WO2018210173A1 - 乳腺癌标志物及应用 - Google Patents

乳腺癌标志物及应用 Download PDF

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WO2018210173A1
WO2018210173A1 PCT/CN2018/086238 CN2018086238W WO2018210173A1 WO 2018210173 A1 WO2018210173 A1 WO 2018210173A1 CN 2018086238 W CN2018086238 W CN 2018086238W WO 2018210173 A1 WO2018210173 A1 WO 2018210173A1
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breast cancer
circtada2a
circrna
circnol10
expression
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陈敏
徐建震
于浩洋
张国君
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深圳市晋百慧生物有限公司
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Priority to CN202211045951.1A priority Critical patent/CN115992201A/zh
Priority to CN201880002722.7A priority patent/CN109415770B/zh
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Definitions

  • the present invention relates to compositions, methods and uses for the diagnosis, prognosis and treatment of breast cancer.
  • the present invention relates to circRNA molecules for diagnosis, prognosis and treatment of breast cancer, kits and microarrays for the diagnosis, prognosis and treatment of breast cancer associated therewith, and related methods and uses.
  • breast cancer is a common malignant tumor and ranks first among women worldwide in the incidence of cancer. In recent years, China's breast cancer morbidity and mortality have increased year by year, seriously endangering the health of Chinese women. From the point of view of molecular changes and clinical relevance, breast cancer is a complex and diverse disease that should be considered as a type of tumor with a different molecular level. There are at least four molecular subtypes of breast cancer in clinical practice today: Luminal A (ER+PR+Her2-), Luminal B (ER+PR-Her2-), Her2 (ER-PR- and Her2+), and TNBC (ER-PR). - and Her2-).
  • Circular RNA is thought to be caused by non-classical splicing of linear pre-mRNA into a circular form. CircRNA is evolutionarily conserved, and circRNA is structurally stable and abundant in C. elegans, zebrafish, fruit flies, mice, and humans. Some circRNAs are as abundant as 200 times their linear counterparts.
  • the circRNA is determined to be a stable structure mainly produced by exons or introns, and is differentially generated by back splicing or lariat intron, and then forms neither 5'
  • the cap also has no covalently closed loop structure of the 3' tail.
  • exon circRNA is very stable in cells, compared to the average half-life of mRNA for 10 h, and the half-life of most species of exon circRNA is over 48 h. Both exon circRNA and intron circRNA play a role in regulating gene expression. Recent studies have shown that circRNA may play its biological functions in several ways, such as actively participating in pre-mRNA splicing, affecting gene expression by binding to proteins, epigenetic miRNA sponges, and RNA transport through epigenetic regulation. And promote the transcription of its maternal mRNA. To date, more than 10,000 human circRNAs have been discovered, widely distributed and diversely present in humans, often with tissue-specific and developmental-specific specificities, and are closely related to molecular biology and molecular oncology.
  • Circular RNA is a widely distributed and diverse endogenous non-coding RNA with enormous regulatory potency. It has been demonstrated that circRNA plays a role in the development of several types of diseases such as atherosclerosis and nervous system disorders. Play an important role. In recent years, the role and function of circRNA in cancer has become a new focus in cancer research. The unique characteristics of circRNA are constantly being revealed and discovered, which will expand the understanding of cancer, especially in the development of cancer and the malignant development of cancer. Abnormal expression of circRNA in human cancers such as esophageal cancer, CRC and gastric cancer.
  • circITCH exhibits anti-tumor function by interacting with miRNAs such as miR-7, miR-17 and miR-214 in esophageal squamous cell carcinoma.
  • miRNAs such as miR-7, miR-17 and miR-214 in esophageal squamous cell carcinoma.
  • Enriched RNA found 312 circRNAs differentially expressed in breast or ovarian cancer.
  • circRNAs have been shown to be aging biomarkers of Drosophila. In gastric cancer and hepatocellular carcinoma (HCC), circRNA is found to be a new cancer biomarker.
  • HCC gastric cancer and hepatocellular carcinoma
  • the Kalari team recently identified new breast cancer-specific circRNAs by analyzing a large number of samples of breast cancer circRNA sequencing (circ-Seq) in the TCGA library, and also classified unique circRNAs for different breast cancer subtypes.
  • circRNA may be a new biomarker for breast cancer diagnosis.
  • circRNA is detectable in human body fluids such as plasma, saliva, and exosomes, suggesting that circRNA may be more clinically useful for cancer diagnosis.
  • the present inventors studied the expression of circRNA in a primary tumor sample of a breast cancer patient, and identified a circRNA which can be used as a breast cancer marker or a breast cancer target.
  • a circRNA for use as a breast cancer marker (e.g., a diagnostic or prognostic marker) or a breast cancer target, the circRNA being selected from one or more of the group consisting of: circTADA2A-E6, circTADA2A- E5/E6, circNOL10, circNSUN2, circCSRNP2, circFAM125B, circCDC27 and circABCC1.
  • a reagent and/or microarray for detecting a circRNA of the first aspect for the preparation of a kit for diagnosis or prognosis of breast cancer.
  • a specific probe for detecting a circRNA of the first aspect for the preparation of a microarray for diagnosis or prognosis of breast cancer.
  • a kit for diagnosis and/or prognosis of breast cancer comprising a reagent or microarray for measuring the expression level of circRNA according to the first aspect 1 is provided.
  • a method of diagnosing whether a subject has breast cancer or is at risk of developing breast cancer or prognosing breast cancer comprising measuring circRNA of said first aspect of the test sample The expression level.
  • the method can include:
  • a reverse transcription product is obtained by reverse transcribed RNA from a test sample obtained from the subject;
  • the circRNA expression level of the test sample is lower than the circRNA expression level of the control sample, indicating that the subject has breast cancer or is at risk of developing breast cancer, or predicts a poor prognosis of breast cancer.
  • circTADA2A-E6 an agent for increasing the expression level of circTADA2A-E6 and/or a nucleic acid expressing circTADA2A-E6 for the preparation of a medicament for inhibiting or treating breast cancer.
  • a method for identifying a breast cancer inhibitor comprising identifying a substance that increases the expression level of circRNA in breast cancer cells.
  • the circRNA is preferably circTADA2A-E6 and/or circNOL10.
  • a breast cancer inhibitor identified by the method of the seventh aspect.
  • the use of the inhibitor of the eighth aspect, in the manufacture of a medicament for inhibiting or treating breast cancer, is provided.
  • a pharmaceutical composition for inhibiting or treating breast cancer comprising circRNA, an agent which increases expression level of circRNA, a nucleic acid expressing circRNA, and/or a breast cancer suppression according to the eighth aspect is provided
  • the pharmaceutical composition optionally further contains other agents for inhibiting or treating breast cancer.
  • the circRNA is preferably circTADA2A-E6 and/or circNOL10.
  • a method for inhibiting or treating breast cancer comprising administering to a patient in need thereof a therapeutically effective amount of a circRNA, an agent which increases the expression level of the circRNA, a nucleic acid expressing the circRNA, and/or Or the breast cancer inhibitor of the eighth aspect, optionally other agents for inhibiting or treating breast cancer.
  • the circRNA is preferably circTADA2A-E6 and/or circNOL10.
  • microarray, kit, method, composition or inhibitor as described above, wherein the breast cancer may be selected from the group consisting of Luminal A and B, HER2+ and triple subtype breast cancer, more preferably Sanyinia. Breast cancer.
  • Figure 1 Profile analysis of circular RNA in human normal breast tissue and breast cancer tissue.
  • FIG. 1 Expression levels of two circTADA2A and prognostic and predictive value of both circTADA2A for TNBC.
  • c-d breast cancer cell line and immortalized mammary gland cell line MCF-10A circTADA2A-E6 (c) and circTADA2A-E5/E6 (d) expression were analyzed by qRT-PCR.
  • DFS disease-free survival
  • OS overall survival
  • FIG. 3 Overexpression of circTADA2A-E6 inhibits breast cancer cell proliferation, colony formation, and invasion.
  • CircTADA2A silencing promotes breast cancer cell proliferation, colony formation, migration and invasion.
  • FIG. 5 Expression levels of circNL10 and prognostic and predictive value of circNL10.
  • (b) Real-time quantitative PCR analysis of the expression level of circNOL10 in breast cancer cell lines and normal breast cells MCF-10A.
  • (c). ROC curve analysis - area under the curve of circNOL10 in different molecular typing breast cancers.
