WO2012161124A1 - Composition contenant un micro arn ou système d'expression associé - Google Patents

Composition contenant un micro arn ou système d'expression associé Download PDF

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WO2012161124A1
WO2012161124A1 PCT/JP2012/062825 JP2012062825W WO2012161124A1 WO 2012161124 A1 WO2012161124 A1 WO 2012161124A1 JP 2012062825 W JP2012062825 W JP 2012062825W WO 2012161124 A1 WO2012161124 A1 WO 2012161124A1
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mir
hsa
microrna
cancer
human
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English (en)
Japanese (ja)
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中城公一
浜川裕之
田中宏史
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Ehime University NUC
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Ehime University NUC
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Priority to JP2013516348A priority Critical patent/JP6011945B2/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the present invention relates to a composition containing microRNA or an expression system thereof and a head and neck cancer marker.
  • Micro RNA is a small molecule RNA consisting of 18 to 25 bases, and inhibits translation into the protein by binding to the target mRNA.
  • miRNA / miRNA is a small molecule RNA consisting of 18 to 25 bases, and inhibits translation into the protein by binding to the target mRNA.
  • miRBase® http://www.miRbase.org/
  • these miRNA expression or functional abnormalities are involved in various diseases.
  • Patent Document 1 In particular, regarding cancer, many miRNAs whose expression is enhanced or suppressed in cancer tissues or cancer cells have been identified by comprehensive expression analysis using miRNA microarrays (Patent Document 1).
  • MiR-21, miR-155, miR-17-5p, miR-19, etc. are known as miRNAs whose expression is enhanced in cancer.
  • miR-19 is necessary and sufficient for cell carcinogenesis, and its target One of them was shown to be a tumor suppressor gene PTEN.
  • PTEN a tumor suppressor gene
  • TS-miR Tumor suppressor miR
  • MiR-137 and miR-193a have been suggested as TS-miR for oral cancer (Non-patent Document 2).
  • a microRNA that not only reduces expression in cancer but also can inhibit the growth of the cancer cell by being expressed in the cancer cell is more advantageous for the treatment and prevention of cancer.
  • microRNA can be used as a cancer marker in serum or saliva, it will be useful for cancer diagnosis.
  • the present invention provides, as one aspect, microRNA capable of inhibiting the growth of head and neck cancer cells and use thereof. Moreover, this invention provides the microRNA which can be utilized as a head and neck cancer marker in a bodily fluid sample as another aspect, and its utilization. Moreover, this invention provides the microRNA which can inhibit the growth of a human cancer cell as another aspect, and its utilization.
  • One aspect of the present invention is a composition comprising microRNA or an expression system capable of expressing microRNA in a cell, wherein the microRNA is hsa-miR-1289, hsa-miR-892b, hsa-miR.
  • the present invention relates to a composition for suppressing the growth of selected human head and neck cancer cells.
  • the present invention provides a method for inhibiting the growth of human head and neck cancer cells, wherein the cancer cell is contacted with a composition comprising microRNA or an expression system capable of expressing microRNA in the cell.
  • Said microRNA is hsa-miR-1289, hsa-miR-892b, hsa-miR-661, hsa-miR-193b *, hsa-miR-671-5p, hsa-miR-124 *, hsa-miR-1908, hsa-miR-876-5p, hsa-miR-192 *, hsa-miR-124, hsa-miR-760, hsa-miR-192, hsa-miR-206, hsa-miR-629 , Hsa-miR-1, hsa-miR-708 *, and hsa-miR-629
  • the present invention further includes, as other embodiments, hsa-miR-1289, hsa-miR-876-5p, hsa-miR-1, hsa-miR-892b, hsa-miR-124, hsa-miR-124 *, hsa -Use of a microRNA selected from the group consisting of miR-206, hsa-miR-193b *, hsa-miR-192 *, and hsa-miR-192, as a head and neck cancer marker in a body fluid sample About the use of.
