WO2021128516A1 - CircRNA PVT1及肽段在肿瘤生长预测、转移预测、预后评估和治疗中的应用 - Google Patents

CircRNA PVT1及肽段在肿瘤生长预测、转移预测、预后评估和治疗中的应用 Download PDF

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WO2021128516A1
WO2021128516A1 PCT/CN2020/071984 CN2020071984W WO2021128516A1 WO 2021128516 A1 WO2021128516 A1 WO 2021128516A1 CN 2020071984 W CN2020071984 W CN 2020071984W WO 2021128516 A1 WO2021128516 A1 WO 2021128516A1
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pvt1
circrna
tumor
cancer
expression
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PCT/CN2020/071984
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French (fr)
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陈地灵
郭殷锐
吴清平
谢意珍
祁龙凯
唐晓萃
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广东省微生物研究所（广东省微生物分析检测中心）
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • A—HUMAN NECESSITIES
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention belongs to the fields of biotechnology and medicine, and specifically relates to the correlation and application of CircRNA PVT1 (hsa_circ_0001821) nucleic acid fragment and circRNA PVT1 translated peptide PVT1-104aa in tumor growth prediction, metastasis prediction, prognosis evaluation, and treatment.
  • CircRNA can be used as a potential biomarker molecule for drugs or clinical diagnosis.
  • Studies have shown that in addition to linear RNA molecules, there are a large number of closed circular RNA molecules in living organisms, which are specifically expressed in individual development, different tissues and diseases. It was identified by sequencing that these circRNA molecules usually consist of 2-4 exons of protein-encoding genes, which are circularized by the complementary combination of the 5'end of the former exon and the 3'end of the latter exon. Due to the circular sealing properties of circRNA, it can escape the action of exonuclease. Therefore, circRNA can exist stably in cells for a long time. It is the latest research hotspot in the RNA field in recent years. It is also because of its stability that it is easy for us to think of whether circRNA can be used as a potential biomarker molecule for drugs or clinical diagnosis.
  • Circular RNA has important physiological functions. Based on the study of the structure and function of circRNAs, it is found that they play a very important role in the development of a variety of diseases, such as atherosclerosis, nervous system disorders, diabetes, and tumors.
  • the main functions are as follows: 1) circRNAs can be used as "sponge" sponges to adsorb miRNAs and inhibit their functions; 2) circRNAs directly regulate the level of other RNAs through base complementary pairing; 3) circRNAs can bind to proteins to inhibit protein activity and recruitment The components of protein complexes or/and regulate the activity of proteins; 4) circRNA can also be used as a template for translation to guide protein synthesis.
  • RNA may have the following important significance: 1) The unique competitive endogenous (ceRNA) characteristics of circRNA can provide new ideas for drug development; 2) the tissue specificity of circRNA And stability, circRNA has become a good biomarker; 3) The study of circRNA can provide a new direction for the evolution of life.
  • ceRNA competitive endogenous
  • Circular RNA is an emerging research hotspot. In recent years, research on its function has also focused on tumors, accounting for 1/3 to 1/2 of the ratio. Compared with other markers, there are fewer studies on circular RNA and its translated protein in tumors.
  • the present invention provides a new approach that can be used for tumor diagnosis, tumor treatment plan selection, and tumor prognosis evaluation.
  • the present invention achieves the above-mentioned objects through the following schemes:
  • CircRNA PVT1 and PVT1-104aa are provided as drug targets for early diagnosis of tumors, treatment of tumors, alleviation or prevention of tumor metastasis, and/or improvement of tumor prognosis.
  • the product test is performed on a mammal or a sample obtained from the mammal that has or is suspected of having a tumor or is at risk of having a tumor or has had a tumor but has been cured.
  • the mammal is selected from: primates, rodents, livestock mammals, mammals and pets, such as humans, apes, orangutans, monkeys, cows, sheep, horses, camels, pigs , Dogs, cats, rabbits, mice, etc.
  • the mammal has been identified as a mammal suffering from a tumor or a tumor that has been cured using methods known in the art.
  • the sample is selected from: a tissue or cell sample obtained from the mammal, such as a breast tissue or cell sample, a cancer tissue or cell sample, a paracancerous tissue or cell sample.
  • the sample is a fresh sample, a frozen sample, a fixed sample (such as a formalin fixed sample), or an embedded sample (such as a paraffin embedded sample).
  • the substance used to detect the expression levels of CircRNA PVT1 and PVT1-104aa is a substance used to detect CircRNA PVT1 and PVT1-104aa at the gene level and/or protein level.
  • the substance used to detect the expression levels of CircRNA PVT1 and PVT1-104aa is a substance used in one or more detection techniques or methods selected from the following group: immunohistochemistry (such as immunofluorescence Analysis, ELISA, immunogold method), Western blotting, Northern blotting, PCR, biochip method.
  • immunohistochemistry such as immunofluorescence Analysis, ELISA, immunogold method
  • Western blotting Western blotting
  • Northern blotting PCR
  • biochip method biochip method
  • the substance used to detect the expression levels of CircRNA PVT1 and PVT1-104aa is selected from substances specific for CircRNA PVT1 and PVT1-104aa, such as an anti-PVT1-104aa antibody or an antigen-binding fragment thereof, preferably a single Cloned antibodies; CircRNA PVT1 specific probes, gene chips, PCR primers, gRNA (guide RNA), etc.
  • the substance used to detect the expression levels of CircRNA PVT1 and PVT1-104aa has a detectable label, for example, the detectable label selected from the group consisting of: radioisotope, fluorophore, chemiluminescence Part, enzyme, enzyme substrate, enzyme cofactor, enzyme inhibitor, dye, metal ion, or ligand (e.g., biotin or hapten).
  • the detectable label selected from the group consisting of: radioisotope, fluorophore, chemiluminescence Part, enzyme, enzyme substrate, enzyme cofactor, enzyme inhibitor, dye, metal ion, or ligand (e.g., biotin or hapten).
  • the tumor is selected from breast cancer, glioma, stomach cancer, kidney cancer, colorectal cancer, esophageal cancer, liver cancer, and the like.
  • the breast cancer is selected from the following types: invasive ductal carcinoma, non-invasive ductal carcinoma; or, luminal type A, luminal type B, luminal-HER2 type, HER2 overexpression type, basal Cell-like type, TNP-non-basal type; alternatively, hormone receptor positive type, HER2/neu receptor positive type, triple negative type.
  • the product includes: compared with a normal control value, the levels of CircRNA PVT1 and PVT1-104aa in the subject or a sample obtained from the subject are increased, indicating that the subject is prone to or has undergone cancer metastasis, Either it indicates that the prognosis of the subject's cancer is poor, or that the subject already has cancer.
  • the normal control value is obtained from a sample of a subject not suffering from a tumor, a sample obtained from a normal tissue of the subject, and the levels of CircRNA PVT1 and PVT1-104aa of a normal subject.
  • the normal control value is: the molecular level of CircRNA PVT1 and PVT1-104aa measured in a non-tumor normal biological sample (such as a sample obtained from a healthy person or normal tissue of a subject to be tested), through statistics The population standard level determined by science, or the standardized level.
  • the product is a test kit.
  • the detection kit further comprises one or more substances selected from the following group: containers, buffers, adjuvants, solvents, negative controls, positive controls, instructions for use.
  • the detection kit is a kit for detecting PVT1-104aa expression in a biological sample by immunohistochemical method based on immunoenzyme labeling method, which includes: blocking solution (such as 10% goat serum), anti-PVT1 -104aa antibody (such as rabbit anti-human or mouse PVT1-104aa monoclonal antibody or polyclonal antibody), secondary antibody (such as anti-rabbit biotinylated secondary antibody), labeled conjugate (such as HRP-labeled streptavidin), Substrate buffer (such as DAB substrate buffer), color developing solution (such as DAB color developing solution) and/or substrate solution.
  • blocking solution such as 10% goat serum
  • anti-PVT1 -104aa antibody such as rabbit anti-human or mouse PVT1-104aa monoclonal antibody or polyclonal antibody
  • secondary antibody such as anti-rabbit biotinylated secondary antibody
  • labeled conjugate such as HRP-labeled streptavidin
  • Substrate buffer such as D
  • the detection kit is a kit for detecting the expression of CircRNA and PVT1 in a biological sample based on fluorescently labeled in situ hybridization technology (FISH technology), which includes: fluorescent molecular probes.
  • FISH technology fluorescently labeled in situ hybridization technology
  • a method for predicting tumor metastasis, evaluating tumor prognosis, or diagnosing tumors comprising: detecting the level of CircRNA PVT1 and PVT1-104aa in a subject or a sample obtained from the subject step.
  • the levels of CircRNA PVT1 and PVT1-104aa in the subject or a sample obtained from the subject are increased, indicating that the subject is prone to or has undergone tumor metastasis, or indicating that the subject’s
  • the prognosis of tumor patients is poor, or it indicates that the subject has cancer.
  • the application of an inhibitor that inhibits CircRNA PVT1 and PVT1-104aa and/or its genes in the preparation of drugs for treating tumors, alleviating or preventing tumor metastasis, and/or improving tumor prognosis is provided.
  • the inhibitor is selected from: inhibitors that directly bind to CircRNA PVT1 and PVT1-104aa, inhibitors that block the binding of CircRNA PVT1 and PVT1-104aa to their receptors or ligands, PVT1 (LINC00079; MIR1204HG ; NCRNA00079; onco-lncRNA-100) and CircRNA PVT1 inhibitors, inhibitors to reduce the expression levels of CircRNA PVT1 and PVT1-104aa, inhibitors to promote the degradation of CircRNA PVT1 and PVT1-104aa, for knockout or knockdown Substances expressed by CircRNA PVT1 and PVT1-104aa, such as related promoter elements, recombinant plasmids, expression vectors and related antibodies that can down-regulate the expression of CircRNA PVT1 and the protein expression level of PVT1-104aa.
  • the inhibitor is selected from: anti-PVT1-104aa antibody, anti-PVT1-104aa receptor antibody, CRISPR/Cas9 system against CircRNA PVT1 and PVT1-104aa, interfering RNA against PVT1 and CircRNA PVT1, Antisense oligonucleotides for PVT1 and CircRNA PVT1, CircRNA PVT1 and PVT1-104aa expression and/or function inhibitory compounds.
  • the CRISPR/Cas9 system for CircRNA PVT1 and PVT1-104aa includes: a vector pCD513B-Cas9 that expresses Cas9 enzyme and a vector psk-U6-sgRNA that expresses guideRNA (gRNA); the expression is for HSPA4
  • the gRNA sequence selected from different sites of the gene is preferably a gRNA having any one of the following.
  • GRNA-NC GGCCTTAAGAGTACTCATC.
  • Cir-PVT1-GRNA1 Cir-PVT1-GRNA1:
  • Cir-PVT1-GRNA1-F1 5’P*CACCGAGTGGTCTGGGGAATAACGC3’,
  • Cir-PVT1-GRNA1-R1 5’P*AAA CGCGTTATTCCCCAGACCACTC3’;
  • Cir-PVT1-GRNA2 Cir-PVT1-GRNA2:
  • Cir-PVT1-GRNA2-F2 5’P*CACCGCGAAGCT CCATGCAGCTGAC3’
  • Cir-PVT1-GRNA2-R2 5’P*AAACGTCAGCTGCATGGAGCTTCGC3’;
  • NC-GRNA-F 5’P*CACCGGCGCCTTAAGAGTACTCATC3’
  • the drug comprises: (a) CircRNA PVT1 and PVT1-104aa and/or an inhibitor of its genes; and (b) a pharmaceutically or health care acceptable carrier.
  • the drug also contains other anti-tumor substances, such as DNA damage chemotherapeutics, multi-targeted tyrosine kinase inhibitors, cell proliferation inhibitors, angiogenesis inhibitors, alkylating agents, and anti-metabolites. , Anti-tumor antibiotics, plant anti-cancer substances, hormones or immunosuppressive agents.
  • the drug is in the form of solid (such as granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid (such as oral liquid, solution or syrup).
  • the form of the drug is suitable for oral, parenteral (such as intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or subcutaneous administration), subcutaneous, intraperitoneal, intrapulmonary, intranasal , Intratumor, local administration.
  • parenteral such as intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or subcutaneous administration
  • subcutaneous, intraperitoneal, intrapulmonary, intranasal , Intratumor, local administration such as intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or subcutaneous administration.