  • (d) The relationship between the expression level of circNOL10 and DFS and OS. The number of cases involved in the analysis was 107 (8 of 115 cases were lost after surgery, and the rate of loss of follow-up was 6.96%).
  • a “therapeutic agent” is an atom, molecule or compound that can be used to treat a disease.
  • therapeutic agents include antibodies, antibody fragments, peptides, drugs, toxins, enzymes, nucleases, hormones, immunomodulators, circular RNA, antisense oligonucleotides, small interfering RNA (siRNA), chelating agents, boron compounds Photosensitizers, dyes and radioisotopes.
  • Cyclic RNA or “circRNA” means a novel class of non-coding RNAs that are widely and diversely present in mammalian cells and have endogenous RNA molecules that regulate gene expression, mainly from exon transcripts and Introns undergo nonlinear reverse splicing, and their ends form a closed loop through covalent bonds, exhibiting different characteristics from linear RNA.
  • a circRNA as a breast cancer marker, wherein the circRNA is selected from one or more of the group consisting of: circTADA2A-E6, circTADA2A-E5/E6, circNOL10, circNSUN2, circCSRNP2, circFAM125B, circCDC27 and circABCC1, preferably selected From one or more of the following: circTADA2A-E6, circTADA2A-E5/E6 and circNOL10.
  • reagent and/or microarray for detecting a circRNA as described in 1 above for the preparation of a kit for the diagnosis of breast cancer, said reagent preferably comprising a specific probe.
  • kits for the diagnosis of breast cancer comprising a reagent or microarray for measuring the level of circRNA according to the above 1, preferably comprising a specific probe.
  • a method of diagnosing whether a subject has breast cancer or is at risk of developing breast cancer comprising measuring a level of circRNA according to above 1 in a test sample, the method preferably comprising:
  • the circRNA level of the test sample is lower than the circRNA level of the control sample, indicating that the subject has or is at risk of developing breast cancer.
  • step (2) comprises hybridizing the reverse transcription product to the circRNA-specific probe or a microarray for detecting the circRNA.
  • a reagent or microarray for measuring circTADA2A-E6 and/or circNOL10 levels in a kit for the prognosis of breast cancer, said reagent preferably comprising a specific probe.
  • a kit for breast cancer prognosis comprising a reagent and/or a microarray for detecting circTADA2A-E6 and/or circNOL10, preferably comprising a specific probe.
  • a method for prognosis of a breast cancer patient comprising measuring a level of circTADA2A-E6 and/or circNOL10 in a test sample, the method preferably comprising:
  • the circTADA2A-E6 and/or circNOL10 levels of the test sample are lower than the circTADA2A-E6 and/or circNOL10 levels of the control sample, indicating that the breast cancer has a poor prognosis.
  • step (2) comprises the specific probe of the reverse transcription product with the circTADA2A-E6 and/or circNOL10 or for detecting the circTADA2A-E6 and/or circNOL10 Microarray hybridization.
  • a circTADA2A-E6 and/or circNOL10 an agent that increases circTADA2A-E6 and/or circNOL10 levels and/or a nucleic acid that expresses circTADA2A-E6 and/or circNOL10 in the manufacture of a medicament for inhibiting or treating breast cancer.
  • a method for identifying a breast cancer suppressor comprising identifying a substance that increases the level of circTADA2A-E6 and/or circNOL10 in breast cancer cells.
  • a breast cancer inhibitor identified by the method of 16 above.
  • a pharmaceutical composition for inhibiting or treating breast cancer comprising circTADA2A-E6, an agent that increases circTADA2A-E6 levels, a nucleic acid expressing circTADA2A-E6, and/or a breast cancer inhibitor according to 17 above,
  • the pharmaceutical composition optionally further contains other agents for inhibiting or treating breast cancer.
  • a method for inhibiting or treating breast cancer comprising administering to a patient in need thereof a therapeutically effective amount of a circTADA2A-E6, an agent that increases circTADA2A-E6 levels, a nucleic acid that expresses circTADA2A-E6, and/or according to the above
  • the breast cancer is selected from the group consisting of Luminal A and B, HER2+ and triple negative subtype breast cancer, Preferred is a triple negative subtype breast cancer.
  • nucleic acid is a vector for expressing circTADA2A-E6 and/or circNOL10.
  • Another 57 TNBCs were collected from Zhejiang Provincial Cancer Hospital and Linyi People's Hospital. The medical ethics committee of Shantou University Medical College, Zhejiang Cancer Hospital and Linyi People's Hospital approved the program, and participants signed written informed consent before surgery.
  • RNA Stabilization Solution RNA Stabilization Solution, Invitrogen, Beijing, China.
  • the tissue was kept at 4 ° C overnight, and then the supernatant was taken out and transferred to -80 ° C for long-term storage.
  • Corresponding adjacent non-tumor tissue was taken 5 cm from the edge of the cancer, and the tissue did not contain significant tumor cells as assessed by an experienced pathologist.
  • Tumors were staged according to the Tumor-lymph node-metastasis (TNM) staging system of the International Union against Cancer. Histological grading was assessed according to the National Comprehensive Cancer Network (NCCN) Oncology Clinical Practice Guidelines (V.1.2011). H&E staining of normal breast tissue was performed by a pathologist.
  • RNA samples were prepared and microarray hybridization were performed based on the Arraystar standard protocol (Arraystar Inc.). Briefly, total RNA was digested with RNase (Epicentre, Inc.) to remove linear RNA and enrich for circular RNA. Then, the enriched circular RNA was amplified and transcribed into a fluorescent cRNA (Arraystar Super RNA Labeling Kit; Arraystar) by random priming. The labeled cRNA was purified using the RNeasy Mini Kit (Qiagen). The concentration and specific activity (pmol Cy3/ ⁇ gcRNA) of the labeled cRNA was determined using a NanoDrop ND-1000. The labeled cRNA was hybridized to an Arraystar Human circRNA Array (8x15K, Arraystar). After washing the slides, the array was scanned with an Agilent Scanner G2505C.
  • Agilent Scanner G2505C Agilent Scanner G2505C.
  • cDNA was prepared from total RNA.
  • the primers used in qRT-PCR were designed as convergent primers to detect circular junctions and span the reverse splicing junctions.
  • the sequences of the three circRNAs were obtained from the database "circinteractome” ( https://circinteractome.irp.nia.nih.gov/) .
  • the data was analyzed by the ⁇ Ct method. All primers were synthesized by Generay Biotech. All results are expressed as the mean ⁇ SD of three independent experiments.
  • the circRNA-miRNA-gene network was enriched by using the validated candidate circRNA as a seed according to TargetScan (http://www.targetscan.org/) in conjunction with miRanda ( http://www.microrna.org/ ) analysis.
  • the circRNA–miRNA–mRNA interaction network of circTADA2A was established using Cytoscape (http://www.cytoscape.org/).
  • the predicted gene functions in the network are annotated with GO and KEGG path analysis.
  • DAVID Database for Annotation, Visualization and Integrated Discovery
  • the FASTA file for the putative miRNA binding site in circRNA was obtained from miRBase release 20.0 (http://www.mirbase.org/). Only mature miRNAs are considered for seed analysis.
  • the miRNA is aligned to a circRNA.
  • the putative target site for miRNA is the long 6 nucleotide sequence of the reverse complement of nucleotides 2-7 representing the mature miRNA sequence in the genome.
  • cDNA was synthesized from total RNA according to the protocol supplied. Based on The relative gene expression of circular RNA was measured by the ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) of the Green method. The qPCR Ct value is automatically calculated using the manufacturer's software. Human-ACTB was used as an internal standard control. Specifically, circRNA abundance was determined using a divergent primer that anneals to the distal end of the circRNA. The PCR product was directly subjected to Sanger sequencing using sequencing primers.
  • the (genomic region) mature sequence of circTADA2A-E6 was synthesized and cloned into pLCDH-ciR-copGFP-Puro (Geneseed, Guangzhou, China). And verified by sequencing.
  • siRNA target sequences are as follows:
  • miRNA mimics and inhibitors were synthesized by Ribobio (Guangzhou, China). Cells with Lipofectamine TM 3000 (invitrogen, USA) were transfected.
  • CCK-8 Proliferation assay
  • Transwell invasion assay Transwell invasion assay. Invasion assays were performed with cell culture inserts (8 [mu]M pore size; BD) and matrigel invasion chamber (BD), respectively. The transfected cells were serum-starved for 24 h, and 1 ⁇ 10 4 MDA-MB-231 cells or 2 ⁇ 10 4 MCF-7 cells in serum-free medium were inoculated into the upper chamber. Cells were fixed with 4% paraformaldehyde at 20 hours (MDA-MB-231) or 48 hours (MCF-7) for invasion assay and then stained with 0.1% crystal violet. Each assay was performed in triplicate. Two investigators counted the number of cells in three fields in each well.