  • the present invention provides a composition comprising microRNA or an expression system capable of expressing microRNA in a cell, wherein the microRNA comprises hsa-miR-1289, hsa-miR-892b, hsa -miR-661, hsa-miR-193b *, hsa-miR-671-5p, hsa-miR-124 *, hsa-miR-1908, hsa-miR-876-5p, hsa-miR-192 *, hsa- Consists of miR-124, hsa-miR-760, hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR-708 *, and hsa-miR-629 *
  • the present invention relates to a composition for suppressing the growth of human cancer
  • a method for inhibiting the growth of human cancer cells wherein a composition comprising microRNA or an expression system capable of expressing microRNA in the cell is contacted with the cancer cells.
  • the microRNA is hsa-miR-1289, hsa-miR-892b, hsa-miR-661, hsa-miR-193b *, hsa-miR-671-5p, hsa-miR-124 *, hsa -miR-1908, hsa-miR-876-5p, hsa-miR-192 *, hsa-miR-124, hsa-miR-760, hsa-miR-192, hsa-miR-206, hsa-miR-629,
  • the present invention relates to a method for suppressing the growth of human cancer cells selected from the group consisting of hsa-miR-892b, hs
  • the present invention can contribute to diagnosis, treatment, prevention, or recurrence prevention of head and neck cancer. Moreover, according to the present invention, for example, it can contribute to the diagnosis, treatment, prevention, or recurrence prevention of cancers other than human head and neck cancer.
  • FIG. 1 is an example of a graph showing the effect of growth inhibition by TS-miR on human oral squamous cell carcinoma cell GFP-SAS.
  • FIG. 2 is an example of a graph showing the effect of growth inhibition by TS-miR on human salivary gland cancer cells GFP-ACCM.
  • FIG. 3 is an example of a graph showing the effects of hsa-miR-629 * growth inhibition on various human cancer cells.
  • FIG. 4 is an example of a graph showing the influence of growth inhibition by hsa-miR-1289 on various human cancer cells.
  • FIG. 5 is an example of a graph showing the effect of hsa-miR-892b growth inhibition on various human cancer cells.
  • FIG. 6A is an example of a graph showing the expression of hsa-miR-1289 in oral squamous cell carcinoma tissue at each stage
  • FIG. 6B is an example of a graph showing the expression of hsa-miR-1289 in salivary gland cancer tissue. It is.
  • FIG. 7A is an example of a graph showing expression of hsa-miR-892b in oral squamous cell carcinoma tissues at each stage
  • FIG. 7B is an example of a graph showing expression of hsa-miR-892b in salivary gland cancer tissues. It is.
  • the present invention includes the following aspects: [1] A composition comprising microRNA or an expression system capable of expressing microRNA in a cell, wherein the microRNA comprises hsa-miR-1289, hsa-miR-892b, hsa-miR-661, hsa- miR-193b *, hsa-miR-671-5p, hsa-miR-124 *, hsa-miR-1908, hsa-miR-876-5p, hsa-miR-192 *, hsa-miR-124, hsa-miR -760, hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR-708 *, and hsa-miR-629 *, human A composition for inhibiting the growth of head and neck cancer cells; [2]
  • a method for suppressing the growth of human cancer cells comprising contacting the cancer cells with a composition comprising microRNA or an expression system capable of expressing microRNA in the cells, Is hsa-miR-1289, hsa-miR-892b, hsa-miR-661, hsa-miR-193b *, hsa-miR-671-5p, hsa-miR-124 *, hsa-miR-1908, hsa- miR-876-5p, hsa-miR-192 *, hsa-miR-124, hsa-miR-760, hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa a method for inhibiting the growth of human cancer cells selected from the group consisting of -miR-708 * and hs
  • microRNA / miRNA refers to a kind of low-molecular non-coding RNA, and has a normal meaning used in the technical field.
  • the microRNA referred to using an ID can refer to a database (for example, miRBase® (http://www.miRbase.org/)).
  • microRNA refers to mature microRNA consisting of 18 to 25 bases.