  • a method for screening a candidate drug for treating tumors, alleviating or preventing tumor metastasis, and/or improving tumor prognosis comprising testing the candidate drug against a subject or a sample obtained from the subject.
  • the impact of CircRNA PVT1 and PVT1-104aa levels in CircRNA PVT1 and PVT1-104aa, where the decrease in CircRNA PVT1 and PVT1-104aa levels after the use of the candidate drug indicates that the candidate drug has the potential to treat tumors, alleviate or prevent tumor metastasis, and/or improve tumor prognosis effect.
  • the reduction refers to the level of CircRNA PVT1 and PVT1-104aa relative to a sample obtained from a subject not suffering from a tumor, a sample obtained from a normal tissue of the subject, and a normal subject.
  • the reduction refers to a normal control value, which is measured from a non-tumor normal biological sample (such as a sample obtained from a normal tissue of a healthy person or a subject to be tested).
  • a non-tumor normal biological sample such as a sample obtained from a normal tissue of a healthy person or a subject to be tested.
  • the obtained CircRNA PVT1 and PVT1-104aa molecular levels, or population standard levels determined by statistics, or standardized levels.
  • a product for predicting tumor growth, metastasis, evaluating tumor prognosis, or diagnosing tumors which includes substances for detecting the levels of CircRNA PVT1 and PVT1-104aa.
  • a pharmaceutical composition which comprises: an inhibitor for inhibiting CircRNA PVT1 and PVT1-104aa and/or genes thereof as a therapeutically active substance, and a pharmaceutically acceptable carrier.
  • the characteristics related to the active substance and performance of the pharmaceutical composition can be as defined or illustrated herein.
  • a method for tumor diagnosis, treatment of tumor, alleviation or prevention of tumor metastasis, and/or improvement of tumor prognosis comprising: administering CircRNA PVT1 and PVT1-104aa to the subject and/ Or an inhibitor of its gene or a drug containing the inhibitor.
  • CircRNA PVT1 of the present invention is preferably the sequence described in SEQ ID NO 1.
  • the PVT1-104aa of the present invention is preferably the sequence described in SEQ ID NO 2.
  • CircRNA PVT1 nucleic acid fragment and peptide PVT1-104aa are useful in tumor growth prediction, metastasis prediction, and prognosis evaluation. And play a significant and stable role in the treatment.
  • Figure 1 is a schematic diagram of the transcription of lncRNA and PVT1 at the position of the representative chromosome chr8 (q24.21).
  • Figure 2 is the identification result of circRNA PVT1.
  • Figure 3 is the sequence of CircRNA PVT1.
  • Figure 4 is an immunohistochemical comparison of PVT1-104aa expression in breast cancer tissue and adjacent non-tumor tissues.
  • Figure 5 is a graph showing the comparison of the expression levels of PVT1-104aa in breast cancer tissues and non-tumor tissues adjacent to cancer.
  • Figure 6 is an immunohistochemical map of PVT1-104aa expression in colorectal cancer tissues and non-tumor tissues adjacent to cancer.
  • Fig. 7 is a comparison diagram of the expression levels of PVT1-104aa in colorectal cancer tissues and adjacent non-tumor tissues.
  • Figure 8 is an immunohistochemical map of the expression of PVT1-104aa in kidney cancer tissue and non-tumor tissue adjacent to the cancer.
  • Figure 9 is a comparison diagram of the expression levels of PVT1-104aa in kidney cancer tissues and adjacent non-tumor tissues.
  • Figure 10 is a graph showing the western blot results of PVT1-104aa and ⁇ -actin in human breast cancer and adjacent tissues.
  • Figure 11 is a graph showing the western blot results of PVT1-104aa and ⁇ -actin in colorectal cancer and adjacent tissues.
  • Figure 12 is a comparison of the expression levels of CircRNA PVT1 in various tumors and adjacent tissues by PCR.
  • Figure 13 is a picture of mice after inoculation of 106 MB-231-NC cells on BALB/c nude mice.
  • Fig. 14 is a picture of mice after inoculation of 106 B-231-siRNA cells on BALB/c nude mice.
  • Figure 15 shows the tumor volume after inoculation of 106 MB-231-NC cells and B-231-siRNA cells on BALB/c nude mice.
  • Figure 16 is an immunositu hybridization map of MB-231 cells and transiently transfected interference plasmid (sicircPVT1) and overexpression plasmid (sscircPVT1).
  • Figure 17 is an immunositu hybridization map of MCF-7 cells and transiently transfected interference plasmid (sicircPVT1) and overexpression plasmid (sscircPVT1).
  • Figure 18 is a microscopic view of the expression of circPVT1 in human breast cancer tissues and adjacent tissues.
  • Figure 19 is a comparison diagram of the tumor growth size of transplanted tumor mice by the overexpression of PVT1-104aa adeno-associated virus packaging system.
  • Figure 20 is a comparison diagram of the tumor growth weight of the transplanted tumor mice by the overexpression PVT1-104aa adeno-associated virus packaging system.
  • Figure 21 is a comparison chart of the survival rate of the overexpression PVT1-104aa adeno-associated virus packaging system in the transplanted tumor mice.
  • Figure 22 is a graph showing the survival rate of gastric cancer patients in different PVT1-104aa groups.
  • Figure 23 is a graph of the survival rate of esophageal cancer patients in different PVT1-104aa groups.
  • Figure 24 is a graph of the survival rate of breast cancer patients in different PVT1-104aa groups.
  • One of the objectives of the present invention is to find molecules that can be effectively used for tumor growth prediction, metastasis prediction, and patient prognosis assessment and provide corresponding detection products.
  • Another object of the present invention is to provide targets, drugs and methods for effective treatment of tumors.
  • CircRNA PVT1 and PVT1-104aa are highly expressed in a variety of tumors (including breast cancer, glioma, gastric cancer, kidney cancer, colorectal cancer, esophageal cancer, etc.). Take breast cancer as an example.
  • the expression of CircRNA PVT1 and PVT1-104aa in the patient's tumor tissue is related to the patient's lymph node metastasis, and is closely related to the tumor size and stage. Its high expression is significantly related to the patient's short overall survival time and poor prognosis.
  • CircRNA PVT1 and PVT1-104aa molecules can significantly inhibit tumor growth and metastasis.
  • CircRNA PVT1 and PVT1-104aa molecules in tumor growth prediction, tumor metastasis prediction, tumor prognosis evaluation, and tumor treatment in subjects, and provides corresponding detection kits.
  • the above progress suggests that CircRNA PVT1 and PVT1-104aa detection and intervention treatment methods are expected to become a new growth point in tumor diagnosis and treatment.
  • CircRNA PVT1 is formed by splicing the second exon of LncRNA PVT1 (Pvt1oncogene, LINC00079, MIR1204HG, NCRNA00079, onco-lncRNA-100), as shown in the figure 1-
  • Pvt1oncogene LINC00079, MIR1204HG, NCRNA00079, onco-lncRNA-100
  • mouse CircRNA PVT1 (mmu_circ_0000604) is:
  • PVT1-104aa protein or polypeptide and “protein or polypeptide encoded by CircRNA PVT1 gene” are used interchangeably, and both refer to the protein or polypeptide encoded by CircRNA PVT1 gene, their conservative variant polypeptides, or Its homologous protein or polypeptide, or its active fragment (for example, PVT1-104aa binding domain).
  • the human PVT1-104aa sequence is (SEQ ID NO 2):
  • mouse PVT1-68aa The sequence of mouse PVT1-68aa is:
  • PVT1-104aa detection substance or “reagent for detecting PVT1-104aa molecule” or “reagent for detecting PVT1-104aa expression” are used interchangeably, and both refer to specific PVT1-104aa molecule, And can be used to directly or indirectly detect the presence and/or content of PVT1-104aa molecules. These test substances can detect PVT1-104aa at the gene level or protein level.
  • CircRNA PVT1 detection substance or “reagents for detecting CircRNA PVT1 molecules” or “reagents for detecting CircRNA PVT1 expression” are used interchangeably, and both refer to specific CircRNA PVT1 molecules and can be used directly Or indirectly detect the presence and/or content of CircRNA PVT1 molecules. These test substances can detect CircRNA PVT1 at the gene level.
  • the sequence of the CircRNA PVT1 molecule is known, a person of ordinary skill in the art can prepare or commercially obtain reagents specific to the CircRNA PVT1 molecule based on conventional methods.
  • the detection reagents that can be used in the present invention include but are not limited to: molecular probes with detection specificity for CircRNA PVT1 molecules, RT-qPCR primers, and the like.
  • the sequence of the PVT1-104aa molecule is disclosed herein, a person of ordinary skill in the art can prepare based on conventional means or obtain a reagent specific to the PVT1-104aa molecule on the market.
  • the detection reagents usable in the present invention include but are not limited to: antibodies with detection specificity for PVT1-104aa molecules.
  • the detection reagent of the present invention may also have detectable labels.
  • the detectable labels include but are not limited to: radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzymes Inhibitors, dyes, metal ions, ligands (such as biotin or hapten), etc.
  • the detection reagent of the present invention may exist in a solution, fixed on a carrier (such as a substrate, an adsorbent), or exist in other conventional ways in the art, as long as the existence method is suitable for detecting CircRNA PVT1 and PVT1-104aa in biological samples. Just check.
  • a carrier such as a substrate, an adsorbent
  • the detection reagent of the present invention is a nucleotide probe, it can exist in the form of a biochip (or "microarray").
  • the present invention also provides a product for predicting tumor growth, predicting tumor metastasis, evaluating tumor prognosis, or diagnosing tumors, which includes: substances for detecting the levels of CircRNA PVT1 and PVT1-104aa, and optionally, Other substances used to predict tumor growth, predict tumor metastasis, evaluate tumor prognosis, or diagnose tumors, such as detection materials for existing tumor markers.
  • the appropriate CircRNA PVT1 and PVT1-104aa detection materials can be selected and made into products suitable for the detection method used, such as a kit.
  • a person of ordinary skill in the art can adjust and change the detection method and the reagents contained in the product according to actual conditions and needs.
  • this article also provides a product (such as a detection kit), which contains: (i) a detection effective amount of one or more reagents for detecting CircRNA PVT1 and PVT1-104aa; (ii) any Optionally, one or more substances selected from the following group: containers, instructions for use, positive controls, negative controls, buffers, adjuvants or solvents, such as solutions for suspending or fixing cells, detectable Labels or tags, solutions that make nucleic acids easy to hybridize, solutions for lysing cells, or solutions for nucleic acid purification.
  • a detection kit which contains: (i) a detection effective amount of one or more reagents for detecting CircRNA PVT1 and PVT1-104aa; (ii) any Optionally, one or more substances selected from the following group: containers, instructions for use, positive controls, negative controls, buffers, adjuvants or solvents, such as solutions for suspending or fixing cells, detectable Labels or tags, solutions that make nucleic acids easy to hybridize, solutions
  • Example 1 provides a detection kit suitable for detecting the expression of PVT1-104aa in a biological sample by immunohistochemical methods.
  • the detection kit may include: blocking solution, such as 10% goat serum; primary antibody, such as rabbit anti-human or mouse PVT1-104aa monoclonal antibody; secondary antibody, such as labeled (such as HRP labeled) or unlabeled goat antibody Rabbit secondary antibody; substrate buffer, such as DAB substrate buffer; color developing solution; and optional containers containing the above reagents and instructions for use.
  • Example 2 provides a detection kit suitable for detecting the expression of PVT1-104aa in a biological sample by Western blot method.
  • the detection kit may include: blocking solution, such as 5% skimmed milk powder; primary antibody, such as rabbit anti-human or mouse PVT1-104aa polyclonal antibody; secondary antibody, such as labeled (such as HRP labeled) or unlabeled goat antibody Rabbit secondary antibody; chemiluminescence detection reagents, such as ECL chemiluminescence detection kits; and optional containers containing the above reagents and instructions for use.
  • Example 3 provides a detection kit suitable for detecting the expression of CircRNA PVT1-104aa in a biological sample by an RT-qPCR method.
  • the detection kit may include: extraction reagents, such as Trizol; DNA removal reagents, such as DNase I; reverse transcription reagents, such as RT Mix, Enzyme Mix, Random hexamers; real time qPCR reagents, such as Green Master Mix, Forward primer, Reverse primer; and optional containers containing the above reagents and instructions for use.