  • 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 appeared. Colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and then counted.
  • Example 1 Screening and validation of circRNA differentially expressed in breast cancer
  • High-throughput circRNA chip sequencing helps identify differentially expressed circRNA between breast cancer and normal breast tissue.
  • the scatter plot shows the differentially expressed circular RNA between the two groups, with a fold difference of more than 1.5 times; the volcano map shows a statistically significant differential expression of the circular RNA between the two groups.
  • a total of 140 up-regulated and 95 down-regulated circular transcripts were found in breast cancer compared to normal breast tissue (Fig. 1c), and this expression profile data is consistent with other studies, indicating that most of the circRNAs are derived from regions containing genes.
  • circRNAs show different expression clusters (Fig. 1d).
  • the interaction between these circRNAs and their target miRNAs can be theoretically predicted by software according to complementary miRNA matching sequences.
  • a total of 465 miRNAs are predicted to bind to circRNA.
  • the complete circRNA-miRNA interaction network map was drawn by Cytoscape software (Fig. 1e). All of these data indicate that there is a wide variety of differentially expressed circRNAs in breast cancer that may be useful in the screening and diagnosis of disease.
  • CircRNAs differentially expressed in breast cancer TNBC tissues were screened by circRNA microarrays, and 8 circRNAs with down-regulated expression folds greater than 1.5-fold were screened. The results are shown in Table 1.
  • Real-time PCR confirms differentially expressed circRNA in triple-negative breast cancer tissues
  • circRNAs were selected (see Table 1), and these differentially expressed circRNAs were further confirmed in 178 breast tissues by real-time fluorescent quantitative PCR (qRT-PCR).
  • the lengths of the eight circRNAs ranged from 158 nt to 843 nt, and all eight circRNAs were exon splicing.
  • the outer-end primers of the exon were designed for the circularization site of the circular RNA transcript (outward-facing primer). ) to distinguish linear products encoded by genes.
  • the circRNA is reverse transcribed into cDNA using random hexamer primers and amplified with the reverse primer to produce the desired circular specific RNA product.
  • circTADA2A-E6 and circTADA2A-E5/E6 circRNAs were significantly lower than those in normal breast tissues, namely: circTADA2A-E6, LA>TNBC>Her-2>LB; circTADA2A-E5/E6, LB>TNBC>Her-2>LA (all groups, p ⁇ 0.01).
  • Example 3 Overexpression or silencing of circTADA2A-E6 inhibits or enhances breast cancer cell proliferation, colony formation, invasion
  • the circTADA2A high expression vector and the siRNA interference sequence for knockdown were constructed (Fig. 3a and Fig. 4a).
  • the CCK-8 assay showed that high expression of circTADA2A reduced cell proliferation (p ⁇ 0.05, Figure 3c), while interference with circTADA2A, cell proliferation was enhanced (p ⁇ 0.05, Figure 4c).
  • Scratch healing experiments showed that high expression of circTADA2A reduced the ability of cells to scratch (Fig. 3e), while knockdown promoted the ability of cells to scratch (Fig. 4f).
  • the results of colony formation experiments showed that the ability of circTADA2A-E6 overexpressing cells to reduce colony formation was reduced (Fig.
  • circNOL10 in breast cancer tissues was detected by real-time fluorescent quantitative PCR (qRT-PCR).
  • qRT-PCR real-time fluorescent quantitative PCR
  • sequencing of the amplified product confirmed that the circular RNA circularization site sequence is identical to the circular RNA sequence from the CircBase with a splice junction.
  • the expression level of circNOL10 in BC tissues was significantly lower than that in normal breast tissues, which was: TNBC>LA>Her-2>LB.
  • circNOL10 was also down-regulated in 10 breast cancer cell lines compared to MCF-10 (a non-tumorigenic epithelial cell line) (see Figure 5b).
  • the area under the circNOL10 curve was 0.9212, respectively.