  • composition containing microRNA or an expression system capable of expressing microRNA in a cell refers to a composition in a form in which a desired microRNA can be introduced into a cell.
  • composition containing microRNA include a composition for directly introducing a single-stranded or double-stranded mature microRNA into a cell, and “a microRNA can be expressed in a cell”.
  • composition comprising a simple expression system include a composition comprising a vector in which a polynucleotide encoding a microRNA precursor is linked so as to allow expression.
  • a known method can be adopted as a method for introducing microRNA or a vector into a cell.
  • “suppression of cell growth” includes inhibition of cell growth and cell death, and includes suppression of cell growth in in vivo, in vitro, and ex vivo.
  • “cell” can include human cells and non-human cells, with human cells being preferred.
  • head and neck cancer refers to cancer that can be formed from the neck, excluding the brain and eyes.
  • oral cancer nasal sinus cancer, lip cancer, and pharyngeal cancer. Including laryngeal cancer, cervical tumor, ear cancer.
  • oral cancer generally refers to cancer that is caused by the mucous membrane of a part constituting the oral cavity, such as gingiva, tongue, cheek, palate, floor of mouth, and salivary gland.
  • human cancer is not particularly limited, and for example, head and neck cancer, malignant melanoma, basal cell cancer, ovarian cancer, breast cancer, non-small cell lung cancer, renal cell cancer, bladder Cancer, recurrent superficial bladder cancer, stomach cancer, prostate cancer, biliary tract cancer, pancreatic cancer, lung cancer, cervical cancer, cervical dysplasia, laryngeal papillomatosis, colon cancer, colorectal Includes cancer and carcinoid tumors.
  • the microRNA relates to a composition selected from the group consisting of hsa-miR-1289, hsa-miR-892b, and hsa-miR-629 *.
  • the sequences of the microRNA (mature type) registered in the database are shown in SEQ ID NOS: 1 to 17 in Table 1 below, respectively.
  • the composition of the present invention is in the form of a “composition containing micromicroRNA”, as one embodiment, the mature microRNA represented by the RNA of Table 1 above is included in a single-stranded or double-stranded state. It is.
  • the mature microRNA is double-stranded, from the viewpoint of cell growth inhibition of head and neck cancer cells, double-stranded RNA (ie, between complementary strands) that assumes the state after the precursor has been cleaved into Dicer It is preferable to use a native or near-native double-stranded RNA) that maintains a mismatch.
  • These microRNAs can be synthesized.
  • composition of the present invention is in the form of “a composition containing an expression system capable of expressing microRNA in a cell”, as one embodiment of “an expression system capable of expressing microRNA in a cell”, RNA Examples thereof include a composition comprising a vector that encodes a precursor or an analogous sequence thereof that expresses the mature microRNA of Table 1 above.
  • a known microRNA expression vector may be used as the expression vector for linking the RNA precursor or a similar sequence thereof.
  • the composition of this embodiment is a composition for suppressing the growth of human head and neck cancer cells, and the expression system capable of expressing the microRNA contained in the composition of this embodiment or the microRNA in the cell is a human head and neck.
  • Introduce into cancer cells The introduction can be performed using a known transfection method.
  • the introduction may be any of in vivo, in vitro, and ex vivo.
  • the present invention provides a pharmaceutical composition for treating or preventing human head and neck cancer, comprising microRNA or an expression system capable of expressing microRNA in a cell, wherein the microRNA comprises: hsa-miR-1289, hsa-miR-892b, hsa-miR-661, hsa-miR-193b *, hsa-miR-671-5p, hsa-miR-124 *, hsa-miR-1908, hsa-miR- 876-5p, hsa-miR-192 *, hsa-miR-124, hsa-miR-760, hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR
  • the present invention relates to
  • the pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier.
  • the pharmaceutical carrier is not particularly limited.
  • a carrier that can increase the efficiency with which a microRNA or an expression system capable of expressing microRNA in a cell enters a target site, tissue, cell, etc. Atelocollagen, liposome, cationic liposome and the like.