  • test kit herein can also be accompanied by an instruction manual for the kit, which records how to use the kit for detection, and how to use the test results to judge tumor metastasis and prognosis, and to select treatment options.
  • the kit may also include other reagents clinically used in the judgment of tumor development in the subject, selection of treatment options, and/or prognosis evaluation to assist or verify the results obtained by detecting CircRNA PVT1 and PVT1-104aa.
  • reagents clinically used in the judgment of tumor development in the subject, selection of treatment options, and/or prognosis evaluation to assist or verify the results obtained by detecting CircRNA PVT1 and PVT1-104aa.
  • CircRNA PVT1 and PVT1-104aa are used in tumor growth prediction, tumor metastasis prediction, prognostic evaluation, and diagnosis. And application in drug screening
  • the levels of CircRNA PVT1 and PVT1-104aa are closely related to tumor growth, tumor metastasis, prognosis and diagnosis, and thus can be used as indicators for tumor growth prediction, metastasis prediction, prognostic evaluation and diagnosis.
  • prognosis refers to predicting the possible course and outcome of a disease, which includes judging the specific consequences of the disease (such as recovery, the appearance or disappearance of other abnormalities such as certain symptoms, signs and complications, and death).
  • the poor prognosis mentioned in this article includes but is not limited to: shortened survival time, prone to tumor metastasis, rapid increase in the number of tumors, accelerated tumor growth, and increased TNM grades. After predicting the patient's prognosis, it can be combined with treatments that reduce the amount of CircRNA PVT1 and PVT1-104aa molecules to improve the patient's prognosis.
  • the following methods can be used for tumor growth prediction, tumor metastasis prediction, prognostic evaluation and/or diagnosis: detect the levels of CircRNA PVT1 and PVT1-104aa molecules in the test subject or samples obtained from the subject, and compare the levels with the control Level comparison; if the comparison result shows that the levels of CircRNA PVT1 and PVT1-104aa molecules in the subject are higher than the control level, it indicates that the subject is prone to tumor metastasis, poor prognosis, or has cancer.
  • the method of the present application may also optionally include: obtaining a test sample from a subject; contacting the test sample with a reagent or kit for detecting CircRNA PVT1 and PVT1-104aa levels.
  • normal control refers to the level of CircRNA PVT1 and PVT1-104aa molecules used as a reference, which includes but is not limited to: a non-tumor normal biological sample obtained from the same subject (for example, obtained from the subject's non-tumor adjacent cancer) CircRNA PVT1 and PVT1-104aa molecular levels measured in tissues or normal tissue samples, population standard levels determined by statistics, or standardized levels.
  • this article also provides a method for screening drug candidates for treating tumors, alleviating or preventing tumor metastasis, and/or improving tumor prognosis.
  • the method includes testing the drug candidates for CircRNA PVT1 in a subject or a sample obtained from the subject.
  • the effect of PVT1-104aa level wherein, after using the candidate drug, the reduction of CircRNA PVT1 and PVT1-104aa levels indicates that the candidate drug has the effect of treating tumors, alleviating or preventing tumor metastasis, and/or improving tumor prognosis.
  • the features involved in the drug candidate screening method of this application can be as defined or illustrated herein.
  • the drug candidate is an inhibitor of CircRNA PVT1 and PVT1-104aa.
  • CircRNA PVT1 and PVT1-104aa inhibitors and/or active substances are used in their broadest sense, including antagonists, blockers, etc., which are negative regulators of CircRNA PVT1 and PVT1-104aa The substance of action.
  • antagonist refers to a substance that interferes with the binding of one molecule to another molecule or interferes with the characteristics of another cell's stimulation of a cell through steric hindrance, configuration change or other biochemical mechanisms (such as Molecules, compounds or drugs), for example, functional antagonism or physiological antagonism that produces opposite effects through different receptors, through competitive binding with agonists, binding with receptor-related intermediates, etc.
  • blocking agent refers to a substance that partially or completely prevents or inhibits a certain effect.
  • antagonist and blocker are not limited to a specific mechanism of action, but generally refer to the functional properties described herein.
  • CircRNA PVT1 and PVT1-104aa in the present invention include, but are not limited to: natural extracts; anti-CircRNA PVT1 and anti-PVT1-104aa, anti-PVT1-104aa receptor antibodies or anti-PVT1-104aa antibodies that bind to their receptors; CircRNA PVT1 source gene LncRNA PVT1 inhibitor (such as siRNA, shRNA, antisense oligonucleotide, CRISPR-Cas9 system); PVT1-104aa source gene CircRNA PVT1 inhibitor (such as siRNA, shRNA, antisense oligonucleotide Glycolic acid, CRISPR-Cas9 system); CircRNA PVT1 and PVT1-104aa binding and/or functional inhibitors, etc. (such as binding inhibitors that compete with PVT1-104aa to bind to their receptors); other signals for CircRNA PVT1 and PVT1-104aa A chemical substance that has inhibitor
  • the inhibitor of the PVT1-104aa signal pathway can be an antibody or an active fragment thereof, such as a monoclonal antibody, a polyclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, an antibody active fragment (such as Fv, Fab, Fab', F(ab') 2 ).
  • the antibodies can be obtained by methods known in the art, for example, reference can be made to Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988), etc. .
  • a monoclonal antibody is used, and its preparation can be accomplished by the hybridoma method or recombinant DNA method first described by Kohler et al. (Nature, 256:495 (1975)).
  • the inhibitor of the present application can significantly inhibit the growth and metastasis of tumor cells, and improve the prognosis of tumor subjects, and thus can be used as an effective drug for tumor treatment.
  • the present invention also provides a new target for tumor treatment, namely CircRNA PVT1 and PVT1-104aa targets.
  • This article also provides a pharmaceutical composition (or medicine), which contains an effective amount of CircRNA PVT1 and PVT1-104aa inhibitors as the active substance or one of the main active substances.
  • the medicament of the present invention can be used to treat diseases, disorders, or signs related to CircRNA PVT1 and PVT1-104aa overexpression or dysfunction.
  • diseases, disorders, or signs related to CircRNA PVT1 and PVT1-104aa overexpression or dysfunction for example, cancers related to overexpression or dysfunction of CircRNA PVT1 and PVT1-104aa, especially breast cancer.
  • the term "pharmaceutically acceptable” ingredient is a substance that is suitable for use in humans and/or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance that has a reasonable benefit/risk ratio.
  • the term “effective amount” refers to an amount that can produce function or activity on humans and/or animals and can be accepted by humans and/or animals.
  • the term "pharmaceutically acceptable carrier” refers to a carrier for the administration of a therapeutic agent, and includes various excipients and diluents.
  • the term refers to such pharmaceutical carriers: they are not essential active ingredients themselves, and they are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • the medicament of the present invention may contain liquid, such as water, saline, glycerol and ethanol and other carriers.
  • auxiliary substances may also be present in these carriers, such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffer substances, and the like.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, and preferably, the pH is about 6-8.
  • unit dosage form refers to the preparation of a product into a dosage form required for single administration for the convenience of taking, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, sustained-release dosage forms .
  • 1 to 6 doses of the products described herein are administered per day, such as 1 to 3 doses.
  • the effective dose of the active substance used may vary with the conditions of the subject to be administered or treated.
  • the specific situation is determined according to the subject's individual situation (for example, the subject's weight, age, physical condition, and desired effect), which is within the scope of the judgment of a skilled professional (such as a physician).
  • the drugs described herein can be solid (such as granules, tablets, freeze-dried powder, suppositories, capsules, sublingual tablets) or liquid (such as oral liquid) or other suitable shapes.
  • the route of administration can be conventional in the art, such as oral, parenteral (such as intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or subcutaneous administration), subcutaneous, intraperitoneal, intrapulmonary, intranasal, tumor Give internally and locally.
  • the products described herein can also contain other active substances used to improve and treat cancer when used as drugs or pharmaceutical compositions, such as DNA damage chemotherapeutics, multi-targeted tyrosine kinase inhibitors, and cell proliferation inhibitors. , Vascular proliferation inhibitors, alkylating agents, anti-metabolites, anti-tumor antibiotics, plant anti-cancer substances, hormones or immunosuppressive agents, etc.
  • CircRNA PVT1 and PVT1-104aa inhibitors described herein can be used in combination with each other, and can also be combined with other drugs and therapeutic methods for the treatment of cancer.
  • a detection kit (including reagent a, reagent b, reagent c, and reagent d) is prepared according to the following composition, and the kit is suitable for detecting the expression of PVT1-104aa in biological samples by immunohistochemical methods.
  • the kit may also include a container containing the above-mentioned reagents and instructions for use.
  • Reagent a blocking solution, 10% goat serum (purchased from Solebold);
  • Reagent b rabbit anti-human or mouse PVT1-104aa polyclonal antibody, antibody name PV-24aa, sequence CSGAQLGLRPDLLAR;
  • Reagent c HRP-labeled goat anti-rabbit secondary antibody (purchased from Soleibao);
  • Reagent d DAB substrate buffer and color developing solution (purchased from Soleibao).
  • Dewaxing Put the slices in xylene 1 15 min; xylene 2 15 min; absolute ethanol 1 5 min; absolute ethanol 2 5 min; 95% ethanol for 10 min; distilled water for 10 min.
  • Serum sealing histochemical stroke circle (to keep the BSA or antibody in the circle), so that the tissue is in the circle. Add 3% BSA dropwise to evenly cover the tissue in the histochemical circle, and seal at room temperature for 30 minutes. (The primary antibody is goat-derived and blocked with rabbit serum, and other sources are blocked with BSA)
  • DAB color development After the slices are dried slightly, freshly prepared DAB color development solution (30 ⁇ L per slice) is added dropwise, and the display time is controlled under the microscope. The positive is brown-yellow, and the section is washed with tap water to stop the color development.
  • FIG. 4 The test results are shown in Figure 4 to Figure 9.
  • Fig. 4, Fig. 6, Fig. 8 are respectively the comparison of immunohistochemistry of PVT1-104aa expression in breast cancer tissue, colorectal cancer tissue, kidney cancer tissue and corresponding non-tumor tissues adjacent to cancer.
  • Figure 5, Figure 7, Figure 9 are the comparison of the expression levels of PVT1-104aa in breast cancer tissue, colorectal cancer tissue, kidney cancer tissue and corresponding adjacent non-tumor tissues.
  • the IHC (immunohistochemical method) analysis results from Figure 4 to Figure 9 show that PVT1-104aa is in a variety of tumor samples (breast cancer, brain glioma, gastric cancer, kidney cancer, nodules) compared with adjacent non-tumor tissues. Rectal cancer, esophageal cancer) are highly expressed.
  • kit of this embodiment can be used to detect the high expression of PVT1-104aa, so as to diagnose or predict a variety of tumors (breast cancer, glioma, gastric cancer, kidney cancer, colorectal cancer, esophageal cancer, etc.).
  • tumors breast cancer, glioma, gastric cancer, kidney cancer, colorectal cancer, esophageal cancer, etc.
  • a detection kit (including reagent a, reagent b, reagent c, and reagent d) is prepared according to the following composition, and the kit is suitable for detecting the expression of PVT1-104aa in biological samples by Western blot method.
  • the kit may also include a container containing the above-mentioned reagents and instructions for use.
  • Reagent a blocking solution, 5% skimmed milk powder (purchased from Soleibao);
  • Reagent b Rabbit anti-human or mouse PVT1-104aa polyclonal antibody (customized), antibody name PV-24aa, sequence CSGAQLGLRPDLLAR;
  • Reagent c HRP-labeled goat anti-rabbit secondary antibody (purchased from Soleibao);
  • Reagent d ECL chemiluminescence detection kit (purchased from Soleibao).
  • 20% glycerin can be added to the product and stored at -20°C to -80°C.
  • Sample protein processing After measuring the protein content, calculate the corresponding volume of 40ug-100 ⁇ g protein and take it out as the loading amount, and then add 5*SDS-PAGE protein loading buffer (add 1 ⁇ L per 4 ⁇ L protein sample Protein loading buffer), boiling in a water bath for 5 minutes or a PCR machine at 95°C for 5 minutes.