  • CircNOL10 is used to predict breast cancer risk assessment
  • circNOL10 may be a potential predictive biomarker for breast cancer progression.
  • circRNAs which are commonly transcribed and play important in a variety of biological processes. Functional role.
  • the inventors found that a total of 140 up-regulations were found in breast cancer compared with normal breast tissue.
  • 95 down-regulated circular RNAs 8 circRNA down-regulations were confirmed by q-RT-PCR in 178 breast cancer patients.
  • circRNAs are new potential prognostic and predictive biomarkers of TNBC, as a target gene for endogenous RNA and miRNA competitively modulate miRNAs, thereby producing a tumor suppressor effect. These findings provide a theoretical and clinical basis for the future diagnosis and treatment of circRNA for breast cancer.

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Abstract

本申请涉及作为乳腺癌标志物的circRNA及其用途，所述circRNA选自circTADA2A-E6、circTADA2A-E5/E6、circNOL10、circNSUN2、circCSRNP2、circFAM125B、circCDC27和circABCC1。本发明还涉及用于乳腺癌诊断、预后和治疗的试剂盒、微阵列、药物以及相关的方法和用途。

Description

乳腺癌标志物及应用 技术领域
本发明涉及乳腺癌诊断、预后和治疗的组合物、方法和用途。具体而言，本发明涉及与乳腺癌诊断、预后和治疗的circRNA分子、与之相关的乳腺癌诊断、预后和治疗的试剂盒和微阵列、以及相关的方法和用途。
背景技术
乳腺癌是一种常见的恶性肿瘤，在全球女性癌症发病率中位居第一。近年来，中国的乳腺癌发病率和死亡率逐年上升，严重危及了中国女性的健康。从分子改变与临床相关的角度来看，乳腺癌是一种复杂而多样的疾病，应当被认为是一类分子水平各不相同的肿瘤。现今临床上乳腺癌至少有4种分子亚型：Luminal A(ER+PR+Her2-)、Luminal B(ER+PR-Her2-)、Her2(ER-PR-和Her2+)和TNBC(ER-PR-和Her2-)。现如今，生物标志物的检测也开始用于乳腺癌的预后和辅助化疗如21基因标记(21-gene signature)(Oncotype DX)和欧洲普通70基因检测MammaPrint(European common 70-gene detection MammaPrint)。因此，发现，鉴定并对乳腺癌的生物标志物进行分组的研究是必须和重要的。
环状RNA(circRNA)据认为是由线性pre-mRNA非经典剪接成环状形式所致。circRNA在进化上保守，在秀丽线虫(C.elegans)、斑马鱼、果蝇、小鼠以及人类中circRNA结构稳定且丰度高。某些circRNA的丰度高达其线性对应物的200倍。circRNA被确定是主要由外显子或内含子产生的稳定结构，并且通过反向剪接(back splicing)或套索内含子(lariat intron)差异产生(differentially generated)，然后形成既无5’帽也无3’尾的 共价闭合环结构。外显子circRNA在细胞中非常稳定，相比之下mRNA的平均半衰期为10h，大多数种类的外显子circRNA半衰期超过48h。外显子circRNA和内含子circRNA在调控基因表达中皆起作用。最近的研究表明：circRNA可能通过以下几种方式发挥其生物学功能，如积极参与pre-mRNA剪接、通过与蛋白结合影响基因表达、通过表观遗传调控miRNA的海绵(epigenetic miRNA sponges)、RNA转运者、以及促进其母体mRNA转录。迄今为止，超过10,000种人类circRNA已被发现，广泛分布且多样化地存在于人类，通常具有组织特异性和发育期特异性，与分子生物学和分子肿瘤学密切相关。
环状RNA(circRNA)作为广泛分布且多样化的、具有巨大调控效能的内源非编码RNA，已有研究证明circRNA在若干类型的疾病(诸如动脉粥样硬化和神经系统病症)的发生中发挥着重要作用。最近几年，circRNA在癌症中所起的作用和功能成为癌症研究领域的新焦点。circRNA的独特特征不断地被揭示和发现，将扩展对癌症的认识，尤其在癌症发生和癌的恶性发展方面。在人类癌症如食管癌、CRC和胃癌中circRNA表达异常。circITCH在食管鳞状细胞癌中通过与miRNA如miR-7、miR-17和miR-214相互作用显示出抗肿瘤功能。Ahmed等人通过Ingenuity IPA工具试剂盒(
Figure PCTCN2018086238-appb-000001
QIAGEN Redwood City,www.qiagen.com/ingenuity)富集RNA发现了在乳腺癌或卵巢癌中差异表达的312种circRNA。
文献报道，circRNA的总体特征是种类繁多且数量庞大、分布广泛、较强的进化保守性、组织特异性、极高的稳定性和高丰度表达。circRNA这种独特的优势，使之成为可用于疾病诊断、预后和预测治疗反应的新生物标志物。首先，circRNAs被证明为果蝇属的衰老生物标志物。在胃癌和肝细胞癌(HCC)中，circRNA发现可作为新的癌症生物标志物。最 近Kalari研究小组通过对TCGA库中大量样本的乳腺癌circRNA测序(circ-Seq)进行分析后，鉴定出新的具有乳腺癌特异性的circRNA，并且还分类出对应不同乳腺癌亚型的独特circRNA组：三阴性(TNBC)、雌激素受体阳性(ER+)和HER2阳性(HER2+)，从此可看出circRNA有可能是用于乳腺癌诊断的新生物标志物。此外，circRNA在人类体液如血浆、唾液和外泌体(exosomes)中可检测到，表明circRNA在临床上应用于癌症诊断的可能更大。
发明内容
本发明人对乳腺癌患者原发性肿瘤样本中的circRNA表达进行了研究，鉴定出可用作乳腺癌标志物或乳腺癌靶标的circRNA。
按照本发明的第一方面，提供用作乳腺癌标志物(例如诊断或预后标志物)或乳腺癌靶标的circRNA，所述circRNA选自以下的一种或更多种：circTADA2A-E6、circTADA2A-E5/E6、circNOL10、circNSUN2、circCSRNP2、circFAM125B、circCDC27和circABCC1。
按照本发明的第二方面，提供用于检测第一方面所述的circRNA的试剂和/或微阵列在制备用于乳腺癌诊断或预后的试剂盒中的用途。
按照本发明的第三方面，提供用于检测第一方面所述的circRNA的特异性探针在制备用于乳腺癌诊断或预后的微阵列中的用途。
按照本发明的第四方面，提供一种乳腺癌诊断和/或预后的试剂盒，其包含用于测量根据第一方面1所述的circRNA表达水平的试剂或微阵列。
按照本发明的第五方面，提供一种诊断受试者是否患有乳腺癌或处于发生乳腺癌风险、或者对乳腺癌进行预后的方法，其包括测量测试样本中第一方面所述的circRNA的表达水平。所述方法可以其包括：
从获自受试者的测试样本逆转录RNA，获得逆转录产物；
检测第一方面所述的circRNA的表达水平；和
将测试样本的所述circRNA表达水平与作为对照的正常乳腺组织的所述circRNA表达水平相比较，
其中测试样本的所述circRNA表达水平低于对照样本的所述circRNA表达水平，预示着受试者患有乳腺癌或处于发生乳腺癌风险，或者预示着乳腺癌预后不良。
按照本发明的第六方面，circTADA2A-E6、提高circTADA2A-E6表达水平的药剂和/或表达circTADA2A-E6的核酸在制备抑制或治疗乳腺癌的药物中的用途。
按照本发明的第七方面，提供一种用于鉴定乳腺癌抑制剂的方法，所述方法包括鉴定提高乳腺癌细胞中circRNA的表达水平的物质。所述circRNA优选为circTADA2A-E6和/或circNOL10。
按照本发明的第八方面，提供用第七方面所述的方法鉴定的乳腺癌抑制剂。
按照本发明的第九方面，提供第八方面所述的抑制剂在制备用于抑制或治疗乳腺癌的药物中的用途。
按照本发明的第十方面，提供一种用于抑制或治疗乳腺癌的药物组合物，其包含circRNA、提高circRNA表达水平的药剂、表达circRNA的核酸和/或第八方面所述的乳腺癌抑制剂，所述药物组合物任选还含有用于抑制或治疗乳腺癌的其他药剂。所述circRNA优选为circTADA2A-E6和/或circNOL10。
按照本发明的第十一方面，提供一种用于抑制或治疗乳腺癌的方法，其包括给有需要的患者施用抑制治疗有效量的circRNA、提高circRNA表达水平的药剂、表达circRNA的核酸和/或第八方面所述的乳腺癌抑制剂，任选施用用于抑制或治疗乳腺癌的其他药剂。所述circRNA优选为 circTADA2A-E6和/或circNOL10。
