WO2020025029A1 - Marqueur de diagnostic pour cancer du col de l'utérus, et procédés de diagnostic et de traitement du cancer du col de l'utérus - Google Patents

Marqueur de diagnostic pour cancer du col de l'utérus, et procédés de diagnostic et de traitement du cancer du col de l'utérus Download PDF

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WO2020025029A1
WO2020025029A1 PCT/CN2019/098920 CN2019098920W WO2020025029A1 WO 2020025029 A1 WO2020025029 A1 WO 2020025029A1 CN 2019098920 W CN2019098920 W CN 2019098920W WO 2020025029 A1 WO2020025029 A1 WO 2020025029A1
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cervical cancer
marker
seq
detecting
expression level
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PCT/CN2019/098920
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English (en)
Chinese (zh)
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杨承刚
唐美兰
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北京泱深生物信息技术有限公司
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Priority claimed from CN201810868349.5A external-priority patent/CN108624694B/zh
Priority claimed from CN201810868385.1A external-priority patent/CN108949986B/zh
Priority claimed from CN201810868577.2A external-priority patent/CN108753983B/zh
Priority claimed from CN201810868586.1A external-priority patent/CN108841963B/zh
Priority claimed from CN201810868578.7A external-priority patent/CN108949987B/zh
Priority claimed from CN201810868376.2A external-priority patent/CN109306378A/zh
Application filed by 北京泱深生物信息技术有限公司 filed Critical 北京泱深生物信息技术有限公司
Publication of WO2020025029A1 publication Critical patent/WO2020025029A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to the field of biomedicine, and more specifically, the present invention relates to the application of MNX1, UPF2, TAF2, CMC2, GPR19, and MLF1 in the preparation of tools for diagnosis and treatment of cervical cancer.
  • Cervical cancer is the most common gynecological malignancy, and it ranks first among female reproductive organ tumors. According to statistics from the World Health Organization in 2014, the annual number of new cases of cervical cancer is approximately 528,000, and the number of annual deaths is approximately 266,000. 85% of these cases occur in developing countries, and rural areas are higher than urban areas. . China is a large country with cervical cancer. According to the latest cancer statistics released by the National Cancer Center, the number of new cervical cancer cases in China in 2015 was about 98,900, and the number of annual deaths was about 30,500. And the trend of younger onset [Chen, W, Zheng, R, Baade, D, et al. Cancer statistics in China, 2015 [J].
  • tumor pathogenesis With the development of modern bioassay technology and the continuous in-depth study of tumor pathogenesis, many molecules in various biological processes involving tumorigenesis, such as nucleic acids, proteins, sugars, lipids, small molecule metabolites and even blood Free tumor cells can be used as important tumor markers to provide clear evidence for clinical prevention, diagnosis and treatment. In cervical cancer, some tumor markers have been found for the clinical prevention of cervical cancer.
  • Ki-67 and p16INK4a are two common molecular markers used to analyze tumor cell proliferation status and tumor malignancy. Ki-67 is not expressed in silent GO phase cells, but is highly expressed in G1, S, G2, and M phase cells. Therefore, it can be widely used to analyze the cell's proliferative activity to evaluate tumor progression [EndlE, Gerdes J. The Ki -67 protein: spectacular forms and unknown functions [J] .Exp CellRes, 2000,257 (2): 231-237]; At the same time, because of its wide expression lineage, it still has certain defects as a specific tumor marker In clinical, other molecular markers are needed to assist diagnosis.
  • p16INK4a can specifically bind to CDK4 and CDK6, thereby inhibiting its activity and phosphorylation of downstream pRb, and regulating cell cycle progression and cell differentiation process.
  • ProExC antibody can specifically recognize the nuclear protein MCM2 and topoisomerase TOP2A complex induced by HPV infection.
  • the S-phase genes induced by E7 oncogenes will promote the high expression of TOP2A and MCM2 in the nucleus.
  • TOP2A can combine with six MCM2 molecules to form a stable structure and stay in the nucleus Santin, D, Zhan, F, Bignotti, E, et al.
  • HPV DNA integrity and stability are mainly maintained by the capsid protein L1 and L2 proteins forming a stable protective shell.
  • L1 protein can also promote the infection of mucosal basement membrane band cells or cervical epithelial cells by recognizing corresponding receptors on host cells.
  • the expression of L1 protein will continue to be detected, but the expression of L1 protein will gradually disappear as the degree of cervical cancer progresses.