  • PVDF polyvinylidene fluoride membrane
  • the PVDF membrane Before using the PVDF membrane, it needs to be activated: the PVDF membrane needs to be soaked in pure methanol for 3-5s, then add transfer buffer to the basin, and put the shelf, glass rod, filter paper, two sponges and The PVDF activated membrane activated by methanol was put into it and soaked for 20 minutes.
  • TBS Tris-HCL buffered salt solution. Tris-HCL buffer is added to the isotonic salt solution and HCl is added to adjust the pH to 7.4; Tween is a non-ionic detergent. The role of refolding antigen can improve specific recognition ability.
  • PVT1-104aa is in a variety of tumor samples (including but not limited to breast cancer, brain glioma, gastric cancer, kidney cancer, colorectal cancer) compared with non-tumor tissues adjacent to cancer. Cancer, esophageal cancer, etc.).
  • Example 3 Using RT-qPCR to detect the expression of CircRNA and PVT1 in human tumor tissues
  • TRIzol Invitrogen, USA was used to extract total RNA from tissues, and qRT-PCR detection was performed on the ABI StepOnePlus TM PCR system (Life Technologies) real-time quantitative PCR instrument.
  • CircRNA PVT1 (hsa_circ_0001821) calculated relative quantification (GAPDH as internal control) using the reference method 2 - ⁇ Ct: Livak, KJ, etc., Analysis ofrelative gene expression data using real -time quantitative PCR and the2 - ⁇ Ct method.Methods.2001; 25: 402 -408.
  • CircRNA PVT1 is in a variety of tumor samples (breast cancer, brain glioma, gastric cancer, kidney cancer, colorectal cancer) compared with adjacent non-tumor tissues. Cancer, esophageal cancer) are highly expressed.
  • mice after inoculation are shown in Figure 13 and Figure 14, respectively, and the calculated tumor volume size comparison is shown in Figure 15.
  • circRNA PVT1 nucleic acid fragments can be used in the treatment of tumors (including but not limited to breast cancer, glioma, gastric cancer, kidney cancer, colorectal cancer, and esophageal cancer), for example, by silencing circRNA PVT1.
  • Example 5 The inhibitory effect of sicircPVT1 on tumors of different cell lines
  • MB-231 cells human breast cancer cells
  • interference plasmid interference plasmid
  • sscircPVT1 overexpression plasmid
  • sscircPVT1 overexpression plasmid
  • a vertical row of hsa_circ PVT1-CY3 indicates the fluorescent probe: CircPVT1-CY3: caaaagatcaggcctcaagccca
  • the second vertical row indicates the fluorescent dye DAPI (4',6-diamidino-2-phenylindole)
  • the third vertical row indicates Make a combination Merge.
  • MCF-7 cells human breast cancer cells
  • sicircPVT1 and sscircPVT1 overexpression plasmids
  • sicircPVT1 overexpression plasmids
  • Figure 17 the immunositu hybridization map as shown in Figure 17, where the first vertical Row hsa_circ PVT1-CY3 indicates fluorescent probe: CircPVT1-CY3: caaaagatcaggcctcaagccca, the second vertical row indicates the fluorescent dye DAPI (4',6-diamidino-2-phenylindole), and the third vertical row indicates combination Merge.
  • the results in Figure 16 show that the interference plasmid (sicircPVT1) and the overexpression plasmid (sscircPVT1) were transiently transfected in MB-231 cells.
  • the results of the immunositu hybridization FISH experiment showed that the transient transfection interference plasmid (sicircPVT1) inhibited the growth of tumor cells.
  • the expression plasmid (sscircPVT1) promotes the growth of tumor cells, which indicates that circPVT1 can be used as a target for the treatment of breast cancer and other tumors.
  • the results in Figure 17 show that the interference plasmid (sicircPVT1) and overexpression plasmid (sscircPVT1) were transiently transfected in MCF-7 cells.
  • the results of the immunositu hybridization FISH experiment showed that the transient transfection interference plasmid (sicircPVT1) inhibited tumor cell growth, transiently Transfection of overexpression plasmid (sscircPVT1) promotes tumor cell growth, which indicates that circPVT1 can be used as a target for breast cancer and other tumor treatments.
  • Tissue fixation After the tissue is taken out and washed, immediately put it in a fixative solution (prepared with DEPC water) and fix it for 2-12 hours.
  • Digestion According to the length of tissue fixation, the slices are boiled in the repair solution for 10-15 minutes and cooled naturally. After the gene stroke circle, according to the different index characteristics of different tissues, proteinase K (20ug/ml) was added dropwise for digestion at 37°C for 20 minutes. After washing with pure water, wash with PBS 3 times ⁇ 5min.
  • Pre-hybridization Add pre-hybridization solution dropwise and incubate at 37°C for 1 hour.
  • Hybridization Pour off the pre-hybridization solution, add dropwise the hybridization solution containing probe circ-PVT1-CY3, and hybridize overnight at 37°C in an incubator.
  • the circ-PVT1-CY3 probe sequence caaaagatcaggcctcaagccca.
  • wash after hybridization wash off the hybridization solution, 2 ⁇ SSC, wash at 37°C for 10 min, 1 ⁇ SSC, wash at 37°C for 2 ⁇ 5 min, and 0.5 ⁇ SSC at room temperature for 10 min. If there are more non-specific hybrids, formamide washing can be added.
  • DAPI counter-staining nuclei DAPI staining solution is added to the slices, and the sections are incubated for 8 minutes in the dark, after washing, the anti-fluorescence quenching mounting tablets are added to the slides.
  • Example 7 The effect of overexpression of PVT1-104aa adeno-associated virus packaging system on tumor growth in transplanted tumor mice experiment
  • the empty vector of the adeno-associated virus packaging system and the over-expressing adeno-associated virus packaging system of PVT1-104aa are injected into the tumor to compare the tumor growth size and tumor Tumor growth and survival rate are shown in Figure 19-21.
  • Example 8 Correlation between the expression of CircRNA PVT1 and PVT1-104aa molecules in breast cancer tissues and the prognosis of patients sexual analysis