对于如上所述的用途、微阵列、试剂盒、方法、组合物或抑制剂，其中所述的乳腺癌可以选自Luminal A和B、HER2+和三阴亚型乳腺癌，更优选为三阴亚型乳腺癌。
附图说明
图1.人正常乳腺组织和乳腺癌组织中环状RNA的概况分析。(a)散点图.X轴：正常乳腺组织(标准化的)，Y轴：TNBC组织(标准化的)。绿色线代表倍数变化。顶部绿色线之上和底部绿色线之下的circRNA表示两组样本之间circRNA表达水平变化超过1.5倍。(b)火山图.X轴：log2(变化倍数)，Y轴：-log10(P值)。垂直绿色线对应于1.5倍上调和下调，水平绿色线表示p值为0.05。图中的红点表示具有统计学意义的差异表达的circRNA。(c)正常乳腺组织和乳腺癌组织(Luminal A和TNBC亚型)之间的circRNA概况比较。(d)乳腺癌组织和人正常乳腺组织之间差异表达的circRNA的聚类热图(clustered heatmap)，行表示circRNA，列表示组织样本。根据Pearson相关，对circRNA进行分类。数值表示各组换算为log10的平均SRPBM。(e)在乳腺癌中差异表达的215种circRNA和它们在乳腺癌中的靶标miRNA的网络。该网络由212种上调的circRNA(红色节点)、93种下调的circRNA(黄色节点)及它们的靶标miRNA(蓝色节点)组成。(f)8种所选circRNA的微阵列数据和qRT-PCR结果之间的比较。
图2.两种circTADA2A的表达水平和这两种circTADA2A对TNBC的预后性和预测性价值。(a-b)相比于正常乳腺组织(n＝16)，以下不同乳腺癌亚型中circTADA2A-E6(a)和circTADA2A-E5/E6(b)的表达水平通过qRT-PCR来进行验证：LA(n＝25)、LB(n＝21)、Her-2(n＝17)和TNBC(n＝115)。*P<0.05,**P<0.01。(c-d)乳腺癌细胞系和无限增殖化 乳腺细胞系MCF-10A中circTADA2A-E6(c)和circTADA2A-E5/E6(d)表达通过qRT-PCR分析。(e-f)分别在TNBC(n＝115)、LA(n＝25)、LB(n＝21)和Her-2(n＝17)中circTADA2A-E6和circTADA2A-E5/E6之ROC分析的AUC。(g-h)circTADA2A-E6和circTADA2A-E5/E6表达与无病生存期(disease-free survival,DFS)(h)和总生存期(overall survival,OS)(g)的相关性。n＝107,115位患者中的8位(6.96％)在首次手术后失访。
图3.circTADA2A-E6的过表达抑制乳腺癌细胞增殖、集落形成、侵袭。(a)慢病毒circTADA2A-E6表达载体plvx-CMV-circTADA2A-E6-EF1-ZsGreen-Puro的构建。(b)在MCF-7和MDA-MB-231细胞中用circTADA2A-E6载体或对照载体转染后，通过qRT-PCR分析异位circTADA2A-E6表达。(c)具有异位circTADA2A-E6表达的MCF-7和MDA-MB-231细胞的CCK-8测定和增殖率。(d)过表达circTADA2A-E6的MCF-7和MDA-MB-231细胞的克隆形成测定。代表性图示于左侧，转化灶/孔的定量示于右侧。误差条表示三个独立实验的平均值±SD。(e)过表达circTADA2A-E6的MCF-7和MDA-MB-231细胞划痕试验的代表性图像。(f)用Boyden小室研究异位circTADA2A-E6表达对细胞侵袭的影响。细胞穿透的形态学比较示于左侧，而穿透细胞的定量示于右侧。误差条表示三个独立实验的平均值±SD。*p<0.05,**p<0.01。(g)E-cad和波形蛋白的蛋白质水平通过过表达circTADA2A-E6的MCF-7和MDA-MB-231细胞的蛋白质印迹进行评价。图中，“vector control”为载体对照。
图4.circTADA2A沉默促进乳腺癌细胞增殖、克隆形成、迁移和侵袭。(a)特异性结合至circTADA2A-E6反向剪接接点的siRNA的示意图和靶标序列。(b)用两种circTADA2A-E6siRNA转染细胞后circTADA2A-E6RNA表达的qRT-PCR分析。(c)通过CCK-8测定，评价circTADA2A-E6 siRNA对MCF-7和MDA-MB-231细胞细胞增殖的影响。(d)对用两种circTADA2A-E6siRNA转染的MCF-7和MDA-MB-231细胞的集落形成测定。代表性图示于左侧，转化灶/孔的定量示于右侧。误差条表示平均值±SD。数据代表三个独立实验。(e)用Boyden小室研究circTADA2A-E6siRNA表达对细胞侵袭的影响。细胞穿透的形态学比较示于左侧，而穿透细胞数的定量示于右侧。(f)被两种circTADA2A-E6siRNA转染的MCF-7和MDA-MB-231细胞的伤口愈合试验。代表性图像示于左侧，对细胞迁移的定量示于右侧。(g)用MCF-7和MDA-MB-231细胞，在用两种circTADA2A-E6siRNA转染之后，通过蛋白质印迹评价E-cad和波形蛋白(Vimentin)的蛋白质水平。
图5.circNL10的表达水平以及circNL10的预后性和预测性价值。(a)实时荧光定量PCR分析不同分子分型乳腺癌组织与正常乳腺组织中circNOL10的表达水平：LA(n＝25),LB(n＝21),Her-2(n＝17)以及TNBC(n＝115)，正常(Normal)(n＝16).*P<0.05,**P<0.01。(b)实时荧光定量PCR分析乳腺癌细胞系以及类正常乳腺细胞MCF-10A中circNOL10的表达水平。(c).ROC曲线分析—circNOL10在不同分子分型乳腺癌中的曲线下面积。(d)circNOL10的表达水平与DFS和OS的关系。参与分析的病例数为107例(115例病例中有8例在手术后失访，失访率为6.96％)。
具体实施方式
在描述本发明方法和组合物之前，应当理解，本发明并不局限于所述的特定方法或组合物，因此当然可能会有所不同。还应当理解的是，本文所使用的术语仅用于描述特定的实施方案，并非限制性的。描述实施例是为本领域的普通技术人员提供如何制造和使用本发明的完整的公开内容和描述，并非旨在限制发明人视为其发明的范围，也并非旨在表示下面的实验是进行的全部或仅有的实验。已经努力确保所用数字(例 如数量、温度等)的准确度，但一些实验误差和偏差应该予以考虑。
除非另有定义，否则本文使用的所有技术和科学术语具有如由本发明所属领域的普通技术人员通常理解的相同的含义。现在描述一些潜在的和优选的方法和材料，尽管类似或等同于本文描述的任何方法和材料可以用于实施或测试本发明。本文提及的所有出版物在此通过引用并入，以公开和描述与所引用的出版物有关的方法和/或材料。可以理解的是，存在矛盾的情况下，以本公开内容取代所引用的出版物中的任何公开内容。
如在阅读本公开内容时对本领域技术人员显而易见的，每个本文所述和说明的单独的实施方案具有分立的组件和特征，其可容易地与其他若干实施方案中的特征分离或结合，而不脱离本发明的范围或实质。可以按所列举事件的顺序或逻辑上可能的任何其他顺序实施任何列举的方法。
除非另外说明，否则本文和所附权利要求中所用的“一个(种)”意指“一个(种)或多个(种)”。
当提供数值范围时，应当理解，该范围的上限和下限之间的每个中间值(至下限单位的十分之一)也被具体地公开，除非上下文另有明确说明。在所述范围中的任何所述值或中间值之间的各个较小的范围以及在所述范围内的其他所述值或中间值包含在本发明之内。这些较小范围的上限和下限可独立地包含在该范围内或排除在该范围之外，并且服从于所述范围中任何所特定地排除的，其中上下限中任一个或两个或无一个包含在所述较小范围内的每个范围也包含在本发明内。当所述范围包含一个或两个界限，除去任一或两个那些包含在内的界限的范围也包含在本发明中。
“治疗剂”是可用于治疗疾病的原子、分子或化合物。治疗剂的实例 包括抗体、抗体片段、肽、药物、毒素、酶、核酸酶、激素、免疫调节剂、环状RNA、反义寡核苷酸、小干扰RNA(siRNA)、螯合剂、硼化合物、光敏剂、染料和放射性同位素。
“环状RNA”或“circRNA”意指一类新型的非编码RNA,广泛且多样地存在于哺乳动物细胞中，具有调控基因表达作用的内源性RNA分子,主要由外显子转录本及内含子经非线性反向剪接，其首尾通过共价键形成闭合的环，表现出与线性RNA不同的特性。
本发明提供以下技术方案：
1.circRNA作为乳腺癌标志物的用途，其中所述circRNA选自以下的一种或更多种：circTADA2A-E6、circTADA2A-E5/E6、circNOL10、circNSUN2、circCSRNP2、circFAM125B、circCDC27和circABCC1，优选选自以下的一种或更多种：circTADA2A-E6、circTADA2A-E5/E6和circNOL10。
2.用于检测上述1中所述的circRNA的试剂和/或微阵列在制备用于乳腺癌诊断的试剂盒中的用途，所述试剂优选包括特异性探针。
3.用于检测上述1中所述的circRNA的特异性探针在制备用于乳腺癌诊断的微阵列中的用途。
4.乳腺癌诊断用的试剂盒，其包含用于测量根据上述1所述的circRNA水平的试剂或微阵列，所述试剂优选包括特异性探针。
5.一种诊断受试者是否患有乳腺癌或处于发生乳腺癌风险的方法，其包括测量测试样本中根据上述1所述的circRNA的水平，所述方法优选包括：
(1)从获自受试者的测试样本逆转录RNA，获得逆转录产物；
(2)测量根据上述1所述的circRNA的水平；和
(3)将测试样本的所述circRNA水平与作为对照的正常乳腺组织 的所述circRNA水平相比较，