  • Mcmurray H, Mccance D J. Human papillomavirus type, E6, activation, TERT, gene, transcription, introduction, c-Myc, and release of USF-mediated reduction [J] .J Virol, 2003,77 (18): 9852-9861]. Therefore, the disappearance of HPV-L1 expression indicates that the viral genome has been integrated into the host genome and can be used for the diagnosis of CIN3 stage of cervical cancer.
  • Lanminin-5 is a tumor marker closely related to tumor invasion.
  • the expression of Lanminin-5 gene in many different types of malignant tumors is often closely related to tumor progression.
  • Laminin-5 is mainly expressed in the early stages of tumors, especially in the small infiltrated skin lesions, so it can be used for the early detection of cervical squamous cell infiltration.
  • MIB-I has a similar expression pattern as Ki-67, that is, tumor cells that are active in proliferative states that are significantly overexpressed in each phase of GINS, and combined with Ki-67 detection can be used to help analyze the cycle status of tumor cells. Therefore, MIB-1 is another important indicator for detecting cell proliferation activity during atypical hyperplasia.
  • tumor markers there are many tumor markers currently used for the diagnosis of cervical cancer, the existing tumor markers are also lacking because of their constitutive expression under normal conditions or in non-malignant tumor diseases. Tumor-specific. Therefore, it is urgent to find tumor-specific antigens as biomarkers for early diagnosis and prognosis of clinical cervical cancer.
  • An object of the present invention is to provide a marker for diagnosing cervical cancer.
  • Another object of the present invention is to provide a method for detecting cervical cancer by detecting a difference in expression of the marker.
  • a third object of the present invention is to provide a method for treating cervical cancer by inhibiting the expression of the marker.
  • a fourth object of the present invention is to provide a method for screening drugs for treating cervical cancer.
  • a fifth object of the present invention is to provide a medicament for treating cervical cancer.
  • the present invention provides a marker for diagnosing cervical cancer, the marker is selected from one or more of the following groups: MNX1, UPF2, TAF2, CMC2, GPR19, MLF1.
  • the invention provides reagents for detecting the aforementioned markers.
  • the reagent for detecting the aforementioned marker includes a reagent for detecting the expression level of the aforementioned marker;
  • the reagent for detecting the aforementioned marker includes a reagent capable of quantifying the transcription level of the aforementioned marker, and / or a reagent capable of quantifying the level of the aforementioned marker protein.
  • the reagent capable of quantifying the aforementioned marker transcription level includes a primer for specifically amplifying the aforementioned marker used in real-time quantitative PCR; the reagent capable of quantifying the aforementioned marker protein level
  • the reagents include antibodies that specifically bind to the aforementioned markers.
  • the invention also provides the application of a reagent for detecting the aforementioned markers in the preparation of a tool for diagnosing cervical cancer.
  • the reagent for detecting the aforementioned marker includes a reagent for detecting the expression level of the aforementioned marker;
  • the reagent for detecting the aforementioned marker includes a reagent capable of quantifying the transcription level of the aforementioned marker, and / or a reagent capable of quantifying the level of the aforementioned marker protein.
  • the reagent capable of quantifying the aforementioned marker transcription level includes a primer for specifically amplifying the aforementioned marker used in real-time quantitative PCR; the reagent capable of quantifying the aforementioned marker protein level
  • the reagents include antibodies that specifically bind to the aforementioned markers.
  • the reagents for quantifying the aforementioned marker transcription levels of the present invention can perform their functions based on known methods using nucleic acid molecules: such as PCR, such as Southern hybridization, Northern hybridization, dot hybridization, fluorescent in situ hybridization (FISH), DNA Microarray, ASO method, high-throughput sequencing platform, etc.
  • the analysis can be performed qualitatively, quantitatively, or semi-quantitatively using this reagent.
  • the PCR method is a known method, for example, an ARMS (Amplification Refractory Mutation System) method, an RT-PCR (Reverse Transcriptase-PCR) method, a nested PCR method, and the like.
  • the amplified nucleic acid can be prepared by dot blot hybridization, surface plasmon resonance (SPR), PCR-RFLP, in situ RT-PCR, PCR-SSO (sequence-specific oligonucleotide), or PCR-SSP.
  • SPR surface plasmon resonance
  • PCR-RFLP in situ RT-PCR
  • PCR-SSO sequence-specific oligonucleotide
  • PCR-SSP PCR-SSP.