  • tissue microarray scanning was used. Scan the PVT1-104aa protein in cancer tumor tissues, and perform H-score score (histochemistry score) to analyze the correlation between PVT1-104aa expression and patient survival rate. The results are shown in Figure 22, Figure 23, and Figure 24.
  • Figure 22 shows the survival curve drawn from the clinical samples of 35 gastric cancer patients with H-score less than 120 as the Low PVT1-104aa group and H-score greater than 120 as the High PVT1-104aa group.
  • the results show that the High PVT1-104aa group is significantly better than the Low PVT1-104aa group.
  • the PVT1-104aa group had a lower survival rate and worse prognosis.
  • Figure 23 shows the clinical samples of 35 patients with esophageal cancer.
  • the H-score is less than 120 as the Low PVT1-104aa group, and the H-score is greater than 120 as the High PVT1-104aa group.
  • the survival curve is drawn. The results show that the High PVT1-104aa group is significantly better than the Low The PVT1-104aa group had a lower survival rate and worse prognosis.
  • Figure 24 shows the clinical samples of 20 breast cancer patients with H-score less than 120 as the Low PVT1-104aa group, and H-score greater than 120 as the High PVT1-104aa group.
  • the survival curve is drawn. The results show that the High PVT1-104aa group is significantly better than the Low The PVT1-104aa group had a lower survival rate and worse prognosis.
  • PVT1-104aa can be used for tumor prognosis assessment.

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Abstract

提供CircRNA PVT1(hsa_circ_0001821)核酸片段及circRNA PVT1翻译的肽段PVT1-104aa在肿瘤生长预测、转移预测、预后评估以及治疗中的相关性及其应用，CircRNA PVT1及PVT1-104aa作为肿瘤早期诊断、治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的药物靶点的应用。

Description

CircRNA PVT1及肽段在肿瘤生长预测、转移预测、预后评估和治疗中的应用 技术领域
本发明属于生物技术和医学领域，具体涉及CircRNA PVT1(hsa_circ_0001821)核酸片段及circRNA PVT1翻译的肽段PVT1-104aa在肿瘤生长预测、转移预测、预后评估以及治疗中的相关性及其应用。
背景技术
CircRNA(环状RNA)可作为药物或临床诊断的潜在生物标记物分子。研究表明，生命体中除了线性的RNA分子，还大量存在着一类闭合环状的RNA分子，他们在个体发育、不同组织和疾病中都具有特异性表达。经测序鉴定，这些circRNA分子通常由蛋白编码基因的2-4个的外显子部分，通过前面外显子的5’端与后面外显子的3’端互补结合环化而来。由于circRNA的环状封闭特性，使其可以逃避核酸外切酶的作用。因此，circRNA在细胞中可以长期稳定存在，是近年RNA领域最新的研究热点，也正是因为它的稳定性很容易让我们联想到：circRNA是否可以作为药物或临床诊断的潜在生物标记物分子。
环状RNA具有重要的生理功能。在对circRNAs结构和功能研究的基础上，发现它们在多种疾病，如动脉粥样硬化，神经系统紊乱，糖尿病和肿瘤等疾病发生发展过程中，发挥非常重要的作用。主要有以下几种功能：1)circRNAs可以作为“sponge”海绵吸附miRNA，抑制其功能；2)circRNA通过碱基互补配对直接调控其他RNA水平；3)circRNA能与蛋白质结合，抑制蛋白质活性、募集蛋白质复合体的组分或/和调控蛋白质的活性；4)circRNA也可以作为翻译的模板指导蛋白质的合成。
虽然其具体功能尚未完全明确，但研究环状RNA可能具有下列重要的意义：1)circRNA独具的竞争性内源(ceRNA)特征可为药物开发提供新的思路；2)circRNA的组织特异性和稳定性，已经使circRNA成为一种良好的生物标志物；3)circRNA的研究可为生命的进化研究提供新的方向。
环状RNA作为新兴的研究热点，近年对其功能等的研究还集中在肿瘤上，占到1/3到1/2的比例。相对于其他标志物，环状RNA及其翻译的蛋白在肿瘤中的研究较少。
目前，国内外尚无报道使用环状RNA翻译的蛋白来作为肿瘤标记物或肿瘤转移及预后相关性和治疗的研究报道。而本领域迫切需要寻找到可有效用于肿瘤诊断、肿瘤治疗方案选择、肿瘤预后评估的重要靶标分子，并将其用于这些用途中。
发明内容
针对以上不足，本发明提供一种可用于肿瘤诊断、肿瘤治疗方案选择、肿瘤预后评估的新途径。
本发明通过以下方案达到上述目的：
在本发明的第一方面，提供CircRNA PVT1及PVT1-104aa作为肿瘤早期诊断、治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的药物靶点的应用。
在本发明的第二方面中，提供了一种用于检测CircRNA PVT1及PVT1-104aa水平的物质在预测肿瘤生长、肿瘤转移、肿瘤预后评估或诊断肿瘤的产品中的应用。
在一些实施方式中，所述产品检测针对罹患或疑似罹患肿瘤或具有患肿瘤风险或曾罹患肿瘤但已治愈的哺乳动物或获自所述哺乳动物的样品进行。
在一些实施方式中，所述哺乳动物选自：灵长类动物、啮齿类动物、畜牧类哺乳动物、哺乳动物宠物等，例如人、猿、猩猩、猴、牛、羊、马、骆驼、猪、狗、猫、兔、鼠等。
在一些实施方式中，所述哺乳动物已采用本领域已知的方式鉴定为罹患肿瘤或已治愈肿瘤的哺乳动物。
在一些实施方式中，所述样品选自获自所述哺乳动物的：组织或细胞样品，如乳腺组织或细胞样品、癌组织或细胞样品、癌旁组织或细胞样品。
在一些实施方式中，所述样品是新鲜样品、冻存样品、固定样品(例如福尔马林固定样品)、包埋样品(例如石蜡包埋样品)。
在一些实施方式中，所述用于检测CircRNA PVT1及PVT1-104aa表达水平的物质是用于在基因水平和/或蛋白质水平上检测CircRNA PVT1及PVT1-104aa的物质。
在一些实施方式中，所述用于检测CircRNA PVT1及PVT1-104aa表达水平的物质是用于选自下组的一种或多种检测技术或方法中的物质：免疫组织化学法(如免疫荧光分析、ELISA、免疫胶体金法)、Western印迹法、Northern印迹法、PCR、生物芯片法。
在一些实施方式中，所述用于检测CircRNA PVT1及PVT1-104aa表达水平的物质选自对CircRNA PVT1及PVT1-104aa具有特异性的物质，例如抗PVT1-104aa抗体或其抗原结合片段，优选单克隆抗体；CircRNA PVT1特异性的探针、基因芯片、PCR引物、gRNA(向导RNA)等。
在一些实施方式中，所述用于检测CircRNA PVT1及PVT1-104aa表达水平的物质带有可检测标记物，例如，所述选自下组的可检测标记物：放射性同位素、荧光团、化学发光部分、酶、酶底物、酶辅因子、酶抑制剂、染料、金属离子、或配体(如，生物素或半抗原)。
在一些实施方式中，所述肿瘤选自：乳腺癌，脑胶质瘤，胃癌，肾癌，结直肠癌、食管癌、肝癌等。
在一些实施方式中，乳腺癌选自如下分型：浸润性导管癌、非侵润性导管癌；或者，管腔A型、管腔B型、管腔-HER2型、HER2过表达型、基底细胞样型、TNP-非基底型；或者，激素受体阳性型、HER2/neu受体阳性型、三阴性型。
在一些实施方式中，所述产品包括：与正常对照值相比，所述对象或获自所述对象的样品中CircRNA PVT1及PVT1-104aa水平提高，表明所述对象易于或已发生癌症转移，或表明所述对象的癌症预后不良，或表明所述对象已患有癌症。
在一些实施方式中，所述正常对照值获自未罹患肿瘤对象的样品、获自所述对象正常组织的样品、正常对象的CircRNA PVT1及PVT1-104aa水平。
在一些实施方式中，所述正常对照值为：由非肿瘤的正常生物样品(如获自健康人或待测对象正常组织的样品)中测得的CircRNA PVT1及PVT1-104aa分子水平、通过统计学确定的群体标准水平、或经标准化的水平。
在一些实施方式中，所述产品为检测试剂盒。
在一些实施方式中，所述检测试剂盒还包含选自下组的一种或多种物质：容器、缓冲剂、助剂、溶剂、阴性对照物、阳性对照物、使用说明书。
在一个实施方式中，所述的检测试剂盒是基于免疫酶标法的免疫组织化学方法检测生物样品中PVT1-104aa表达的试剂盒，其中包含：封闭液(如10％山羊血清)、抗PVT1-104aa抗体(例如兔抗人或小鼠PVT1-104aa单克隆抗体或多克隆抗体)、二抗(如抗兔生物素化二抗)、标记的结合物(如HRP标记链亲和素)、底物缓冲液(如DAB底物缓冲液)、显色液(如DAB显色液)和/或底物溶液。
在一个实施方式中，所述的检测试剂盒是基于荧光标记的原位杂交技术(FISH技术)检测生物样品中CircRNA PVT1表达的试剂盒，其中包含：荧光分子探针。
在本发明的第三方面，还提供了用于预测肿瘤转移、对肿瘤预后评估或诊断肿瘤的方法，所述方法包括：检测对象或获自对象的样品中CircRNA PVT1及PVT1-104aa的水平的步骤。
在一些实施方式中，与正常对照相比，所述对象或获自所述对象的样品中CircRNA PVT1及PVT1-104aa水平提高，表明所述对象易于或已发生肿瘤转移，或表明所述对象的肿瘤患者预后不良，或表明所述对象已患有癌症。
在本发明的第四方面中，提供了抑制CircRNA PVT1及PVT1-104aa和/或其基因的抑制剂在制备用于治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的药物中的应用。
在一些实施方式中，所述抑制剂选自：与CircRNA PVT1及PVT1-104aa直接结合的抑制剂、阻断CircRNA PVT1及PVT1-104aa与其受体或配体结合的抑制剂、PVT1(LINC00079；MIR1204HG；NCRNA00079；onco-lncRNA-100)和CircRNA PVT1表达水平的抑制剂、降低CircRNA PVT1及PVT1-104aa表达水平的抑制剂、促进CircRNA PVT1及PVT1-104aa降解的抑制剂、用于敲除或敲减CircRNA PVT1及PVT1-104aa表达的物质，例如能够下调CircRNA PVT1的表达及PVT1-104aa的蛋白表达水平的相关启动子元件、重组质粒、表达载体和相关抗体。
在一些实施方式中，所述抑制剂选自：抗PVT1-104aa抗体、抗PVT1-104aa受体的抗体、针对CircRNA PVT1及PVT1-104aa的CRISPR/Cas9系统、针对PVT1及CircRNA PVT1的干扰RNA、针对PVT1及CircRNA PVT1的反义寡核苷酸、CircRNA PVT1及PVT1-104aa表达和/或功能抑制化合物。
在一些实施方式中，所述针对CircRNA PVT1及PVT1-104aa的CRISPR/Cas9系统包含：表达有Cas9酶的载体pCD513B-Cas9和表达有guide RNA(gRNA)的载体psk-U6-sgRNA；表达针对HSPA4基因不同位点选取的gRNA序列优选为具有下述任一项所示的gRNA。
Guide#1：AGTGGTCTGGGGAATAACGC；
Guide#2：CGAAGCTCCATGCAGCTGAC；
GRNA-NC：GCGCCTTAAGAGTACTCATC。
Cir-PVT1-GRNA1:
Cir-PVT1-GRNA1-F1：5’P*CACCGAGTGGTCTGGGGAATAACGC3’，
Cir-PVT1-GRNA1-R1：5’P*AAA CGCGTTATTCCCCAGACCACTC3’；
Cir-PVT1-GRNA2:
Cir-PVT1-GRNA2-F2：5’P*CACCGCGAAGCT CCATGCAGCTGAC3’，
Cir-PVT1-GRNA2-R2：5’P*AAACGTCAGCTGCATGGAGCTTCGC3’；
GRNA-NC:
NC-GRNA-F：5’P*CACCGGCGCCTTAAGAGTACTCATC3’，
NC-GRNA-R：5’P*AAACG ATGAGTACTCTTAAGGCGCC3’。
在一些实施方式中，所述药物包含：(a)CircRNA PVT1及PVT1-104aa和/或其基因的抑制剂；和(b)药学上或保健品学可接受的载体。
