulating primer) are designed to the outside of the circular RNA transcript at both ends of the exon, to distinguish from the linear product encoded by the gene. Next, the circRNA is reverse transcribed into cDNA using random hexamer primers and amplified using reverse primers to yield the desired circular specific RNA product. Finally, sequencing of the amplified product confirmed that the circular RNA circularization site sequence was identical to the circular RNA sequence with splice junctions from CircBase. Finally, we used the divergent primer set (divergent primer set) to determine the expression level of circRNA in triple negative breast cancer tissue and normal breast tissue samples (n=16). All of these circrnas were confirmed for differential expression, demonstrating the accuracy of the circular RNA chip data (see fig. 1 f).

Example 2 expression levels of two types of circTADA2A and prognostic and predictive value of TNBC by the two types of circTADA2A

Two circular RNAs that are down-regulated in breast cancer tissue

First, the expression of two types of circTADA2A-E6 and circTADA2A-E5/E6, both of which originate from the TADA2A gene, in breast cancer tissues of different molecular types was identified. As shown in fig. 2c and 2d, qRT-PCR analysis showed that: the expression level of the two types of circTADA2A in BC tissues is significantly lower than that of normal breast tissues, and is: the circlTADA 2A-E6, LA > TNBC > Her-2> LB; circTADA2A-E5/E6, LB > TNBC > Her-2> LA (all groups, p < 0.01). We found that the expression levels of circTADA2A-E6 (see FIG. 2 c) and circTADA2A-E5/E6 (see FIG. 2 d) were lower in 10 breast cancer cell lines compared to MCF-10, a non-tumorigenic epithelial cell line.

Correlation between expression of two circTADA2A and clinical case profile

To investigate the potential correlation between expression of circTADA2A-E6 and circTADA2A-E5/E6 and clinical parameters, we analyzed the TNM (tumor size, lymph node metastasis and distant metastasis) status, and the results showed that the decrease in expression level of the tumor circTADA2A-E6 was significantly correlated with more lymph node metastasis (p=0.012) and high clinical stage (p=0.022) (fig. 2E and 2 f), but that there was no obvious correlation between circTADA2A-E5/E6 and TNM stage. These results suggest: there is a correlation between the down-regulation of the expression of circTADA2A-E6 and the invasive character of TNBC.

The results are summarized in tables 2 and 3.

TABLE 2 correlation between clinical pathological factors and expression of circTADA2A-E6 in TNBC tissue (n=115)

AJCC,American Joint Committee on Cancer

# 4.35% loss of patient information

# 9, 56% of patient information was lost

*p<0.05

TABLE 3 correlation between clinical pathological factors and expression of circTADA2A-E5/E6 in TNBC tissue (n=115)

AJCC,American Joint Committee on Cancer

# 4.35% loss of patient information

# 9, 56% of patient information was lost

*p<0.05

Diagnostic value of the expression of circTADA2A-E6 and circTADA2A-E5/E6 in breast cancer

To assess whether two circTADA2A can be used as a diagnostic index for triple negative (n=115) breast cancer, ROC curves were made using a total of 16 normal breast tissues as controls. In the ROC curves of TNBC compared to the normal group, the areas under the curves of the circTADA2A-E6 and the circTADA2A-E5/E6 are 0.8554 (95% ci= 0.9089-1.016, p <0.0001, fig. 2E) and 0.9366 (95% ci= 0.9089-1.016, p <0.001, fig. 2 f), respectively. In addition, it was found that: the areas under all curves in the circTADA2A-E6 and circTADA2A-E5/E6 in LA (n=25), LB (n=21) and Her-2 (n=17) were higher than 0.84 (FIG. 2E/f). These results suggest: two types of circTADA2A can be used as potential diagnostic biomarkers for breast cancer.