  • Method AMPFLP (amplifiable fragment length polymorphism) method
  • MVR-PCR method and PCR-SSCP (single-strand conformation polymorphism) method.
  • the reagent capable of quantifying the aforementioned marker transcription level may be a specific primer for the marker gene or transcript, or may be a specific recognition probe for the marker gene or transcript, or may include both Primers and probes.
  • primers for the marker gene or transcript described above include primers that specifically amplify the marker used in real-time quantitative PCR.
  • primer sequence is as follows:
  • the primer sequences of MNX1 are shown in SEQ ID NO.1 and SEQ ID NO.2;
  • the primer sequences of UPF2 are shown in SEQ ID NO. 3 and SEQ ID NO. 4;
  • TAF2 The primer sequences of TAF2 are shown in SEQ ID NO.5 and SEQ ID NO.6;
  • the primer sequences of CMC2 are shown in SEQ ID NO.7 and SEQ ID NO.8;
  • the primer sequences of GPR19 are shown in SEQ ID NO. 9 and SEQ ID NO. 10;
  • the primer sequences of MLF1 are shown in SEQ ID NO.11 and SEQ ID NO.12.
  • Primers can be prepared by chemical synthesis, appropriately designed by referring to known information using methods known to those skilled in the art, and prepared by chemical synthesis.
  • Probes can be prepared by chemical synthesis, appropriately designed by referring to known information using methods known to those skilled in the art, and prepared by chemical synthesis, or can be prepared by preparing genes containing desired nucleic acid sequences from biological materials, and using the design Primers for amplifying a desired nucleic acid sequence are prepared by amplifying it.
  • the reagent for quantifying the aforementioned marker protein level of the present invention may perform its function based on a known method using an antibody: for example, it may include ELISA, radioimmunoassay, immunohistochemistry, Western blot, and the like.
  • the reagent for quantifying the aforementioned marker protein level of the present invention includes an antibody or a fragment thereof that specifically binds the aforementioned marker.
  • An antibody or fragment thereof of any structure, size, immunoglobulin class, origin, etc. can be used as long as it binds the target protein.
  • the antibodies or fragments thereof included in the detection reagent of the present invention may be monoclonal or polyclonal.
  • An antibody fragment refers to a portion of the antibody (partial fragment) or a peptide containing a portion of the antibody that retains the binding activity of the antibody to the antigen.
  • the antibody fragment may include F (ab ′) 2 , Fab ′, Fab, single-chain Fv (scFv), disulfide-bonded Fv (dsFv) or a polymer thereof, a dimerized V region (diabody), or containing CDR peptide.
  • the reagent for quantifying the aforementioned markers of the present invention may include an isolated nucleic acid encoding an antibody or an amino acid sequence encoding an antibody fragment, a vector comprising the nucleic acid, and a cell carrying the vector.
  • Antibodies can be obtained by methods known to those skilled in the art. For example, a mammalian cell expression vector that retains all or part of a target protein or incorporates a polynucleotide encoding them is prepared as an antigen. After immunizing the animals with the antigen, immune cells are obtained from the immunized animals and fused with myeloma cells to obtain hybridomas. Antibodies were then collected from the hybridoma culture. Finally, a monoclonal antibody directed against the aforementioned marker can be obtained by subjecting the obtained antibody to antigen-specific purification using the aforementioned marker or a part thereof used as an antigen.
  • a polyclonal antibody can be prepared by immunizing an animal with the same antigen as above, collecting a blood sample from the immunized animal, separating the serum from the blood, and then performing antigen-specific purification of the serum using the above-mentioned antigen.
  • the antibody fragment can be obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.
  • Binding of a label to an antibody or a fragment thereof can be performed by a method generally known in the art.
  • the protein or peptide can be fluorescently labeled as follows: the protein or peptide is washed with a phosphate buffer, a dye prepared with DMSO, a buffer, or the like is added, and then the solution is mixed and left at room temperature for 10 minutes.