其中测试样本的所述circRNA水平低于对照样本的所述circRNA水平，预示着受试者患有乳腺癌或处于发生乳腺癌风险。
6.根据上述5所述的方法，其中步骤(2)包括将所述逆转录产物与所述circRNA特异性探针或用于检测所述circRNA的微阵列杂交。
7.根据上述5所述的方法，所述方法包括通过qRT-PCR测量所述circRNA的水平。
8.根据上述1所述的circRNA作为乳腺癌预后标志物的用途。
9.用于测量circTADA2A-E6和/或circNOL10水平的试剂或微阵列在制备乳腺癌预后用的试剂盒中的用途，所述试剂优选包括特异性探针。
10.circTADA2A-E6和/或circNOL10的特异性探针在制备乳腺癌预后用的微阵列中的用途。
11.用于乳腺癌预后的试剂盒，其包含用于检测circTADA2A-E6和/或circNOL10的试剂和/或微阵列，所述试剂优选包括特异性探针。
12.一种用于乳腺癌患者预后的方法，其包括测量测试样本中circTADA2A-E6和/或circNOL10的水平，所述方法优选包括：
(1)从获自受试者的测试样本逆转录RNA，以获得逆转录产物；
(2)检测circTADA2A-E6和/或circNOL10的水平；和
(3)将测试样本的所述circTADA2A-E6和/或circNOL10水平与作为对照的正常乳腺组织的所述circTADA2A-E6和/或circNOL10水平相比较，
其中测试样本的所述circTADA2A-E6和/或circNOL10水平低于对照样本的所述circTADA2A-E6和/或circNOL10水平，预示着所述乳腺癌预后较差。
13.根据上述12所述的方法，其中步骤(2)包括将所述逆转录产物与 所述circTADA2A-E6和/或circNOL10的特异性探针或用于检测所述circTADA2A-E6和/或circNOL10的微阵列杂交。
14.根据上述12所述的方法，所述方法包括通过qRT-PCR测量所述circTADA2A-E6和/或circNOL10的水平。
15.circTADA2A-E6和/或circNOL10、提高circTADA2A-E6和/或circNOL10水平的药剂和/或表达circTADA2A-E6和/或circNOL10的核酸在制备抑制或治疗乳腺癌的药物中的用途。
16.一种用于鉴定乳腺癌抑制剂的方法，所述方法包括鉴定提高乳腺癌细胞中circTADA2A-E6和/或circNOL10的水平的物质。
17.用根据上述16所述的方法鉴定的乳腺癌抑制剂。
18.根据上述17所述的抑制剂在制备用于抑制或治疗乳腺癌的药物中的用途，其中所述药物任选还含有用于抑制或治疗乳腺癌的其他药剂。
19.一种用于抑制或治疗乳腺癌的药物组合物，其包含circTADA2A-E6、提高circTADA2A-E6水平的药剂、表达circTADA2A-E6的核酸和/或根据上述17所述的乳腺癌抑制剂，所述药物组合物任选还含有用于抑制或治疗乳腺癌的其他药剂。
20.一种用于抑制或治疗乳腺癌的方法，其包括给有需要的患者施用抑制治疗有效量的circTADA2A-E6、提高circTADA2A-E6水平的药剂、表达circTADA2A-E6的核酸和/或根据上述17所述的乳腺癌抑制剂，任选施用用于抑制或治疗乳腺癌的其他药剂。
21.根据上述1-20中任一项的用途、微阵列、试剂盒、方法、组合物或抑制剂，其中所述乳腺癌选自Luminal A和B、HER2+和三阴亚型乳腺癌，更优选为三阴亚型乳腺癌。
22.根据上述15和19-21中任一项的用途、组合物或方法，其中所 述核酸为用于表达circTADA2A-E6和/或circNOL10的载体。
实施例
下面参考附图和以下具体实施例，进一步描述本发明。应当理解，本发明并不限于这些具体的实施例。
材料和方法
临床人群和样本采集
从中国汕头大学医学院附属肿瘤医院2009年6月至2015年12月之间的乳腺癌手术样本，采集了总共121个乳腺癌组织(LA,n＝25,LB n＝21,Her-2,n＝17,TNBC,n＝58)和16个正常乳腺组织。分别从中国浙江省肿瘤医院和临沂人民医院采集了另外57个TNBC。汕头大学医学院、浙江省肿瘤医院和临沂人民医院的医学伦理委员批准了该方案，并且参与者在手术之前签署了书面知情同意书。
所有的新鲜肿瘤组织样本被立即保存在
Figure PCTCN2018086238-appb-000002
RNA稳定溶液(
Figure PCTCN2018086238-appb-000003
RNA Stabilization Solution,Invitrogen,Beijing,China)中。为了让该溶液充分渗透所述组织，将组织保持在4℃过夜，然后取出清上清液，将其转移至-80℃以长期储存。从距离癌边缘5cm处取对应的相邻非肿瘤组织，经有经验的病理学家评估，该组织不含明显的肿瘤细胞。根据国际抗癌联合会(International Union Against Cancer)的肿瘤-淋巴结-转移(TNM)分期系统，对肿瘤进行分期。按照美国国家综合癌症网络(National Comprehensive Cancer Network(NCCN))肿瘤学临床实践指南(V.1.2011)，对组织学分级进行评估。对正常乳腺组织进行H&E染色并由病理学家来验证。
circRNA微阵列杂交
样本制备和微阵列杂交基于Arraystar标准方案(Arraystar Inc.)进行。简言之，总RNA用RNA酶(Epicentre,Inc.)消化，除去线性RNA并富集 环状RNA。然后，对所富集的环状RNA进行扩增，用随机引发法将其转录为荧光cRNA(Arraystar Super RNA Labeling Kit；Arraystar)。经标记的cRNA用RNeasy Mini Kit(Qiagen)纯化。用NanoDrop ND-1000测定标记cRNA的浓度和比活(pmol Cy3/μgcRNA)。将标记的cRNA杂交到Arraystar Human circRNA Array(8x15K,Arraystar)上。洗涤玻片后，阵列用Agilent Scanner G2505C扫描。
circRNA微阵列数据分析
用Agilent Feature Extraction软件(version 11.0.1.1)提取数据。用R软件包进行一系列数据处理，包括分位数标准化(quantile normalization)。根据倍数变化截止值(FC>＝1.5)和具有统计学意义的P值(p值<＝0.05)，选择差异表达的circRNA。应用基于TargetScan42和miRanda43的Arraystar自制计算机程序，预测circRNA的miRNA靶标和circRNA-miRNA相互作用。为集中所靶向的miRNA概况，用miRNA支持载体回归(mirSVR)算法，对所预测的miRNA靶标的功效进行计分和排序(rank)。相应地，对于每种circRNA，鉴定出mirSVR计分最高的5种miRNA，来建立“前5”circRNA-miRNA网络(1种circRNA与5种miRNA相连)。
用qRT-PCR验证候选circRNA
用Trizol试剂(Life Technologies,Carlsbad,USA)，按照厂商的说明，从乳腺癌组织和正常乳腺组织提取组织总RNA。RNA样本的纯度和浓度用NanoDrop ND-1000(Thermo Fisher Scientific,Wilmington,DE)测定。通过在变性琼脂糖凝胶上电泳评价RNA完整性。制备RNA并将其储存于-80℃以供验证实验用。
用
Figure PCTCN2018086238-appb-000004
II First Strand cDNASynthesis Kit(Geneseed,China)，由总RNA制备cDNA。qRT-PCR中所用的引物被设计为汇聚性引物 (convergentprimer)，用以探测环接点(circular junction)并跨越反向剪接接点。从数据库“circinteractome”( https://circinteractome.irp.nia.nih.gov/) 获取3种circRNA的序列。数据用ΔCt法分析。所有引物皆由Generay Biotech合成。所有的结果皆以三个独立实验的平均值±SD表示。
经验证的候选circTADA2A-E6的注释和功能预测
按照TargetScan(http://www.targetscan.org/)联合miRanda( http://www.microrna.org/)分析，将经验证的候选circRNA用作种子，来富集circRNA-miRNA-基因网络。应用Cytoscape(http://www.cytoscape.org/)建立circTADA2A的circRNA–miRNA–mRNA相互作用网络。所述网络中预测的基因功能用GO和KEGG路径分析进行注释。通过Database for Annotation,Visualization and Integrated Discovery(DAVID； http://www. david.abcc.ncifcrf.gov/)，鉴定出由Kyoto Encyclopedia of Genes and Genomes(KEGG)、Biocarta和Reactome(http://www.genome.jp/kegg/)限定的生物学路径。
检测推定的miRNA结合位点
circRNA中推定miRNA结合位点的FASTA文件由miRBase release20.0(http://www.mirbase.org/)获取。仅考虑成熟miRNA用于种子分析。将所述miRNA与circRNA进行比对。miRNA的推定靶位点是基因组中代表成熟miRNA序列的核苷酸2-7的反向互补物的长6个核苷酸的序列。
用于mRNA和circRNA的cDNA合成和qRT-PCR