在一些实施方式中，所述药物还包含其他抗肿瘤物质，例如DNA损伤类化疗药、多靶向酪氨酸激酶抑制剂、细胞增殖抑制剂、血管增生抑制剂、烷化剂、抗代谢物质、抗肿瘤抗 生素、植物类抗癌物质、激素或免疫抑制剂。
在一些实施方式中，所述药物为固态(如颗粒剂、片剂、冻干粉、栓剂、胶囊、舌下含片)或液态(如口服液、溶液剂或糖浆剂)形式。
在一些实施方式中，所述药物的形式适于口服、胃肠外(如静脉内、动脉内、腹膜内、肌肉内、真皮内或皮下给药)、皮下、腹膜内、肺内、鼻内、瘤内、局部给药。
在本发明的第五方面，提供了一种筛选治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的候选药物的方法，所述方法包括测试所述候选药物对对象或获自对象的样品中CircRNA PVT1及PVT1-104aa水平的影响，其中，在使用所述候选药物后，CircRNA PVT1及PVT1-104aa水平降低表明所述候选药物具有治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的效果。
在一些实施方式中，所述降低是指相对于获自未罹患肿瘤对象的样品、获自所述对象正常组织的样品、正常对象的CircRNA PVT1及PVT1-104aa水平。
在一些实施方式中，所述降低是指相对于正常对照值而言，所述正常对照值为：由非肿瘤的正常生物样品(如获自健康人或待测对象正常组织的样品)中测得的CircRNA PVT1及PVT1-104aa分子水平、或通过统计学确定的群体标准水平、或经标准化的水平。
在本申请的第六方面，提供了一种用于预测肿瘤生长、转移、对肿瘤预后评估或诊断肿瘤的产品，其包含：用于检测CircRNA PVT1及PVT1-104aa水平的物质。
在本发明的第七方面，还提供了一种药物组合物，其包含：作为治疗活性物质的抑制CircRNA PVT1及PVT1-104aa和/或其基因的抑制剂以及药学上可接受的载体。
该药物组合物中的活性物质以及性能等所涉及的特征可如本文中所限定或阐述。
在本发明的第八方面，还提供了一种肿瘤诊断、治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的方法，所述方法包括：给予所述对象CircRNA PVT1及PVT1-104aa和/或其基因的抑制剂或包含所述抑制剂的药物。
本发明所述的CircRNA PVT1优选为SEQ ID NO 1所述的序列。
本发明所述的PVT1-104aa优选为SEQ ID NO 2所述的序列。
本发明所述的circRNA的组织特异性和稳定性，已经使circRNA成为一种良好的生物标志物，经试验证实，CircRNA PVT1核酸片段及肽段PVT1-104aa在肿瘤生长预测、转移预测、预后评估和治疗中发挥显著而稳定的作用。
附图说明
下面结合附图对本发明作进一步说明，其中这些显示仅为了图示说明本发明的实施方案，而不是为了局限本发明的范围。
图1是代表染色体chr8(q24.21)位置转录lncRNA PVT1的示意图。
图2是circRNA PVT1的鉴定结果。
图3是CircRNA PVT1的序列。
图4是乳腺癌组织和癌旁非肿瘤组织中PVT1-104aa表达的免疫组化图对比。
图5是乳腺癌组织和癌旁非肿瘤组织中PVT1-104aa的表达量对比图。
图6是结直肠癌组织和癌旁非肿瘤组织中PVT1-104aa表达的免疫组化图。
图7是结直肠癌组织和癌旁非肿瘤组织中PVT1-104aa的表达量对比图。
图8是肾癌组织和癌旁非肿瘤组织中PVT1-104aa表达的免疫组化图。
图9是肾癌组织和癌旁非肿瘤组织中PVT1-104aa的表达量对比图。
图10是人乳腺癌及癌旁组织的PVT1-104aa及β-actin的western blot结果图。
图11是结直肠癌及癌旁组织的PVT1-104aa及β-actin的western blot结果图。
图12是PCR法在各种肿瘤及癌旁组织中CircRNA PVT1的表达量对比。
图13是在BALB/c裸鼠上接种106的MB-231-NC细胞后的小鼠图。
图14是在BALB/c裸鼠上接种106的B-231-siRNA细胞后的小鼠图。
图15是在BALB/c裸鼠上接种106的MB-231-NC细胞和B-231-siRNA细胞后的肿瘤体积。
图16是MB-231细胞及瞬时转染干扰质粒(sicircPVT1)和过表达质粒(sscircPVT1)后的免疫原位杂交图。
图17是MCF-7细胞及瞬时转染干扰质粒(sicircPVT1)和过表达质粒(sscircPVT1)后的免疫原位杂交图。
图18是人乳腺癌组织和癌旁组织中的circPVT1表达的显微镜观察图。
图19是过表达PVT1-104aa腺相关病毒包装体系对移植瘤小鼠肿瘤生长大小对比图。
图20是过表达PVT1-104aa腺相关病毒包装体系对移植瘤小鼠肿瘤生长重量对比图。
图21是过表达PVT1-104aa腺相关病毒包装体系对移植瘤小鼠的生存率对比图。
图22是不同PVT1-104aa组别的胃癌患者生存率曲线图。
图23是不同PVT1-104aa组别的食管癌患者生存率曲线图。
图24是不同PVT1-104aa组别的乳腺癌患者生存率曲线图。
具体实施方式
本发明的目的之一在于寻找到可有效用于肿瘤生长预测、转移预测以及患者预后评估的分子并提供相应的检测产品。
本发明的另一目的在于提供有效治疗肿瘤的靶标、药物及方法。
我们在研究中发现，CircRNA PVT1及PVT1-104aa在多种肿瘤(包括乳腺癌，脑胶质瘤，胃癌，肾癌，结直肠癌，食管癌等)中高表达，以乳腺癌为例，乳腺癌患者的肿瘤组织中CircRNA PVT1及PVT1-104aa的表达与患者淋巴结转移相关，并与肿瘤大小、分期密切相关，其高表达与患者的总生存时间短、预后差显著相关。干扰小鼠乳腺癌细胞CircRNA PVT1及PVT1-104aa分子，能够显著抑制肿瘤生长及转移。由此本申请提供了CircRNA PVT1及PVT1-104aa分子在用于对象中肿瘤生长预测、肿瘤转移预测以及肿瘤预后评估、肿瘤治疗中的新用途，并提供了相应的检测试剂盒。以上进展提示CircRNA PVT1及PVT1-104aa的检测及干预治疗方法有望成为肿瘤诊断及治疗新的增长点。
本文中提供的所有数值范围旨在清楚地包括落在范围端点之间的所有数值及它们之间的数值范围。可对本发明提到的特征或实施例提到的特征进行组合。本说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
如本文所用，“含有”、“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
1.CircRNA PVT1及PVT1-104aa
如本文所用，术语“CircRNA PVT1”与“hsa_circ_0001821”可互换使用，CircRNA PVT1是由LncRNA PVT1(Pvt1oncogene，LINC00079，MIR1204HG，NCRNA00079，onco-lncRNA-100)第二外显子剪切形成，如图1-图2所示，其序列(如图3所示)为：
Figure PCTCN2020071984-appb-000001
小鼠CircRNA PVT1(mmu_circ_0000604)序列为：
Figure PCTCN2020071984-appb-000002
如本文所用，术语“PVT1-104aa蛋白或多肽”与“CircRNA PVT1基因编码的蛋白质或多肽”可互换使用，均是指由CircRNA PVT1基因编码的蛋白质或多肽、它们的保守性变异多肽、或其同源蛋白或多肽、或其活性片段(例如PVT1-104aa结合结构域)。
人PVT1-104aa序列为(SEQ ID NO 2)：
MHVPSGAQLGLRPDLLARRRLKRCPSRWLCLSAAWSFVQVFSEPDGFTVIFSGLGNNAGGTMHWNDTRPAHFRILKVVLREAVAECLMDSYSLDVHGGRRTAAG。
小鼠PVT1-68aa序列为：
MPSISRPCLLGPLWRLPEPCGFTVRCSFLGSNSGGYKFRSITPGLPGDVTSSWMDSSAWMPTENKDRN。
2.检测物质
如本文所用，术语“PVT1-104aa检测物质”、或“检测PVT1-104aa分子的试剂”或“检测PVT1-104aa表达量的试剂”可互换使用，均是指特异性针对PVT1-104aa分子，且可用于直接或间接检测出PVT1-104aa分子的存在和/或含量的物质。这些检测物质可在基因水平或蛋白质水平上检测PVT1-104aa。
如本文所用，术语“CircRNA PVT1检测物质”、或“检测CircRNA PVT1分子的试剂”或“检测CircRNA PVT1表达量的试剂”可互换使用，均是指特异性针对CircRNA PVT1分子，且可用于直接或间接检测出CircRNA PVT1分子的存在和/或含量的物质。这些检测物质可在基因水平水平上检测CircRNA PVT1。
由于CircRNA PVT1分子的序列已知，本领域普通技术人员可基于常规手段制备或通过市售获得特异性针对CircRNA PVT1分子的试剂。例如，本发明中可用的检测试剂包括但不限于：对CircRNA PVT1分子具有检测特异性的分子探针、RT-qPCR引物等。
由于PVT1-104aa分子的序列在本文中公开，本领域普通技术人员可基于常规手段制备或通过市售获得特异性针对PVT1-104aa分子的试剂。例如，本发明中可用的检测试剂包括但不限于：对PVT1-104aa分子具有检测特异性的抗体。
并且，为了便于检测，本发明的检测试剂还可带有可检测标记，所述可检测标记包括但不限于：放射性同位素、荧光团、化学发光部分、酶、酶底物、酶辅因子、酶抑制剂、染料、金属离子、配体(如生物素或半抗原)等。
本发明的检测试剂可存在于溶液中、固定于载体(如基片、吸附物)上或以其它本领域中常规的方式存在，只要该存在方式适于对生物样品中CircRNA PVT1及PVT1-104aa检测即可。例如，当本发明的检测试剂为核苷酸探针时，其可以生物芯片(或称“微阵列”)的形式存在。
3.检测产品
本发明中还提供了一种用于预测肿瘤生长、预测肿瘤转移、对肿瘤预后评估或诊断肿瘤的产品，其包含：用于检测CircRNA PVT1及PVT1-104aa水平的物质，以及可任选的，用于预测肿瘤生长、预测肿瘤转移、对肿瘤预后评估或诊断肿瘤的其他物质，例如现有肿瘤标志物的检测物质。
根据所用检测方法的需要，可选择适当的CircRNA PVT1及PVT1-104aa检测物质，并将其制成适于所用检测方法的产品，如试剂盒。本领域普通技术人员可根据实际条件和需要对检测方式和产品中所含试剂进行调整和改变。
由此，本文中还提供了一种产品(如检测试剂盒)，其包含：(i)检测有效量的用于检测CircRNA PVT1及PVT1-104aa的一种或多种试剂；(ii)可任选地，选自下组的一种或多种物质：容器、使用说明书、阳性对照物、阴性对照物、缓冲剂、助剂或溶剂，例如用于混悬或固定细胞的溶液，可检测的标签或标记，使核酸易于杂交的溶液，用于裂解细胞的溶液，或用于核酸纯化的溶液。
在实施例1中，本文提供了一种适于通过免疫组织化学方法检测生物样品中PVT1-104aa的表达的检测试剂盒。该检测试剂盒可包含：封闭液，例如10％山羊血清；一抗，例如兔抗人或小鼠PVT1-104aa单克隆抗体；二抗，例如标记(如HRP标记的)或未标记的羊抗兔二抗；底物缓冲液，例如DAB底物缓冲液；显色液；以及可选的装有上述试剂的容器及使用说明书。
在实施例2中，本文提供了一种适于通过Western blot方法检测生物样品中PVT1-104aa的表达的检测试剂盒。该检测试剂盒可包含：封闭液，例如5％脱脂奶粉；一抗，例如兔抗人或小鼠PVT1-104aa多克隆抗体；二抗，例如标记(如HRP标记的)或未标记的羊抗兔二抗；化学发光检测试剂，例如ECL化学发光法检测试剂盒；以及可选的装有上述试剂的容器及使用说明书。
在实施例3中，本文提供了一种适于通过RT-qPCR方法检测生物样品中CircRNA  PVT1-104aa的表达的检测试剂盒。该检测试剂盒可包含：提取试剂，例如Trizol；去除DNA试剂，例如DNase I；反转录试剂，例如RT Mix、Enzyme Mix、Random hexamers；real time qPCR试剂，例如
Figure PCTCN2020071984-appb-000003
Green Master Mix、Forward primer、Reverse primer；以及可选的装有上述试剂的容器及使用说明书。
本文的检测试剂盒中还可附有试剂盒的使用说明书，其中记载了如何采用试剂盒进行检测，以及如何利用检测结果对肿瘤转移及预后情况进行判断、对治疗方案进行选择。
当然，试剂盒还可包含临床上用于对象中肿瘤发展的判断、治疗方案的选择和/或预后评估的其它试剂，以辅助或验证通过检测CircRNA PVT1及PVT1-104aa所得到的结果。本领域普通技术人员可根据具体需要进行常规选择。
4.CircRNA PVT1及PVT1-104aa在肿瘤生长预测、肿瘤转移预测、预后评估、诊断以 及药物筛选中的应用
根据本发明中所公开的内容，CircRNA PVT1及PVT1-104aa的水平与肿瘤的生长、肿瘤的转移、预后和诊断密切相关，从而可作为肿瘤生长预测、转移预测、预后评估以及诊断的指标。
如本文所用，术语“预后”是指预测疾病的可能病程和结局，其包括判断疾病的特定后果(如康复，某种症状、体征和并发症等其它异常的出现或消失及死亡)。本文中所述的预后不良包括但不限于：生存期缩短、易发肿瘤转移、肿瘤数量增加快、肿瘤变大加快、TNM分级上升等。在预测了患者预后情况后，可结合降低CircRNA PVT1及PVT1-104aa分子的量的治疗方法改善患者的预后。
通常，可采用如下方法进行肿瘤生长预测、肿瘤转移预测、预后评估和/或诊断：检测待测对象或获自该对象的样品中CircRNA PVT1及PVT1-104aa分子的水平，并将该水平与对照水平相比较；若比较结果显示对象中的CircRNA PVT1及PVT1-104aa分子的水平高于对照水平，则提示所述对象易发生肿瘤转移、预后不良、或已罹患癌症。在一些实施方式中，本申请的方法还可选地包括：从对象获得待测样品；使待测样品与检测CircRNA PVT1及PVT1-104aa水平的试剂或试剂盒接触。