  • labeling kits such as biotin labeling kits, such as biotin labeling kits-NH2, biotin labeling kits-SH (Dojindo Laboratories); alkaline phosphatase labeling kits such as alkaline phosphate Enzyme labeling kit-NH2, alkaline phosphatase labeling kit-SH (Dojindo Laboratories); peroxidase labeling kits such as peroxidase labeling kit-NH2, peroxidase labeling kit-NH2 (Dojindo Laboratories); Phycobiliprotein labeling kits such as phycobiliprotein labeling kit-NH2, phycobiliprotein labeling kit-SH, B-phycoerythrin labeling kit-NH2, B-phycoerythrin labeling kit-SH, R-phycoerythrin labeling kit-NH2, R-phycoerythrin labeling kit SH (DojindoLaboratories); fluorescent labeling kits such as fluorescein labeling kit-NH2, HiL
  • the present invention also provides a tool for diagnosing cervical cancer, the tool comprising the aforementioned reagent; and an instruction manual or label, wherein the tool is used for diagnosing cervical cancer.
  • the reagent is present in a suitable container.
  • Each of the primers or probes can be adjusted to a concentration of at least one required amount using a diluent such as deionized water, and divided into containers.
  • the tools for diagnosing cervical cancer include, but are not limited to, chips, kits, test strips, or high-throughput sequencing platforms; high-throughput sequencing platforms are a special tool.
  • high-throughput sequencing platforms are a special tool.
  • the kit of the present invention may further include a reagent for extracting nucleic acid, a reagent for PCR, a reagent for staining or color development, and the like.
  • these reagents include, but are not limited to, extraction solutions, amplification solutions, hybridization solutions, color development solutions, washing solutions, and the like.
  • the kit according to the present invention may contain a variety of different reagents suitable for practical use (such as for different detection methods), and is not limited to the currently listed reagents, as long as it is based on the detection of the aforementioned markers to diagnose the cervix Cancer agents are included within the scope of the invention.
  • the invention also provides a method for diagnosing or assisting the diagnosis of cervical cancer.
  • the method includes the step of detecting the expression level of the aforementioned marker.
  • the method includes the following steps:
  • the method includes the following steps:
  • the reference value is the expression level of the aforementioned marker in a non-cervical cancer sample.
  • the non-cervical cancer sample includes a sample of a person who does not have cervical cancer, or a non-cervical cancer tissue sample of a patient with cervical cancer.
  • the obtaining of the test sample of the present invention is a conventional technique in the art, and it is preferred to obtain the test sample by a method that is non-invasive or minimally invasive.
  • the test sample may be (but not limited to): tissue, body fluid.
  • the body fluid includes, but is not limited to, peripheral blood, bone marrow, lymph nodes, peritoneal washes, urine, and sweat.
  • the test sample is from a tissue of a subject.
  • the method of detecting the expression level of the marker described in the step (4) includes a method of detecting the transcription level of the marker described above, and / or a method of detecting the level of the marker protein described above.
  • Methods for detecting the transcription level of the aforementioned markers include PCR, Southern hybridization, Northern hybridization, dot hybridization, fluorescence in situ hybridization (FISH), DNA microarray, ASO method, and high-throughput sequencing platforms; detecting the aforementioned markers
  • Methods for protein levels include ELISA, radioimmunoassay, immunohistochemistry, and Western blotting.
  • the present invention also provides a medicament for preventing or treating cervical cancer, the medicament comprising a substance that inhibits the aforementioned marker.
  • the substance for inhibiting the aforementioned markers of the present invention is not limited as long as the substance can inhibit the expression or activity of the aforementioned markers or substances involved in upstream or downstream pathways of the markers, and is useful for treating the cervix It is sufficient for cancer.
  • the invention also provides the application of the aforementioned markers in the preparation of a medicament for treating cervical cancer.
  • the medicament includes an interfering RNA targeting the expression of the aforementioned marker, or a negatively regulated miRNA, a negatively regulated transcriptional regulatory factor, or an inhibitory targeted small molecule compound.
  • the medicament of the present invention can be used by formulating a pharmaceutical composition in any manner known in the art.
  • This composition contains the active ingredient, plus one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form designed.
  • Inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyhydroxy compounds such as polyethylene glycol, water, Sucrose, ethanol, glycerin and the like, various preservatives, lubricants, dispersants, flavoring agents.
  • Moisturizing tinctures, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like can also be added to them, these substances are used as needed to help the stability of the formula or to help increase the activity or its biological effectiveness or Produces an acceptable mouthfeel or odor when taken orally.
  • the formulations that can be used in this composition can be in the form of the original compound itself, or optionally in the form of a pharmaceutically acceptable salt thereof.
  • the medicament of the invention It can be administered alone, or in various combinations, and in combination with other therapeutic agents.
  • the composition thus formulated may be administered with any suitable means known to those skilled in the art as required.