  • the dosage form of the pharmaceutical composition of this embodiment is not particularly limited, and examples thereof include injections, creams, ointments, tablets, suspensions and the like.
  • the administration method is not particularly limited, and examples thereof include injection, oral, topical, intranasal, rectal, intravenous, and intraarterial administration.
  • known dosage forms of microRNA and / or siRNA supplements can be used as the dosage form of the pharmaceutical composition of the present invention.
  • the composition of the present invention the growth of human head and neck cancer cells can be suppressed. Therefore, this invention relates to the method of suppressing the growth of the cancer cell including making the composition of this invention contact the object human cancer cell as another aspect.
  • the contact method is not particularly limited, and a method that can be introduced into a cancer cell intended for the microRNA in the composition or an expression system capable of expressing the microRNA in the cell can be employed.
  • One embodiment of introducing a microRNA or an expression system capable of expressing microRNA in a cell into a cancer cell is a lipofection method.
  • the dosage of the microRNA or an expression system capable of expressing the microRNA in the cell during the lipofection method and other introduction methods is, for example, 10 to 25 nM.
  • composition of the present invention it is possible to treat or prevent human head and neck cancer. Therefore, this invention relates to the method of suppressing the growth of the cancer cell including administering the pharmaceutical composition of this invention to object as another aspect.
  • the dosage form of the composition to be administered is not particularly limited, and examples thereof include injections, creams, ointments, tablets, suspensions and the like.
  • the administration method is not particularly limited, and examples thereof include injection, oral, topical, intranasal, rectal, intravenous, and intraarterial administration. Administration will depend on the severity and responsiveness of the condition being treated and the course of treatment.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the subject's body.
  • the optimal dose may vary depending on the relative effectiveness of the individual oligonucleotides. In general, it can be estimated based on the EC50 found to be effective in in vitro and in vivo animal models. Generally, the dose is 0.01 ⁇ g to 1 g / kg body weight, preferably 0.01 to 100 mg / kg body weight, more preferably 1 to 10 mg / kg body weight.
  • the administration frequency may be one or more times per day, one week, one month or one year, or once every 2 to 10 years, or by continuous infusion for several hours to several months. The number of repeated administrations can be estimated based on the measured drug concentration in the body fluid or tissue and the residence time. After successful treatment, it may be desirable for the subject to receive maintenance therapy to prevent recurrence of the condition.
  • the present invention relates to a head and neck cancer marker (hereinafter also referred to as “the head and neck cancer marker of the present invention”) in a body fluid sample, which is composed of a part of the microRNA of Table 1.
  • Head and neck cancer markers of the present invention are hsa-miR-1289, hsa-miR-876-5p, hsa-miR-1, hsa-miR-892b, hsa-miR-124, hsa-miR-124 *, hsa It consists of a microRNA selected from the group consisting of -miR-206, hsa-miR-193b *, hsa-miR-192 *, and hsa-miR-192.
  • the present invention provides hsa-miR-1289, hsa-miR-876-5p, hsa-miR-1, hsa-miR-892b, hsa-miR-124, hsa-miR-124 *, hsa in body fluid samples.
  • -Use of a microRNA selected from the group consisting of miR-206, hsa-miR-193b *, hsa-miR-192 *, and hsa-miR-192 as a head and neck cancer marker in a body fluid sample About the use of.
  • the “body fluid sample” includes a blood sample and / or a saliva sample.
  • the “blood sample” include whole blood, plasma, and serum.
  • the blood sample is preferably serum because it easily functions as a head and neck cancer marker.
  • the head and neck cancer marker of the present invention is an indicator of the presence or absence of head and neck cancer.
  • the present invention is a method for analyzing the presence or absence of head and neck cancer, comprising detecting microRNA in a body fluid sample and comparing the measured value with a reference value, MicroRNAs are hsa-miR-1289, hsa-miR-876-5p, hsa-miR-1, hsa-miR-892b, hsa-miR-124, hsa-miR-124 *, hsa-miR-206, hsa
  • the present invention relates to a method for analyzing the presence or absence of head and neck cancer, which is at least one selected from the group consisting of -miR-193b *, hsa-miR-192 *, and hsa-miR-192.