如本文所用，术语“正常对照”是指用作参照的CircRNA PVT1及PVT1-104aa分子的水平，其包括但不限于：由同一对象的非肿瘤正常生物样品(例如获自该对象非肿瘤癌旁组织或正常组织的样品)中测得的CircRNA PVT1及PVT1-104aa分子水平、通过统计学确定的群体标准水平、或经标准化的水平。
此外，本文还提供了一种筛选治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的候选 药物的方法，所述方法包括测试所述候选药物对对象或获自对象的样品中CircRNA PVT1及PVT1-104aa水平的影响，其中，在使用所述候选药物后，CircRNA PVT1及PVT1-104aa水平降低表明所述候选药物具有治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的效果。本申请候选药物筛选方法所涉及的各特征可如本文中所限定或阐述。在一些实施方式中，候选药物为CircRNA PVT1及PVT1-104aa的抑制剂。
5.CircRNA PVT1及PVT1-104aa的抑制剂
如本文所用，术语CircRNA PVT1及PVT1-104aa“抑制剂”和/或“活性物质”以其最广义使用，其包括拮抗剂、阻断剂等，是对CircRNA PVT1及PVT1-104aa起到负调节作用的物质。
术语“拮抗剂”是指通过空间位阻、构型改变或其它生物化学机制以干扰一种分子与另一种分子的结合或干扰另一种细胞对一种细胞的刺激的特性的物质(如分子、化合物或药物)，例如通过不同受体产生相反效应的功能性拮抗或生理性拮抗、通过与激动剂竞争性结合、与受体相关的中间体结合等方式。术语“阻断剂”是指部分或全部阻止或抑制某一作用的物质。术语“拮抗剂”和“阻断剂”不局限于具体的作用机制，而是泛泛地指本文所述的功能特性。
本发明中CircRNA PVT1及PVT1-104aa的抑制剂包括但不限于：天然提取物；抗CircRNA PVT1及抗PVT1-104aa、抗PVT1-104aa受体的抗体或抗PVT1-104aa与其受体结合的抗体；CircRNA PVT1的源基因LncRNA PVT1的抑制剂(如siRNA、shRNA、反义寡核苷酸、CRISPR-Cas9系统)；PVT1-104aa的源基因CircRNA PVT1的抑制剂(如siRNA、shRNA、反义寡核苷酸、CRISPR-Cas9系统)；CircRNA PVT1及PVT1-104aa结合和/或功能抑制剂等(如与PVT1-104aa竞争性结合其受体的结合抑制剂)；其他对CircRNA PVT1及PVT1-104aa信号途径成员具有抑制活性的化学物质。
在一些实施方式中，PVT1-104aa信号途径的抑制剂可为抗体或其活性片段，例如单克隆抗体、多克隆抗体、人源化抗体、人抗体、嵌合抗体、抗体活性片段(如Fv、Fab、Fab'、F(ab') 2)。所述抗体可通过本领域中已知的方法获得，例如可参考Harlow和Lane，《抗体：实验室手册》(Antibodies:A Laboratory Manual)，冷泉港实验室(Cold Spring Harbor Laboratory)(1988)等。优选采用单克隆抗体，其制备可采用最先由Kohler等(Nature，256：495(1975))描述的杂交瘤方法或重组DNA法完成。
本申请的抑制剂能发挥显著抑制肿瘤细胞生长及转移，改善肿瘤对象的预后，由此可作为肿瘤治疗的有效药物。并且，本发明还为肿瘤治疗提供了一种新的靶标，即CircRNA PVT1及PVT1-104aa靶标。
6.包含CircRNA PVT1及PVT1-104aa抑制剂的药物组合物
本文中还提供了一种药物组合物(或称药物)，其中含有有效量的CircRNA PVT1及PVT1-104aa抑制剂作为活性物质或主要活性物质之一。
在一些实施方式中，本发明的药物可用于治疗与CircRNA PVT1及PVT1-104aa过表达或功能异常相关的疾病、病症或体征。例如与CircRNA PVT1及PVT1-104aa过表达或功能异常相关的癌症，尤其是乳腺癌。
如本文所用，术语“药学上可接受的”成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的，即有合理的效益/风险比的物质。如本文所用，术语“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。该术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在《雷明顿药物科学》(Remington’s Pharmaceutical Sciences，Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。
本发明的药物可含有液体，如水、盐水、甘油和乙醇等载剂。另外，这些载体中还可能存在辅助性的物质，如填充剂、崩解剂、润滑剂、助流剂、泡腾剂、润湿剂或乳化剂、矫味剂、pH缓冲物质等。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8。
如本文所用，术语“单位剂型“是指为了服用方便，将产品制备成单次服用所需的剂型，包括但不限于各种固体剂(如片剂)、液体剂、胶囊剂、缓释剂。
在一些实施方式中，每天施用1～6剂本文所述的产品，如施用1～3剂。
应理解，所用活性物质(即HSPA4抑制剂)的有效剂量可随待施用或治疗的对象的情况而变化。具体情况根据对象的个体情况(例如对象体重、年龄、身体状况、所需达到的效果)来决定，这在熟练专业人员(如医师)可以判断的范围内。
本文所述的药物可以为固态(如颗粒剂、片剂、冻干粉、栓剂、胶囊、舌下含片)或液态(如口服液)或其它合适的形状。给予途径可采用本领域常规的方式，例如口服、胃肠外(如静脉内、动脉内、腹膜内、肌肉内、真皮内或皮下给药)、皮下、腹膜内、肺内、鼻内、瘤内、局部给予。
此外，本文所述的产品在作为药物或药物组合物时还可含有用于改善和治疗癌症的其他活性物质，例如DNA损伤类化疗药、多靶向酪氨酸激酶抑制剂、细胞增殖抑制剂、血管增 生抑制剂、烷化剂、抗代谢物质、抗肿瘤抗生素、植物类抗癌物质、激素或免疫抑制剂等。
本文所述的CircRNA PVT1及PVT1-104aa抑制剂相互间可以联合应用，还可以与其它药物和治疗手段联合，用于癌症的治疗。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。本领域技术人员可对本发明做出适当的修改、变动，这些修改和变动都在本发明的范围之内。
下列实施例中未注明具体条件的实验方法，可采用本领域中的常规方法，例如参考《分子克隆实验指南》(第三版，纽约，冷泉港实验室出版社，New York：Cold Spring Harbor Laboratory Press，1989)或按照供应商所建议的条件。DNA的测序方法为本领域常规的方法，也可由商业公司提供测试。
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1：一种检测试剂盒的制备
试剂盒：
本实施例中按如下组成制备检测试剂盒(包括试剂a、试剂b、试剂c、试剂d)，该试剂盒适于以免疫组织化学方法检测生物样品中PVT1-104aa的表达。该试剂盒还可以包括装有上述试剂的容器及使用说明书。
试剂a：封闭液，为10％山羊血清(购自索莱宝公司)；
试剂b：兔抗人或小鼠PVT1-104aa多克隆抗体，抗体名称PV-24aa，序列CSGAQLGLRPDLLAR；
试剂c：HRP标记羊抗兔二抗(购自索莱宝公司)；
试剂d：DAB底物缓冲液及显色液(购自索莱宝公司)。
检测方法：
使用本实施例中的检测试剂盒进行检测的步骤如下：
1、脱蜡：依次将切片放入二甲苯①15min；二甲苯②15min；无水乙醇①5min；无水乙醇②5min；95％乙醇10min；蒸馏水10min。
2、抗原修复
(1)微波抗原修复：组织切片置于盛满柠檬酸抗原修复缓冲液(pH＝6.0)的修复盒中于微波炉内进行抗原修复，中火8min至沸，停火8min保温，再转中低火5-7min，此过程中应 防止缓冲液过度蒸发，切勿干片。(覆盖一层保鲜膜，扎孔，橡胶扎紧)。自然冷却后将玻片置于PBS(pH＝7.4)中在脱色摇床上晃动洗涤2-3次，每次5min。
(2)高压修复：修复盒盛满柠檬酸抗原修复缓冲液(pH＝6.0)放于有一定量水的高压锅内，电磁炉加热至气孔开始喷气，停止加热，释放压力。将切片置于修复盒内，电磁炉加热至气孔喷气，5min后关闭电磁炉，此过程中应防止缓冲液中在脱色摇床上晃动洗涤2-3次，每次5min。
3、阻断内源性过氧化物酶：切片放入3％双氧水溶液，室温避光孵育10-20min，将玻片置于PBS(pH＝7.4)中脱色摇床上晃动洗涤2次，每次5min。
4、血清封闭：组化笔画圈(使BSA或抗体留在圈内)，使组织在圈内。在组化圈内滴加3％BSA均匀覆盖组织，室温封闭30min。(一抗是山羊来源的用兔血清封闭，其他来源的用BSA封闭)
5、一抗：轻轻甩掉封闭液，加一抗(30μL每片)，切片平放于湿盒内4℃过夜或37℃3小时(湿盒内加少量水防止抗体蒸发)，玻片置于PBS(pH＝7.4)中在脱色摇床上晃动洗涤2-3次，每次5min。
6、二抗：切片稍甩干后再圈内滴加与一抗相应种属的二抗(HRP标记)覆盖组织，室温孵育50min，玻片置于PBS(pH＝7.4)中在脱色摇床上晃动洗涤2-3次，每次5min。
7、DAB显色：切片稍甩干后再滴加新鲜配置的DAB显色液(30μL每片)，显微镜下控制显示时间。阳性为棕黄色，自来水冲洗切片终止显色。
检测结果如图4至图9所示。其中，图4、图6、图8分别是乳腺癌组织、结直肠癌组织、肾癌组织和相应的癌旁非肿瘤组织中PVT1-104aa表达的免疫组化图对比。图5、图7、图9分别是乳腺癌组织、结直肠癌组织、肾癌组织和相应的癌旁非肿瘤组织中PVT1-104aa的表达量对比。
从图4-图9的IHC(免疫组织化学方法)分析结果表明，与癌旁非肿瘤组织相比，PVT1-104aa在多种肿瘤样本(乳腺癌，脑胶质瘤，胃癌，肾癌，结直肠癌，食管癌)中均高表达。
这说明，可以采用本实施例试剂盒检测PVT1-104aa的高表达，从而诊断、或预测多种肿瘤(乳腺癌，脑胶质瘤，胃癌，肾癌，结直肠癌，食管癌等)。
实施例2：另一种检测试剂盒的制备
试剂盒：
按如下组成制备检测试剂盒(包括试剂a、试剂b、试剂c、试剂d)，该试剂盒适于以 Western blot方法检测生物样品中PVT1-104aa的表达。该试剂盒还可以包括装有上述试剂的容器及使用说明书。
试剂a：封闭液，为5％脱脂奶粉(购自索莱宝公司)；
试剂b：兔抗人或小鼠PVT1-104aa多克隆抗体(定制)，抗体名称PV-24aa，序列CSGAQLGLRPDLLAR；
试剂c：HRP标记羊抗兔二抗(购自索莱宝公司)；
试剂d：ECL化学发光法检测试剂盒(购自索莱宝公司)。
检测方法：
使用本实施例中的检测试剂盒进行检测的步骤如下：
1、样品制备：
(1)取少量的组织(如小鼠脾脏取1/2)于2ml的离心管中，加入1mL PBS后放入组织研磨仪中研磨1min，离心，去除PBS，再用1mlPBS洗涤一次，离心(12000r，10min)弃去PBS(冰上操作)(0.1g组织以1:10加入含PMSF/RIPA)1mL RIPA(0.99RIPA+0.01PMSF)
(2)加入0.5-1mL的预冷的RIPA裂解液，再放入组织研磨仪中研磨3-5次，使细胞完全破碎，然后将离心管冰浴15min，每隔5min涡旋混合仪振荡30s，保证细胞完全裂解。
(3)充分裂解后，12000g，4℃离心10min，将上清液转移到新的1.5mL的离心管中，即得组织总蛋白产物。
(4)产物中可加入20％的甘油，保存于-20℃至-80℃。
2、蛋白浓度测定：(BAC法)
(1)购买BCA蛋白浓度测定试剂盒测定样品总蛋白，目的是为了保证上样量蛋白在20-100μg之间和尽可能使上样蛋白总量接近。
(2)样品蛋白处理：测完蛋白含量后,计算出40ug-100μg蛋白的对应体积取出作为上样量，再在其中加入5*SDS-PAGE蛋白上样缓冲液(按照每4μL蛋白样品加入1μL蛋白上样缓冲液)，沸水浴5min或者PCR仪95℃加热5min。
3、SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)
(1)清洗玻璃板：先用洗洁精轻轻刷洗玻璃板，然后用自来水冲洗，再用蒸馏水洗浄后晾干；梳子用蒸馏水洗干净，临用前用乙醇擦拭晾干；
(2)制胶与灌胶、上样：玻璃板对齐后放入夹中卡紧，然后垂在卡在架子上准备灌胶。配制好分离胶后往其中加入TEMED(四甲基乙二胺，促凝剂)立即摇晃均匀即可灌胶，灌胶时掌握速度，沿着玻璃板面灌胶，避免产生气泡，灌好后在胶上封一层水，使胶凝结的更 快些。30-40min后，把胶上层的水倒掉，并用吸水纸把残余水吸干；再往配制好的浓缩胶中加入TEMED，立即摇晃均匀即可灌胶，用浓缩胶罐满剩余空间，并将梳子水平插入到浓缩胶中，保证没有气泡。上样前加入足够的电液缓冲液，设定符合实际的电压和电泳时间。(浓缩胶75V，分离胶120V)，电泳至溴酚蓝刚跑出时即可终止电泳。
4、转膜(湿转)：
(1)准备一张适宣大小的聚偏二氟乙烯膜(PVDF)以及滤纸6片。将胶浸泡于转移缓冲液中10min(如检测小分子蛋白，即可免去这一步骤，因小分子蛋白容易扩散出胶)
(2)在使用PVDF膜之前需进行活化：PVDF膜需在纯甲醇中浸泡3-5s，再于盆里加入转移缓冲液，并把转膜用的架子、玻璃棒、滤纸、两块海绵和经过甲醇活化的PVDF活化的膜放入其中浸泡20min。
(3)装配转膜“三明治”：海绵/3层滤纸/胶/膜/3层滤纸/海绵，每层放好后，用玻璃棒排除气泡。再将转移槽置于冰浴中，放于装配好的“三明治”，加入转移缓冲液，通电，恒压70-100V，或者恒流250-300mA，时间2小时。注意：不同仪器和不同分子量蛋白的转膜条件不同，以下条件仅供参考。
5、封闭
用PBS将转好的膜洗净，漂洗1-2min，在室温条件下，将膜转移至含有封闭液的平皿中(5％脱脂牛奶，0.5％TBST)置于脱色摇床上脱色，封闭1h。