使用
Figure PCTCN2018086238-appb-000005
II First Strand cDNA Synthesis Kit(Geneseed,China)，按照所供应的方案由总RNA合成cDNA。用基于
Figure PCTCN2018086238-appb-000006
Green法的ABI Prism 7500序列检测系统(Applied Biosystems,Foster City,CA,USA)，测量环状RNA的相对基因表达。qPCR Ct值用厂商的软件自动计算。用 Human-ACTB作为内标对照。具体而言，使用退火至circRNA远末端的发散性引物(divergent primer)测定circRNA丰度。使用测序引物，直接对PCR产物进行Sanger测序。
用于miRNA的cDNA合成和qRT-PCR
为了定量测定成熟miRNA的量，用Mir-X TM miRNA First-Strand Synthesis(Takara,China)对成熟miRNA进行逆转录，使用Mir-X miRNA qRT-PCR SYBR Kit，在ABI Prism 7500序列检测系统(Applied Biosystems,Foster City,CA,USA)上，用小核U6B(RNU6B)RNA作为内标对照，进行PCR扩增。用2 -ΔΔCt法进行相对定量。
circTADA2A载体的构建
为了对circRNA进行概述，合成circTADA2A-E6的(基因组区)成熟序列，将其克隆到pLCDH-ciR-copGFP-Puro(Geneseed,Guangzhou,China)中。且通过测序进行验证。
circTADA2A siRNA干扰
circTADA2A的siRNA由Ribobio(Guangzhou,China)设计和合成，靶向circTADA2A序列的接合区(junction region)。siRNA靶序列如下：
1：5’-CCATTTCACTGCAGGATGT-3’；
2：5’-CACTGCAGGATGTAGCCAA-3’；
3：5’-TTCCATTTCACTGCAGGAT-3’。
寡核苷酸转染
miRNA模拟物和抑制剂由Ribobio(Guangzhou,China)合成。细胞用Lipofectamine TM 3000(invitrogen,USA)转染。
蛋白质印迹分析
蛋白质提取和蛋白质印迹按照先前所述方法进行。
增殖、细胞周期、凋亡和细胞侵袭测定、体外肿瘤发生测定
增殖测定(CCK-8).用CCK-8试剂盒(Doindo,Japan)测试MCF-7和MDA-MB-231细胞的增殖。将经转染的约1x10 3细胞/孔一式三份在96孔板中孵育。24、48、72、96小时时，向各孔加入CCK-8试剂，于37℃孵育1.5h。用自动微量滴定板读板仪(Synergy4；BioTek,Winooski,VT,USA)测量450nm光密度。
细胞迁移侵袭测定(Transwell invasion assay).分别用细胞培养物插入物(8μM孔径；BD)和matrigel侵袭小室(BD)进行侵袭测定。将经转染的细胞血清饥饿24h，将无血清培养基中的1×10 4MDA-MB-231细胞或2×10 4MCF-7细胞接种到上室中。分别在20小时(MDA-MB-231)或48小时(MCF-7)时用4％多聚甲醛固定细胞，以进行侵袭测定，然后用0.1％结晶紫染色。每个测定一式三份进行。由两位调查人员对各孔中3个视野的细胞数进行计数。
集落形成测定.对于集落形成测定，将100个MDA-MB-231细胞或200个MCF-7细胞一式三份铺板于12孔板中，让其生长至出现可见集落。集落用4％多聚甲醛固定，用0.1％结晶紫染色，然后计数。
统计学分析
所有统计学数据用Statistical Product and Service Solutions SPSS software 16.0(SPSS,Chicago,IL)、GraphPad Prism 5.0(GraphPad Software,La Jolla,CA)和SigmaPlot 10.0(SigmaPlot Software,La Jolla,CA)进行分析。乳腺癌组织和正常乳腺组织之间的circRNA水平差异用t检验对成对数据来测定。通过单因素方差分析(ANOVA)，对circRNA水平和临床病理学因素之间的关联进行进一步分析。建立受试者工作特征(ROC)曲线以评估其诊断价值。circRNA的截止值用SigmaPlot 12.3分析。实验再现性通过Pearson相关检验(Pearson correlation test)来确定。所有实验一式三份进行。数据以平均值±SEM表示(除非另有说明)，用双侧Student t检验 进行统计学分析。P＜0.05被认为有统计学意义。
实施例1.在乳腺癌中差异表达的circRNA的筛选及验证
高通量circRNA芯片测序有助于鉴别乳腺癌和正常乳腺组织之间差异表达的circRNA。通过芯片技术分析8个乳腺癌组织(TNBC,n＝4,luminal A,n＝4)和三个正常乳腺组织的circRNA的差异表达，得到了乳腺癌与正常组织的差异表达谱。散点图展示了两组之间差异表达的环状RNA，差异倍数达到1.5倍以上；火山图展示了两组之间具有统计学意义的差异表达的环状RNA。与正常乳腺组织相比，在乳腺癌中发现总共140种上调和95种下调的环状转录物(图1c)，而且该表达谱数据与其它研究一致，表明circRNA大多数来自含基因区域的外显子和内含子。值得注意的是，对于原发性肿瘤和正常乳腺组织，circRNA显示出不同的表达簇(图1d)。这些circRNA与其靶标miRNA之间的相互作用可以通过软件按照互补miRNA匹配序列在理论上进行预测。预测总共465种miRNA可能会与circRNA结合。通过Cytoscape软件绘制完整的circRNA-miRNA相互作用网络图(图1e)。所有这些数据表明：乳腺癌中存在种类繁多的差异表达的circRNA，可能可用于疾病的筛选和诊断。
通过circRNA微阵列筛选在乳腺癌TNBC组织中差异表达的circRNA，筛选出在下调表达差异倍数在1.5倍以上的8种circRNA。结果示于表1。
表1.通过circRNA微阵列筛选在乳腺癌TNBC组织中差异表达的circRNA
Figure PCTCN2018086238-appb-000007
荧光定量PCR证实三阴性乳腺癌组织中差异表达的circRNA
选择上述8种下调的circRNA(见表1)，又通过实时荧光定量PCR(qRT-PCR)在178例乳腺组织中进一步证实了这些差异表达的circRNA。8种circRNA的长度范围为158nt至843nt，所有这8种circRNA都为外显子剪接，针对环状RNA转录物的环化位点设计了外显子两端向外的引物(outward-facing primer)，以区别于基因编码的线性产物。接下来，用随机六聚体引物将circRNA逆转录成cDNA，用反向引物扩增产生预期的环状特异性RNA产物。最后，对扩增产物进行测序，证实了环状RNA环化位点序列与来自CircBase具有剪接点的环状RNA序列完全一致。最后，我们使用发散性引物组(divergent primer set)，以测定三阴型乳腺癌组织和正常乳腺组织样本中的circRNA表达水平(n＝16)。所有这些circRNA的差异表达都得到了证实，这证明环状RNA芯片数据的精确性(参见图1f)。
实施例2.两种circTADA2A的表达水平和这两种circTADA2A对TNBC的预后性和预测性价值
两种在乳腺癌组织中下调的环状RNA
首先鉴定了circTADA2A-E6和circTADA2A-E5/E6两种circRNA在不同分子分型乳腺癌组织中的表达，这两种circRNA均起源于TADA2A基因。如图2c和2d所示，qRT-PCR分析显示：这两种circTADA2A在BC组织中的表达水平显著低于正常乳腺组织的水平，为：circTADA2A-E6，LA>TNBC>Her-2>LB；circTADA2A-E5/E6，LB>TNBC>Her-2>LA(所有组别,p<0.01)。我们发现与MCF-10(一个非致瘤的上皮细胞株)相比，10株乳腺癌细胞株中的circTADA2A-E6(参见图2c)和circTADA2A-E5/E6(参见图2d)的表达水平较低。
两种circTADA2A的表达与临床病例特征之间的关联
为了研究circTADA2A-E6和circTADA2A-E5/E6的表达和临床参数之间的潜在关联，我们分析了TNM(肿瘤大小、淋巴结转移以及远处转移)状况，结果显示肿瘤circTADA2A-E6表达水平降低与淋巴结转移更多(p＝0.012)和高临床分期(p＝0.022)显著相关(图2e和图2f)，但是circTADA2A-E5/E6与TNM分期没有明显的相关关系。这些结果提示：circTADA2A-E6表达下调与TNBC的侵袭性特征之间有关联。
结果概述于表2和表3中。
表2.临床病理学因素和TNBC组织(n＝115)中circTADA2A-E6表达之间的关联
Figure PCTCN2018086238-appb-000008
AJCC,American Joint Committee on Cancer
#,4.35％患者信息丢失
##,9.56％患者信息丢失
*p<0.05
表3.临床病理学因素和TNBC组织(n＝115)中circTADA2A-E5/E6表达之间的关联
Figure PCTCN2018086238-appb-000009
AJCC,American Joint Committee on Cancer
#,4.35％患者信息丢失
##,9.56％患者信息丢失
*p<0.05
circTADA2A-E6和circTADA2A-E5/E6的表达在乳腺癌中的诊断价值