  • the measurement value includes a measurement value of the amount or concentration of the microRNA in the analysis target body fluid sample, and may be an absolute amount or a relative value.
  • the method for measuring the amount or concentration of microRNA is not particularly limited, and can be performed by, for example, a quantitative PCR method or a DNA microarray method.
  • a measurement value obtained by measuring the amount or concentration of each microRNA of a healthy subject and / or a head and neck cancer patient in the same manner as the analysis target can be used.
  • hsa-miR-1289, hsa-miR-876-5p, and hsa-miR-1 are not detected in body fluid samples of healthy individuals, A type of marker that is detected in a body fluid sample.
  • a type of marker that is detected in a body fluid sample for example, if 0 is set as the reference value, and the measured value of the analysis target body fluid sample is higher than the reference value, it can be determined that there is a possibility that head and neck cancer exists.
  • hsa-miR-124 * and hsa-miR-206 are not detected in a body fluid sample of a head and neck cancer patient, but are detected in a body fluid sample of a healthy person It is a marker.
  • a marker for this type of marker, for example, if the reference value is set to 0 or less than the detection limit, and the measurement value of the analysis target body fluid sample is the same as the reference value, head and neck cancer may be present. Can be judged.
  • hsa-miR-892b, hsa-miR-124, hsa-miR-193b *, sa-miR-192 *, and hsa-miR-192 are It is a type of marker that is detected in both patients but has a significant difference in its measurements.
  • hsa-miR-892b and hsa-miR-124 are markers whose measured values are increased in body fluid samples of head and neck cancer patients compared to healthy individuals.
  • This type of marker for example, sets a measurement value of a body fluid sample of a healthy person and a head and neck cancer patient as the reference value, and the measurement value of the analysis body fluid sample is higher than the measurement value of the healthy person. If it is close to or more than the measured value, it can be determined that head and neck cancer may be present.
  • the previous measured value of the individual to be measured is set as a reference value, and the measured value of the body fluid sample to be analyzed is higher than the reference value (for example, 1.2 times or more, preferably 1.3 times or more, more preferably Is 1.4 times or more), it can be determined that head and neck cancer may be present.
  • Hsa-miR-193b *, hsa-miR-192 *, and hsa-miR-192 increase in measured values in body fluid samples of healthy subjects compared to head and neck cancer patients.
  • This type of marker for example, sets a measurement value of a body fluid sample of a healthy person and a head and neck cancer patient as the reference value, and the measurement value of the analysis body fluid sample is higher than the measurement value of the healthy person. If it is close to or less than the measured value, it can be determined that head and neck cancer may be present.
  • the previous measured value of the individual to be measured is set as a reference value, and the measured value of the body fluid sample to be analyzed is lower than the reference value (for example, 0.8 times or less, preferably 0.7 times or less, more preferably Is 0.6 times or less), it can be determined that head and neck cancer may be present.
  • the present invention is also referred to as a head or neck cancer determination or diagnostic kit (hereinafter referred to as “the determination or diagnosis kit of the present invention”) that includes a reagent for detecting the head and neck cancer marker of the present invention. )
  • the determination or diagnosis kit of the present invention can employ, for example, a known microarray method or quantitative PCR method.
  • the microarray method is not particularly limited as long as the expression level of the microRNA that is the head and neck cancer marker of the present invention can be measured, but the RNA extracted from the body fluid sample is labeled with a label (preferably a fluorescent label). Then, the RNA is brought into contact with a microarray on which a polynucleotide consisting of a nucleic acid sequence complementary to the microRNA to be identified (preferably DNA) or a probe comprising a part thereof is immobilized, and then hybridization is performed. A method of washing the microarray and measuring the expression level of the microRNA remaining on the microarray can be exemplified.
  • the nucleotide type of the nucleic acid sequence is not particularly limited as long as it can specifically hybridize to the microRNA that is the head and neck cancer marker of the present invention, but is preferably DNA because of its high stability.