TBST：为TBS+Tween的缩写，TBS是Tris-HCL缓冲盐溶液，为等渗的盐溶液中加入Tris-HCL缓冲液加入HCl调节PH为7.4；Tween为一种非离子型去污剂，有复性抗原的作用,可提高特异性识别能力。
6、孵育抗体
(1)封闭1h后，用PBS将PVDF膜洗净3次后，每次5min，用稀释的一抗溶解的5％脱脂牛奶，磷酸化蛋白使用TBST溶解的5％BSA，将PVDF膜充分浸没后4℃冰箱孵育过夜。
(2)在室温条件下，然后用TBST将PVDF膜放于脱色摇床上洗5min，连续洗三遍，然后将PVDF膜用相应的二抗充分浸没，室温孵育30min后，在室温下用TBST将PVDF膜放于脱色摇床上洗5min，连续洗三遍，最后置于PBS溶液中浸泡待测。
7、化学发光显色
(1)现配现用ECL试剂工作液(A液：B液＝1:1)，等体积混合于离心管中，将此混合液与朝上的膜蛋白面充分接触，接触1-2min后，将膜上的残液去尽，并放八暗匣中曝光。
(2)最后用显影、定影试剂对膜进行显影和定影；根据不同的光强度调整曝光条件。将 胶片进行扫描并存档，使用Image J软件处理系统分析计算目标带的光密度值。
结果如图10-图11所示。
从图10和图11的Western blot分析结果发现，与癌旁非肿瘤组织相比，PVT1-104aa在多种肿瘤样本(包括但不限于乳腺癌，脑胶质瘤，胃癌，肾癌，结直肠癌，食管癌等)中均高表达。
实施例3：采用RT-qPCR检测CircRNA PVT1在人肿瘤组织中的表达
选取人肿瘤样本(乳腺癌、肺癌、胰腺癌、结肠癌)，对照为各自肿瘤的癌旁组织。采用TRIzol(美国Invitrogen公司)抽提组织总RNA，qRT-PCR检测在ABI StepOnePlus TM PCR系统(Life Technologies)实时定量PCR仪上完成。
使用的引物及产物大小如下表所示：
Figure PCTCN2020071984-appb-000004
CircRNA PVT1(hsa_circ_0001821)相对定量使用2 -ΔΔCt法计算(GAPDH为内参)参照：Livak，KJ.等，Analysis ofrelative gene expression data using real-time quantitative PCR and the2 -ΔΔCt method.Methods.2001；25：402-408。
结果如图12所示，从图12的RT-qPCR分析结果发现，与癌旁非肿瘤组织相比，CircRNA PVT1在多种肿瘤样本(乳腺癌，脑胶质瘤，胃癌，肾癌，结直肠癌，食管癌)中均高表达。
实施例4：sicircPVT1对小鼠肿瘤的抑制影响
在BALB/c裸鼠上接种10 6的MB-MDA-231-NC细胞(对照MB-MDA-231乳腺癌细胞)和MB-MDA-231-siRNA细胞(沉默了circPVT1的MB-MDA-231乳腺癌细胞)，接种后小鼠分别如图13和图14所示，并且计算肿瘤体积大小对比如图15所示。
从图13至图15的结果表明，在BALB/c裸鼠上接种10 6的MB-231-NC细胞和MB-231-siRNA细胞，MB-231-siRNA肿瘤体积明显减少，这说明，沉默circRNA PVT1起到抑制肿瘤生长的作用。
这说明，circRNA PVT1核酸片段可以用于肿瘤(包括但不限于乳腺癌，脑胶质瘤，胃癌，肾癌，结直肠癌，食管癌)的治疗，例如通过沉默circRNA PVT1。
实施例5：sicircPVT1对不同细胞系肿瘤的抑制作用
MB-231细胞(人乳腺癌细胞)作为对照及向MB-231细胞中分别瞬时转染干扰质粒(sicircPVT1)和过表达质粒(sscircPVT1)，观察免疫原位杂交图如图16所示，其中第一竖 排hsa_circ PVT1-CY3表示荧光探针：CircPVT1-CY3：caaaagatcaggcctcaagccca，第二竖排表示荧光染料DAPI(4',6-二脒基-2-苯基吲哚)，并且第三竖排表示进行组合Merge。
MCF-7细胞(人乳腺癌细胞)作为对照及向MCF-7细胞瞬时转染干扰质粒(sicircPVT1)和过表达质粒(sscircPVT1)，观察免疫原位杂交图如图17所示，其中第一竖排hsa_circ PVT1-CY3表示荧光探针：CircPVT1-CY3：caaaagatcaggcctcaagccca，第二竖排表示荧光染料DAPI(4',6-二脒基-2-苯基吲哚)，并且第三竖排表示进行组合Merge。
图16的结果表明，在MB-231细胞瞬时转染干扰质粒(sicircPVT1)和过表达质粒(sscircPVT1)，免疫原位杂交FISH实验结果表明，瞬时转染干扰质粒(sicircPVT1)抑制肿瘤细胞生长，过表达质粒(sscircPVT1)促进肿瘤细胞生长，这表明，circPVT1可作为乳腺癌等肿瘤治疗的靶标。
图17的结果表明，在MCF-7细胞瞬时转染干扰质粒(sicircPVT1)和过表达质粒(sscircPVT1)，免疫原位杂交FISH实验结果表明，瞬时转染干扰质粒(sicircPVT1)抑制肿瘤细胞生长，瞬时转染过表达质粒(sscircPVT1)促进肿瘤细胞生长，这表明，circPVT1可作为乳腺癌等肿瘤治疗的靶标。
上述结果表明，circPVT1可作为肿瘤治疗的靶标。
实施例6：circPVT1FISH原位检测试验
石蜡切片荧光探针原位杂交实验步骤：
1、组织固定：组织取出洗净后立即放入固定液(DEPC水配制)中固定2-12h。
2、脱水：组织固定完成后经梯度酒精脱水后浸蜡，包埋。
3、切片：石蜡经切片机切片，摊片机捞片，62℃烤箱烤片2h。
4、石蜡切片脱蜡至水：依次将切片放入二甲苯Ⅰ15min-二甲苯Ⅱ15min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min，自然晾干，DEPC水浸泡。
5、消化：根据组织固定时间长短，切片于修复液中煮沸10-15分钟，自然冷却。后基因笔画圈，根据不同组织不同指标特性，滴加蛋白酶K(20ug/ml)37℃消化20min。纯水冲洗后PBS洗3次×5min。
6、预杂交：滴加预杂交液，37℃孵育1h。
7、杂交：倾去预杂交液，滴加含探针circ-PVT1-CY3杂交液,恒温箱37℃度杂交过夜。其中circ-PVT1-CY3探针序列：caaaagatcaggcctcaagccca。
8、杂交后洗涤：洗去杂交液，2×SSC，37℃洗10min，1×SSC，37℃洗2×5min，0.5×SSC室温洗10min。若非特异杂交体较多，可以增加甲酰胺洗涤。
9、DAPI复染核：切片滴加DAPI染液，避光孵育8min，冲洗后滴加抗荧光淬灭封片剂封片。
10.镜检拍照：切片于尼康正置荧光显微镜下观察并采集图像。(紫外激发波长330-380nm，发射波长420nm，发蓝光；FAM(488)绿光激发波长465-495nm，发射波长515-555nm，发绿光；CY3红光激发波长510-560，发射波长590nm，发红光。)结果如图18所示。
从图18的结果可以看出，人乳腺癌组织中的circPVT1在乳腺癌高表达，在对应的癌旁组织低表达，表明circPVT1可作为肿瘤标志物，用于肿瘤生长预测、转移预测、预后评估等。
实施例7：过表达PVT1-104aa腺相关病毒包装体系对移植瘤小鼠肿瘤生长的促进作用 实验
在BALB/c裸鼠上接种106的MB-231细胞，待肿瘤长到米粒大小，瘤内注射腺相关病毒包装体系空载体和过表达PVT1-104aa腺相关病毒包装体系，比较肿瘤生长大小及肿瘤生长肿瘤及生存率，如图19-图21所示。
图19-图21的结果表明，过表达PVT1-104aa腺相关病毒包装体系加快肿瘤生长，肿瘤体积明显增大，小鼠的生存率降低，并且与过表达体系的加入剂量正相关，表明PVT1-104aa具有促癌作用。
实施例8：乳腺癌组织中CircRNA PVT1及PVT1-104aa分子的表达与患者预后的相关 性分析
对48例胃癌和35例食管癌患者、20例乳腺癌患者，采用组织芯片扫描方法。对癌肿瘤组织中PVT1-104aa蛋白进行扫描，并且进行H-score的评分即histochemistry score(组织化学评分)，分析PVT1-104aa表达与患者生存率的相关性。结果如图22、图23和图24所示。
图22为35例胃癌患者的临床样本以H-score小于120为Low PVT1-104aa组，H-score大于120为High PVT1-104aa组，绘制的生存曲线，结果可知High PVT1-104aa组明显比Low PVT1-104aa组生存率更低，预后更差。
图23为35例食管癌患者的临床样本以H-score小于120为Low PVT1-104aa组，H-score大于120为High PVT1-104aa组，绘制生存曲线，结果可知High PVT1-104aa组明显比Low PVT1-104aa组生存率更低，预后更差。
图24为20例乳腺癌患者的临床样本以H-score小于120为Low PVT1-104aa组，H-score大于120为High PVT1-104aa组，绘制生存曲线，结果可知High PVT1-104aa组明显比Low PVT1-104aa组生存率更低，预后更差。
上述结果表明，胃癌、食管癌、乳腺癌肿瘤组织中高表达PVT1-104aa的患者的总生存率 相对于低表达PVT1-104aa的患者显著降低。
以上结果提示，肿瘤组织中PVT1-104aa的表达是一个显著独立的预测乳腺癌患者总体生存时间的危险因素。
这表明，PVT1-104aa可以用于肿瘤的预后评估。
本领域的技术人员可对前述的技术方案和技术特征进行任意组合而不脱离本发明的发明构思和保护范围。本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. CircRNA PVT1及PVT1-104aa作为肿瘤早期诊断、治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的药物靶点的应用。
  2. 一种用于检测CircRNA PVT1及PVT1-104aa水平的物质在预测肿瘤生长、肿瘤转移、肿瘤预后评估或诊断肿瘤的产品中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述用于检测CircRNA PVT1及PVT1-104aa表达水平的物质是用于在基因水平和/或蛋白质水平上检测CircRNA PVT1及PVT1-104aa的物质；
    优选的，所述用于检测CircRNA PVT1及PVT1-104aa表达水平的物质是用于选自下组的一种或多种检测技术或方法中的物质：免疫组织化学法、Western印迹法、Northern印迹法、PCR、生物芯片法；
    或，优选的，所述用于检测CircRNA PVT1及PVT1-104aa表达水平的物质选自对CircRNA PVT1及PVT1-104aa具有特异性的物质，例如抗PVT1-104aa抗体或其抗原结合片段，优选单克隆抗体；CircRNA PVT1特异性的探针、基因芯片、PCR引物、gRNA等。
  4. 根据权利要求2所述的应用，其特征在于，所述肿瘤选自：乳腺癌，脑胶质瘤，胃癌，肾癌，结直肠癌、食管癌、肝癌等。
  5. 根据权利要求2所述的应用，其特征在于，所述产品包括：与正常对照值相比，所述对象或获自所述对象的样品中CircRNA PVT1及PVT1-104aa水平提高，表明所述对象易于或已发生癌症转移，或表明所述对象的癌症预后不良，或表明所述对象已患有癌症。
  6. 抑制CircRNA PVT1及PVT1-104aa和/或其基因的抑制剂在制备用于治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的药物中的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述抑制剂选自：与CircRNA PVT1及PVT1-104aa直接结合的抑制剂、阻断CircRNA PVT1及PVT1-104aa与其受体或配体结合的抑制剂、PVT1(LINC00079；MIR1204HG；NCRNA00079；onco-lncRNA-100)和CircRNA PVT1表达水平的抑制剂、降低CircRNA PVT1及PVT1-104aa表达水平的抑制剂、促进CircRNA PVT1及PVT1-104aa降解的抑制剂、用于敲除或敲减CircRNA PVT1及PVT1-104aa表达的物质，例如能够下调CircRNA PVT1的表达及PVT1-104aa的蛋白表达水平的相关启动子元件、重组质粒、表达载体和相关抗体；
    或，
    所述抑制剂选自：抗PVT1-104aa抗体、抗PVT1-104aa受体的抗体、针对CircRNA PVT1及PVT1-104aa的CRISPR/Cas9系统、针对PVT1及CircRNA PVT1的干扰RNA、针对PVT1 及CircRNA PVT1的反义寡核苷酸、CircRNA PVT1及PVT1-104aa表达和/或功能抑制化合物。
  8. 一种筛选治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的候选药物的方法，其特征在于，所述方法包括测试所述候选药物对对象或获自对象的样品中CircRNA PVT1及PVT1-104aa水平的影响，其中，在使用所述候选药物后，CircRNA PVT1及PVT1-104aa水平降低表明所述候选药物具有治疗肿瘤、缓解或防止肿瘤转移和/或改善肿瘤预后的效果。
  9. 一种用于预测肿瘤生长、转移、对肿瘤预后评估或诊断肿瘤的产品，其特征在于，其包含：用于检测CircRNA PVT1及PVT1-104aa水平的物质。
  10. 一种药物组合物，其特征在于，其包含：作为治疗活性物质的抑制CircRNA PVT1及PVT1-104aa和/或其基因的抑制剂以及药学上可接受的载体。
PCT/CN2020/071984 2019-12-25 2020-01-14 CircRNA PVT1及肽段在肿瘤生长预测、转移预测、预后评估和治疗中的应用 WO2021128516A1 (zh)

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