为了评估两种circTADA2A能否用作三阴型(n＝115)乳腺癌的诊断指标，使用总共16个正常乳腺组织作为对照制作了ROC曲线。在TNBC与正常组相比的ROC曲线中，circTADA2A-E6和circTADA2A-E5/E6的曲线下面积分别为0.8554(95％CI＝0.9089-1.016,P<0.0001,图2e)和0.9366(95％CI＝0.9089-1.016,P<0.001,图2f)。另外，发现：circTADA2A-E6和circTADA2A-E5/E6在LA(n＝25)、LB(n＝21)和Her-2(n＝17)中circTADA2A-E6和circTADA2A-E5/E6中的所有曲线下面积皆 高于0.84(图2e/f)。这些结果提示：两种circTADA2A可作为乳腺癌的潜在诊断性生物标志物。
circTADA2A-E6和circTADA2A-E5/E6用于预测乳腺癌风险的评估
在评价circTADA2A-E6和circTADA2A-E5/E6的表达在乳腺癌中的预后价值之前，对115例TNBC病人进行了单因素和多因素Cox比例风险回归分析(Cox proportional hazards regression analysis)。结果表明TNM分期和肿瘤大小是与TNBC病人的总生存期(overall survival,OS)和无病生存期(disease-free survival,DFS)相关的两个独立因素(结果未显示)。重要的是，发现：circTADA2A-E6低表达组的10年总生存期显著短于circTADA2A-E6高表达组(图2h,p＝0.0179)，circTADA2A-E6低表达组的10年DFS显著短于circTADA2A-E6高表达组的10年DFS(图2g)，p值为0.0715。然而，circTADA2A-E5/E6表达与TNBC病人的OS(图2h)和DFS(图2g)并不显著相关。这些分析数据提示：circTADA2A-E6是乳腺癌进展的潜在预测性生物标志物。
实施例3.circTADA2A-E6的过表达或沉默抑制或增强乳腺癌细胞增殖、集落形成、侵袭
circTADA2A-E6抑制细胞增殖和侵袭
构建了circTADA2A高表达载体以及用于敲减的siRNA干扰序列(图3a和图4a)。CCK-8测定结果显示：高表达circTADA2A可以降低细胞的增殖能力(p<0.05,图3c)，而干扰circTADA2A，细胞增殖能力增强(p<0.05,图4c)。划痕愈合实验表明：高表达circTADA2A可以降低细胞的划痕愈合能力(图3e)，而敲减可以促进细胞的划痕愈合能力(图4f)。克隆形成实验结果表明：circTADA2A-E6过表达细胞中克隆形成能力降 低(图3d)，而在circTADA2A-E6敲减细胞中克隆形成能力增加(图4d)。此外，侵袭实验结果显示：细胞侵袭率在circTADA2A-E6过表达细胞中细胞侵袭率降低3.5倍(图3f)，在circTADA2A-E6敲减细胞中增加至2.88倍(图4e)。另外，circTADA2A-E6在MDA-MB-231细胞中过表达显著增加了上皮细胞标记E-钙粘蛋白(E-cadherin)的表达(图3g)，而E-Cad的表达在circTADA2A-E6敲减细胞中则是降低的(图4g)。相比之下，间充质标记波形蛋白(mesenchymal marker vimentin)的表达在MDA-MB-231circTADA2A-E6过表达细胞中降低(图3g)，在circTADA2A-E6敲减细胞中增加(图4g)。综合而言，这些结果表明：circTADA2A-E6在乳腺癌细胞中发挥抑癌的作用，与临床病理结果在很大程度上是一致的，这就是说circTADA2A-E表达水平与肿瘤发生发展相关。
实施例4.circNOL10的表达水平和circNOL10对TNBC的预后性和预测性价值
用与实施例3类似的方法，对circNOL10的表达和临床参数之间的潜在关联进行了分析。
qRT-PCR证实circNOL10在三阴性乳腺癌组织中明显下调
通过实时荧光定量PCR(qRT-PCR)检测circNOL10在乳腺癌组织中的表达。在此过程中，我们针对环状RNA转录物的环化位点设计了发散型引物(outward-facing primer)，以区别于基因编码的线性产物。接下来，我们用随机六聚体引物将circRNA逆转录成cDNA，用反向引物扩增产生预期的环状特异性RNA产物。最后，对扩增产物进行测序，证实了环状RNA环化位点序列与来自CircBase具有剪接点的环状RNA序列完全一致。最后，通过实时荧光定量PCR(qRT-PCR)在178例乳腺组织(TNBC＝115,LA＝25,LB＝17,Her2＝21)中进一步证实了circNOL10乳 腺癌组织中下调(图5a)。circNOL10在BC组织中的表达水平显著低于正常乳腺组织的水平，为：TNBC>LA>Her-2>LB。我们发现与MCF-10(一个非致瘤的上皮细胞株)相比，10株乳腺癌细胞株中的circNOL10也是下调的(参见图5b)。
circNOL10的表达与临床病例特征之间的关联
为了研究circNOL10的表达和临床参数之间的潜在关联，我们分析了TNM(肿瘤大小、淋巴结转移以及远处转移)状况，结果显示肿瘤circNOL10表达水平降低与淋巴结转移更多(p＝032)和高临床分期(p＝0.018)显著相关(表4)。这些结果提示：circNOL10表达下调与TNBC的侵袭性特征之间有关联。
表4.临床病理学因素和TNBC组织(n＝115)中circNOL10表达之间的关联
Figure PCTCN2018086238-appb-000010
circNOL10的表达在乳腺癌中的诊断价值
为了评估circNOL10能否用作三阴型(n＝115)乳腺癌的诊断指标，我们使用总共16个正常乳腺组织作为对照制作了ROC曲线。在TNBC与正常组相比的ROC曲线中，circNOL10曲线下面积分别为0.9212。另外，我们发现：circNOL10在LA(n＝25)、LB(n＝21)和Her-2(n＝17)中的曲线下面积分别是0.9275、0.7619和0.9154(图5c)。这些结果提示：circNOL10可作为乳腺癌的潜在诊断性生物标志物。
circNOL10用于预测乳腺癌风险的评估
circNOL10的生存分析结果显示circNOL10低表达组的10年总生存期显著短于circTADA2A-E6高表达组(图5d),p＝0.027，低表达组的10年DFS显著短于circTADA2A-E6高表达组的10年DFS(图5d)，p值为0.032。这些分析数据提示：circNOL10可能是乳腺癌进展的潜在预测性生物标志物。
随着近年来深度测序(deep sequencing)和微阵列技术的快速发展，越来越多的证据证明哺乳动物基因组可编码circRNA，这些circRNA普遍被转录并且在各种各样的生物过程中发挥重要的功能性作用。本发明人通过对乳腺肿瘤(n＝8)和正常乳腺组织样本(n＝3)的circRNA微阵列数据的分析中，发现：与正常乳腺组织相比，在乳腺癌中发现总共140种上调和95种下调的环状RNA。通过q-RT-PCR在178例乳腺癌患者样本中确证8种circRNA下调。进一步的研究发现，某些circRNA为新的潜在的TNBC预后性和预测性生物标志物，作为一种内源性RNA与miRNA竞争性调控miRNA的靶基因，从而产生抑瘤效应。这些发现为未来circRNA应用于乳腺癌的诊断和治疗提供了理论和临床依据。
                       *   *   *
根据本公开内容，不需要过度实验即可实施本发明的实施方案。尽管已经就优选实施方案描述了本发明，但本领域技术人员应显而易见，在不背离本发明的理念、精神和范围的情况下可以对本文中所描述的试剂盒、药物组合物、用途和方法以及方法中的步骤或步骤顺序加以变化。对本领域技术人员显而易见的等同的替代和修改都在由所附权利要求所限定的本发明精神、范围和理念之内。

Claims (10)

  1. circRNA作为乳腺癌标志物的用途，其中所述circRNA选自以下的一种或更多种：circTADA2A-E6、circTADA2A-E5/E6、circNOL10、circNSUN2、circCSRNP2、circFAM125B、circCDC27和circABCC1，优选选自以下的一种或更多种：circTADA2A-E6、circTADA2A-E5/E6和circNOL10。
  2. 用于检测根据权利要求1中所述的circRNA的试剂和/或微阵列在制备用于乳腺癌诊断或预后的试剂盒中的用途，所述试剂优选包括特异性探针。
  3. 用于检测权利要求1中所述的circRNA的特异性探针在制备用于乳腺癌诊断或预后的微阵列中的用途。
  4. 乳腺癌诊断或预后用的试剂盒，其包含用于测量根据权利要求1所述的circRNA水平的试剂或微阵列，所述试剂优选包括特异性探针。
  5. 一种诊断受试者是否患有乳腺癌或处于发生乳腺癌风险、或者对乳腺癌进行预后的方法，其包括测量测试样本中根据权利要求1所述的circRNA的水平。
  6. 根据权利要求1所述的circRNA作为乳腺癌诊断或预后标志物的用途。
  7. 用于鉴定乳腺癌抑制剂的方法，所述方法包括鉴定提高乳腺癌细胞中circTADA2A-E6和/或circNOL10的水平的物质。
  8. 用根据权利要求7所述的方法鉴定的乳腺癌抑制剂。
  9. 根据权利要求8所述的抑制剂在制备用于抑制或治疗乳腺癌的药物中的用途。
  10. 用于抑制或治疗乳腺癌的药物组合物，其包含circTADA2A-E6、提高circTADA2A-E6水平的药剂、表达circTADA2A-E6的核酸和/或根据权利要求8所述的乳腺癌抑制剂，所述药物组合物任选含有用于抑制或治疗乳腺癌的其他药剂。
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