  • a safe and effective amount of the medicament of the present invention is administered to a human, and the specific dosage should also take into consideration factors such as the route of administration, the health status of the patient, etc., which are all within the skill of a skilled physician.
  • the medicine of the present invention can be prepared into various dosage forms as required. These include, but are not limited to, tablets, solutions, granules, patches, patches, capsules, aerosols or suppositories for transdermal, mucosal, nasal, cheek, sublingual or oral use.
  • the route of administration of the drug of the present invention is not limited as long as it can exhibit the desired therapeutic or preventive effect, including but not limited to intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, Intramuscularly, intratracheally, subcutaneously, through the skin, through the pleura, topically, by inhalation, through the mucosa, skin, gastrointestinal, intra-articular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumor. In some cases, it can be administered systemically. In some cases, it is administered topically.
  • the dose of the medicament of the present invention is not limited as long as a desired therapeutic effect or preventive effect is obtained.
  • the dose of the therapeutic or preventive agent of the present invention can be determined using, for example, a therapeutic effect or a preventive effect on a disease as an index.
  • the present invention also provides a method for preventing or treating cervical cancer, the method comprising administering the aforementioned medicament for preventing or treating cervical cancer.
  • the invention also provides a method for screening drugs for preventing or treating cervical cancer.
  • the method includes the step of detecting the expression level of the aforementioned marker.
  • the method includes the following steps:
  • the candidate substance can reduce the expression level of the aforementioned marker, it indicates that the candidate drug is a potential drug for preventing or treating cervical cancer.
  • step (1) includes: adding a candidate drug to a system expressing or containing the aforementioned marker in the test group; and / or, step (2) includes: detecting the aforementioned in the system of the test group The expression level of the marker is compared with a control group, wherein the control group is a system that does not add the expression of the drug candidate or contains the aforementioned marker; if the expression of the aforementioned marker in the test group The level is statistically lower than the control group, which indicates that the candidate drug is a potential drug for the prevention and treatment of cervical cancer.
  • the system includes a cell system, a solution system, a tissue system, an organ system, or an animal system.
  • the "MNX1 gene” (Chromosome 7, NC_000007.14 (157004853..157010653, completion)) sequence of the present invention can be queried in the NCBI database.
  • the "CMC2 gene” (Chromosome 16, NC_000016.10 (80975795..81007036, completion)) sequence of the present invention can be queried in the NCBI database.
  • the "UPF2 gene” (Chromosome 10, NC_000010.11 (11920022 .. 12043170, completion)) sequence of the present invention can be queried in the NCBI database.
  • the "TAF2 gene” (Chromosome 8, NC_000008.11 (119730773..119832858, completion)) sequence of the present invention can be queried in the NCBI database.
  • the "GPR19 gene” (Chromosome 12, NC_000012.12 (12659691..12717786, completion)) sequence of the present invention can be queried in the NCBI database.
  • the "MLF1 gene” (Chromosome 3, NC_000003.12 (158571163..158606460)) sequence of the present invention can be queried in the NCBI database.
  • a "reference value” refers to a value that is statistically related to a particular result when compared to the results of the analysis.
  • the reference value is determined based on statistical analysis of studies of the expression of the aforementioned markers and known clinical outcomes. Some such studies are shown in the Examples section herein. However, research from the literature and user experience with the methods disclosed herein can also be used to produce or adjust reference values. Reference values can also be determined by considering conditions and outcomes that are particularly relevant to a patient's medical history, genetics, age, and other factors.
  • sample refers to material that is specifically associated with a subject, from which specific information related to the subject can be determined, calculated, or inferred.
  • the sample may consist entirely or in part of biological material from the subject. Samples can also be materials that have been in contact with the subject in a manner that allows testing of the sample to provide information about the subject.
  • the sample may also be a material that has been in contact with other materials. This other material is not the subject's, but enables the first material to be subsequently tested to determine information about the subject, such as a probe or anatomy. Knife cleaning fluid.
  • the sample may be a source of biological material other than the contact subject, as long as a person skilled in the art can still determine the subject-related information from the sample.
  • expression includes the production of mRNA from a gene or gene portion, and includes the production of a protein encoded by the RNA or gene or gene portion, and also includes the presence of a detection substance associated with expression.