Assessment of the risk of breast cancer by the use of circTADA2A-E6 and circTADA2A-E5/E6

Prior to assessing the prognostic value of the expression of circTADA2A-E6 and circTADA2A-E5/E6 in breast cancer, a one-factor and multi-factor Cox proportional risk regression analysis (Cox proportional hazards regression analysis) was performed on 115 TNBC patients. The results indicate that TNM stage and tumor size are two independent factors related to total survival (OS) and disease-free survival (DFS) of TNBC patients (results not shown). Importantly, it was found that: the total 10 year survival of the circTADA2A-E6 low expression group was significantly shorter than that of the circTADA2A-E6 high expression group (FIG. 2h, p=0.0179), and the 10 year DFS of the circTADA2A-E6 low expression group was significantly shorter than that of the circTADA2A-E6 high expression group (FIG. 2 g), with p-value 0.0715. However, the circTADA2A-E5/E6 expression was not significantly associated with the OS (FIG. 2 h) and DFS (FIG. 2 g) of TNBC patients. These analytical data suggest: circTADA2A-E6 is a potential predictive biomarker for breast cancer progression.

EXAMPLE 3 overexpression or silencing of circTADA2A-E6 inhibits or enhances breast cancer cell proliferation, colony formation, invasion

circTADA2A-E6 inhibits cell proliferation and invasion

A circTADA2A high expression vector was constructed and siRNA interference sequences for knockdown (fig. 3a and fig. 4 a). CCK-8 assay results showed: high expression of circTADA2A reduced the proliferative capacity of the cells (p <0.05, fig. 3 c), while interfering with circTADA2A increased the proliferative capacity of the cells (p <0.05, fig. 4 c). Scratch healing experiments showed that: high expression of circTADA2A reduced the scratch healing capacity of the cells (fig. 3 e), whereas knockdown promoted the scratch healing capacity of the cells (fig. 4 f). The results of the clone formation experiments showed that: the clonality was reduced in the cells overexpressing circTADA2A-E6 (FIG. 3 d), whereas the clonality was increased in the cells knocked down by circTADA2A-E6 (FIG. 4 d). Furthermore, the results of the invasive experiments showed that: the cell invasion rate was reduced 3.5-fold in the cells overexpressing circTADA2A-E6 (FIG. 3 f) and increased 2.88-fold in the cells knocked down by circTADA2A-E6 (FIG. 4E). In addition, overexpression of the circTADA2A-E6 in MDA-MB-231 cells significantly increased the expression of the epithelial cell marker E-cadherin (E-cadherein) (FIG. 3 g), whereas E-Cad expression was decreased in the circTADA2A-E6 knockdown cells (FIG. 4 g). In contrast, expression of the mesenchymal marker vimentin (mesenchymal marker vimentin) was decreased in MDA-MB-231circTADA2A-E6 overexpressing cells (FIG. 3 g) and increased in circTADA2A-E6 knockdown cells (FIG. 4 g). Taken together, these results demonstrate that: the circTADA2A-E6 plays a role in inhibiting cancer in breast cancer cells, and is consistent with clinical pathological results to a large extent, that is to say that the expression level of the circTADA2A-E is related to the development of tumorigenesis.

Example 4 expression level of circNOL10 and prognostic and predictive value of circNOL10 for TNBC

Potential associations between expression of circNOL10 and clinical parameters were analyzed in a similar manner to example 3.

qRT-PCR demonstrated significant down-regulation of circNOL10 in triple negative breast cancer tissues

The expression of circNOL10 in breast cancer tissues was detected by real-time fluorescent quantitative PCR (qRT-PCR). In this process, we designed divergent primers (outward-generating primers) for the circularization site of the circular RNA transcript to distinguish from the linear product encoded by the gene. Next, we reverse transcribed the circRNA into cDNA with random hexamer primers and amplified with reverse primers to yield the desired circular specific RNA product. Finally, sequencing of the amplified product confirmed that the circular RNA circularization site sequence was identical to the circular RNA sequence with splice junctions from CircBase. Finally, downregulation in circNOL10 breast cancer tissue was further confirmed by real-time fluorescent quantitative PCR (qRT-PCR) in 178 breast tissues (tnbc=115, la=25, lb=17, her2=21) (fig. 5 a). The expression level of circNOL10 in BC tissue is significantly lower than that of normal breast tissue, and is: TNBC > LA > Her-2> LB. We found that circNOL10 was also down-regulated in 10 breast cancer cell lines compared to MCF-10, a non-tumorigenic epithelial cell line (see FIG. 5 b).

correlation between expression of circNOL10 and clinical case characteristics

To investigate the potential correlation between expression of circNOL10 and clinical parameters, we analyzed the TNM (tumor size, lymph node metastasis and distant metastasis) status, and the results showed that the decrease in tumor circNOL10 expression levels was significantly correlated with more lymph node metastasis (p=032) and high clinical stage (p=0.018) (table 4). These results suggest: there is a correlation between the down-regulation of circNOL10 expression and the invasive character of TNBC.

TABLE 4 correlation between clinical pathology factors and circNOL10 expression in TNBC tissue (n=115)

Diagnostic value of expression of circNOL10 in breast cancer

To assess whether circNOL10 can be used as a diagnostic index for triple negative (n=115) breast cancer, we generated ROC curves using a total of 16 normal breast tissues as controls. In the ROC curve of TNBC compared to the normal group, the area under the circNOL10 curve was 0.9212, respectively. In addition, we found that: the curves of circNOL10 in LA (n=25), LB (n=21) and Her-2 (n=17) are 0.9275, 0.7619 and 0.9154, respectively (FIG. 5 c). These results suggest: circNOL10 can be used as a potential diagnostic biomarker for breast cancer.

Assessment of circNOL10 for predicting breast cancer risk

Survival analysis of circNOL10 showed that the 10 year total survival of the circNOL10 low expression group was significantly shorter than that of the circTADA2A-E6 high expression group (FIG. 5 d), p=0.027, and the 10 year DFS of the low expression group was significantly shorter than that of the circTADA2A-E6 high expression group (FIG. 5 d), with a p-value of 0.032. These analytical data suggest: circNOL10 may be a potential predictive biomarker of breast cancer progression.

With the rapid development of deep sequencing and microarray technology in recent years, there is growing evidence that mammalian genomes can encode circrnas that are commonly transcribed and play an important functional role in a wide variety of biological processes. The inventors found from analysis of circRNA microarray data of breast tumor (n=8) and normal breast tissue samples (n=3) that: a total of 140 up-regulated and 95 down-regulated circular RNAs were found in breast cancer compared to normal breast tissue. 8 circRNA downregulation was confirmed by q-RT-PCR in 178 breast cancer patient samples. Further studies have found that certain circrnas are novel potential TNBC prognostic and predictive biomarkers as a target gene for the competitive modulation of mirnas by endogenous RNAs and mirnas, thereby producing an anti-tumor effect. These findings provide theoretical and clinical basis for future application of circRNA in diagnosis and treatment of breast cancer.

***

Embodiments of the invention can be practiced in accordance with the present disclosure without undue experimentation. Although the present invention has been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the kits, pharmaceutical compositions, uses and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. Equivalent substitutions and modifications apparent to one skilled in the art are intended to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

Use of circRNA as a marker for breast cancer, wherein the circRNA is selected from one or more of the following: the materials are selected from the group consisting of CircNSUN2, circTADA2A-E6, circTADA2A-E5/E6, circNOL10, circCSRNP2, circFAM125B, circCDC and CircABCC1.
2. Use of a reagent and/or microarray for detecting the circRNA according to claim 1, preferably comprising a specific probe, for the preparation of a kit for breast cancer diagnosis or prognosis.
3. Use of a specific probe for detecting the circRNA as defined in claim 1 for the preparation of a microarray for the diagnosis or prognosis of breast cancer.
4. Kit for the diagnosis or prognosis of breast cancer comprising reagents or microarrays for measuring the level of circRNA according to claim 1, preferably comprising specific probes.
5. Use of the circRNA according to claim 1 as a diagnostic or prognostic marker for breast cancer.
6. The use or kit according to any one of the above 1-6, wherein the breast cancer is selected from the group consisting of luminel a and B, HER2+ and triple negative subtype breast cancer.