  • a detection substance associated with expression For example, cDNA, binding of a binding ligand (such as an antibody) to a gene or other oligonucleotide, protein or protein fragment, and a colored portion of the binding ligand are included within the scope of the term "expression”. Therefore, the increase in half-dot density on immunoblots, such as western blots, is also within the scope of the term "expression” based on biological molecules.
  • diagnosing cervical cancer includes determining whether a subject already has cervical cancer, determining whether a subject is at risk of having cervical cancer, or determining that a patient with cervical cancer has relapsed.
  • treatment encompasses a treatment-related disease or condition in a mammal having a related disease or condition, such as a human, and includes:
  • treatment generally relates to the treatment of a human or animal (e.g., as applied by a veterinarian), where certain desired therapeutic effects are achieved, e.g., inhibiting the development of a condition (including reducing the rate of development, stopping development), improving the condition, and curing Illness. It also includes treatment as a precautionary measure, such as prevention. The use of patients who have not yet developed the disorder but are at risk of developing the disorder is also included in the term "treatment.”
  • the present invention finds and confirms for the first time that the expressions of MNX1, UPF2, TAF2, CMC2, GPR19, and MLF1 of the present invention are closely related to the occurrence of cervical cancer, and the number of verified samples is large and the results are accurate.
  • the proposed correlation provides a new way for the diagnosis and treatment of cervical cancer.
  • FIG. 1 shows a statistical graph for detecting differential expression of a marker of the present invention in cervical cancer tissues and normal control tissues by QPCR; wherein, A: MNX1, B: UPF2, C: TAF2, D: CMC2, E: GPR19, F : MLF1;
  • FIG. 2 shows a statistical diagram for detecting differential expression of a marker of the present invention in cervical cancer tissues and normal control tissues by using a Western blot experiment; wherein: A: MNX1, B: UPF2, C: TAF2, D: CMC2, E: GPR19 , F: MLF1;
  • FIG. 3 shows a statistical graph for detecting the marker expression inhibition rate of the present invention by Western blot experiment, wherein A: MNX1, B: UPF2, C: TAF2, D: CMC2, E: GPR19, F: MLF1.
  • cervical cancer tissues provided by the hospital's obstetrics and gynecology department were collected from patients diagnosed with cervical cancer pathologically after hysterectomy (cone or total resection). The surrounding normal tissue was used as the control group.
  • RNAase-free water Take 20 ⁇ L RNAase-free water to dissolve the precipitate, use it after quantification or freeze it at -80 °C refrigerator for later use.
  • TopHat v1.3.1 was used to match the clean fragments to the UCSC H.sapiens reference genome (hg19).
  • the pre-built index of the H.sapiens UCSC hg19 version was downloaded from the TopHat homepage
  • each read default to 20
  • TopHat builds a library of possible splice sites based on exon regions and GT-AG splice signals. Based on these splice site libraries, reads that are not mapped to the genome are mapped to the genome. We use the system default parameters of the TopHat method.
  • the matching read file is processed by Cufflinks v1.0.3.
  • Cufflinks v1.0.3 normalizes the number of RNA-seq fragments to calculate the relative abundance of transcripts.
  • the FPKM value refers to the number of fragments that match a 1 kb exon region of a particular gene in each million sequenced fragments.
  • the Bayesian reasoning method is used to calculate the confidence interval of the FPKM estimate.
  • the referenced GTF annotation file used by Cufflinks was downloaded from the Ensembl database (Homo_sapiens.GRCh37.63.gtf).
  • Cuffdiff uses the original match file to re-evaluate the expression abundance of the transcripts listed in the GTF file and detect the differential expression. Only a q value of ⁇ 0.01 in the Cuffidff output, the test showed that a successful comparison was considered a differential expression.
  • RNA-seq results showed that, compared with normal control tissues, 351 genes were highly expressed in cervical cancer tissues, and 294 genes were under-expressed. The differences were statistically significant (P ⁇ 0.05).
  • Example 1 Forty cases of cervical cancer tissues and corresponding normal control tissues were collected according to the criteria of Example 1.
  • primer sequences for real-time quantitative PCR used in this application are as follows (designed and synthesized by Biotech Engineering (Shanghai) Co., Ltd.):
  • Upstream primer 5'-AGGTGAAGATTTGGTTCCA-3 '(SEQ ID NO. 1);
  • Upstream primer 5'-GAAGAAGAAGAAGAAGGT-3 '(SEQ ID NO. 3);
  • Upstream primer 5'-ACATACCACATCATACCTTCA-3 '(SEQ ID NO. 5);
  • Upstream primer 5'-TGACTTATCTCCACACTTG-3 '(SEQ ID NO.7);
  • Upstream primer 5'-CTACACTGTCATCCACTTCT-3 '(SEQ ID NO. 9);
  • Upstream primer 5'-CATAATCGTAGAGGACAT-3 '(SEQ ID NO.11);
  • Upstream primer 5'-GACCTGACCTGCCGTCTA-3 '(SEQ ID NO. 13);
  • Downstream primer 5'-AGGAGTGGGTGTCGCTGT-3 '(SEQ ID NO. 14).
  • Takara reverse transcription kit (containing genomic DNA removal enzyme) was used for reverse transcription.
  • the specific reaction system is as follows:
  • the reaction was performed at 42 ° C for 5 minutes.
  • the reaction was carried out at 37 ° C for 15 min, and at 85 ° C for 5 s.
  • MNX1 reaction conditions pre-denaturation at 95 °C for 30s;
  • TAF2 reaction conditions 95 ° C pre-denaturation for 30s;
  • MLF1 reaction conditions pre-denaturation at 95 °C for 30s;
  • the 2- ⁇ Ct relative quantitative method was used to analyze the expression level of the marker of the present invention.
  • Ct is the intensity value of the fluorescent signal in the reaction system detected by the thermal cycler.
  • the analysis of experimental data was performed by Bio-RAD analysis software.
  • MNX1 gene was significantly increased in 38 of the 40 cervical cancer tissues.
  • the statistical results are shown in Figure 1A.
  • MNX1 gene was significantly increased in cervical cancer tissues, and the difference was statistically significant (P ⁇ 0.05).
  • TAF2 gene expression in 40 cases of cervical cancer tissues was significantly increased.
  • the statistical results are shown in Figure 1C.
  • TAF2 gene was significantly increased in cervical cancer tissues, and the difference was statistically significant (P ⁇ 0.05).
  • MLF1 gene expression was significantly increased in 35 of the 40 cervical cancer tissues.
  • the statistical results are shown in Figure 1F.
  • MLF1 gene was significantly increased in cervical cancer tissues, and the difference was statistically significant (P ⁇ 0.05).
  • ⁇ -actin was used as an internal reference. After 50 ⁇ g of total protein was separated by SDS-PAGE, it was electrically transferred to a PVDF membrane and blocked with 1 ⁇ TBST containing 5% skimmed milk powder at room temperature for 1 h. Shake at room temperature; add the target protein monoclonal antibody and ⁇ -actin monoclonal antibody, respectively, overnight at 4 ° C; Wash the membrane 4 times with TBST, add secondary antibody, and incubate for 1 hour at room temperature.
  • the gray value of the protein band was analyzed using Image J software, and ⁇ -actin was used as an internal reference to normalize the gray value of the marker protein band of the present invention.
  • the results were expressed as mean ⁇ standard deviation, and statistical analysis was performed using SPSS13.0 statistical software. The difference between the two was tested by t test, which was considered statistically significant when P ⁇ 0.05.
  • MNX1 protein levels were significantly increased in 38 of 40 cervical cancer tissues, and the difference was statistically significant.
  • the statistical results are shown in Figure 2A.
  • MNX1 protein levels in cervical cancer tissues were significantly increased, and the difference was statistically significant (P ⁇ 0.05).
  • TAF2 protein levels in 40 cases of cervical cancer tissues were significantly increased, and the difference was statistically significant.
  • the statistical results are shown in Figure 2C.
  • the level of TAF2 protein in cervical cancer tissues was significantly increased, and the difference was statistically significant (P ⁇ 0.05).
  • MLF1 protein levels in 35 cases of 40 cervical cancer tissues were significantly increased, and the difference was statistically significant.
  • the statistical results are shown in Figure 2F.
  • the MLF1 protein level in cervical cancer tissues was significantly increased, and the difference was statistically significant (P ⁇ 0.05).
  • siRNA-NC negative control siRNA
  • the sense chain is 5’-ACUUGUUGAGCUUGAACUGGU-3 ’(SEQ ID NO. 15);
  • the antisense strand is 5'-CAGUUCAAGCUCAACAAGUAC-3 '(SEQ ID NO. 16),
  • the sense chain is 5’-UCUUCUUGCUGACGUUUGGCC-3 ’(SEQ ID NO. 17);
  • the antisense strand is 5'-CCAAACGUCAGCAAGAAGAAG-3 '(SEQ ID NO.18),
  • siRNA-TAF2 siRNA-TAF2
  • the sense chain is 5'-UUCUAUUCAAGUUUGCAACUG-3 '(SEQ ID NO. 19);
  • the antisense strand is 5’-GUUGCAAACUUGAAUAGAAUC-3 ’(SEQ ID NO. 20),
  • siRNA-CMC2 siRNA-CMC2
  • the sense chain is 5'-UCAACAUCAUUACAAUAACCA-3 '(SEQ ID NO.21);
  • the antisense strand is 5’-GUUAUUGUAAUGAUGUUGAUC-3 ’(SEQ ID NO.22),
  • the sense chain is 5'-AAUUCCAUCAGGUAUUGGCUU-3 '(SEQ ID NO.23);
  • the antisense strand is 5’-GCCAAUACCUGAUGGAAUUAA-3 ’(SEQ ID NO.24),
  • siRNA-MLF1 siRNA-MLF1
  • the sense chain is 5’-AUAUAGUUUCUCAUAUUUGAC-3 ’(SEQ ID NO. 25);
  • the antisense strand is 5’-CAAAUAUGAGAAACUAUAUGC-3 ’(SEQ ID NO. 26),
  • the cells were cultured in a 1640 medium containing double antibodies and 10% fetal bovine serum in a 37 ° C, 5% CO 2 incubator, and the culture medium was changed every 24 hours and passaged once every 48 hours. Cells in logarithmic growth phase were taken for subsequent experiments.
  • the cells were digested one day before transfection, and the cells were seeded in a 6-well plate with about 2 ⁇ 10 5 cells per well and cultured overnight.
  • the degree of cell fusion (about 50-70%) was observed for transient transfection of cellular siRNA.
  • the specific process is as follows:
  • results showed that the OD value of cells in the negative control group (transfected siRNA-NC) was 0.988 ⁇ 0.142, and the OD value of cells in the experimental group (transfected siRNA-MLF1) was 0.486 ⁇ 0.087.
  • the above results indicate that inhibition of MLF1 expression inhibits the proliferation of cervical cancer cells, and the difference is statistically significant (P ⁇ 0.05).

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Abstract

La présente invention concerne un marqueur de diagnostic pour le cancer du col de l'utérus, et des procédés de diagnostic et de traitement du cancer du col de l'utérus. Par la détection de la teneur de MNX1, UPF2, TAF2, CMC2, GPR19 et MLF1 dans les tissus cervicaux chez un sujet, le fait que le sujet possède un cancer du col de l'utérus peut être déterminé, ou, le fait que le sujet présente un risque d'être affecté d'un cancer du col de l'utérus peut être diagnostiqué. De plus, en étudiant l'indice de prolifération des cellules du cancer du col de l'utérus cultivées in vitro, MNX1, UPF2, TAF2, CMC2, GPR19 et MLF1 se sont avérés comme étant des cibles médicamenteuses de traitement du cancer du col de l'utérus.
PCT/CN2019/098920 2018-08-02 2019-08-01 Marqueur de diagnostic pour cancer du col de l'utérus, et procédés de diagnostic et de traitement du cancer du col de l'utérus WO2020025029A1 (fr)

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CN201810868586.1 2018-08-02
CN201810868349.5A CN108624694B (zh) 2018-08-02 2018-08-02 Cmc2作为宫颈癌诊治标志物的用途
CN201810868376.2 2018-08-02
CN201810868349.5 2018-08-02
CN201810868577.2 2018-08-02
CN201810868385.1A CN108949986B (zh) 2018-08-02 2018-08-02 用于诊治宫颈癌的分子标志物-upf2基因及其表达产物
CN201810868578.7 2018-08-02
CN201810868385.1 2018-08-02
CN201810868577.2A CN108753983B (zh) 2018-08-02 2018-08-02 宫颈癌的诊治标志物
CN201810868586.1A CN108841963B (zh) 2018-08-02 2018-08-02 诊治宫颈癌的mlf1基因及其应用
CN201810868578.7A CN108949987B (zh) 2018-08-02 2018-08-02 Gpr19作为诊治宫颈癌的靶标
CN201810868376.2A CN109306378A (zh) 2018-08-02 2018-08-02 Taf2在制备诊断宫颈癌的产品以及治疗宫颈癌的药物中的应用

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