  • the length of a part of the polynucleotide is not particularly limited as long as it can specifically hybridize to the microRNA which is the head and neck cancer marker of the present invention, but a viewpoint of ensuring the stability of hybridization. Therefore, it is preferably 10 to 100 mer, more preferably 10 to 40 mer.
  • said polynucleotide or its one part can be obtained by chemically synthesizing using a well-known method in the said technical field.
  • the array for immobilizing the polynucleotide or a part thereof is not particularly limited, but a glass substrate, a silicon substrate, and the like can be preferably exemplified, and a glass substrate can be more suitably exemplified.
  • the method for immobilizing the above polynucleotide or a part thereof on the array is not particularly limited, and a known method can be used.
  • the determination or diagnosis kit of the present invention includes, in addition to the above-described microarray, for example, a reagent used for RNA labeling reaction, a reagent used for hybridization reaction, a reagent used for washing, a reagent used for RNA extraction from tissue, etc. Any element such as a reagent used in the microarray method may be further included.
  • the quantitative PCR method is a method using a primer set that can amplify the sequence of the microRNA that is the head and neck cancer marker of the present invention, and in particular, as long as the expression level of the microRNA can be measured.
  • a normal quantitative PCR method such as an agarose electrophoresis method, a SYBR green method, or a fluorescent probe method can be used, but the fluorescent probe method is most preferable in terms of accuracy and reliability of quantification.
  • the primer set in the quantitative PCR method means a combination of primers (polynucleotides) capable of amplifying the microRNA sequence that is the head and neck cancer marker of the present invention.
  • the primer is not particularly limited as long as the sequence of the present microRNA can be amplified, but a primer (forward primer) comprising a partial sequence on the 5 ′ side of the sequence of the microRNA that is the head and neck cancer marker of the present invention.
  • a primer set comprising a primer (reverse primer) composed of a sequence complementary to a partial sequence on the 3 ′ side of the sequence of the microRNA.
  • the fluorescent probe is not particularly limited as long as it contains a polynucleotide consisting of a nucleic acid sequence complementary to the sequence of the present microRNA or a part thereof.
  • the TaqMan (registered trademark) probe method using the FRET principle
  • fluorescent probes that can be used in the cycling probe method can be preferably exemplified, and in particular, fluorescent probes that can be used in the TaqMan (registered trademark) probe method (including those using the FRET principle) are more preferably exemplified. can do.
  • the primer set and the fluorescent probe can be obtained by chemical synthesis or the like using a method well known in the art based on the sequence information.
  • a method of quantitative PCR using the above primer set and fluorescent probe a method of using TaqMan (registered trademark) microRNA Assays (Applied Biosystems) according to the attached protocol is preferably exemplified. Can do.
  • the head and neck tumor determination kit of the present invention provided with a primer set and a fluorescent probe further includes an optional element such as a reagent used in a quantitative PCR reaction such as polymerase in addition to the primer set described above. May be.
  • the present invention provides a composition comprising microRNA or an expression system capable of expressing microRNA in a cell, wherein the microRNA comprises hsa-miR-1289, hsa-miR-892b, hsa -miR-661, hsa-miR-193b *, hsa-miR-671-5p, hsa-miR-124 *, hsa-miR-1908, hsa-miR-876-5p, hsa-miR-192 *, hsa- Consists of miR-124, hsa-miR-760, hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR-708 *, and hsa-miR-629 *
  • the present invention comprises hsa-miR-1289, hsa
  • the target cancer to suppress growth is a human cancer selected from the group consisting of lung cancer, pancreatic cancer, and prostate cancer.
  • the microRNA that can be included in the second composition of the invention is selected from the group consisting of hsa-miR-1289, hsa-miR-892b, and hsa-miR-629 *.
  • the second composition of the present invention is a microRNA selected from the group consisting of hsa-miR-1289, hsa-miR-892b, and hsa-miR-629 *, or a microRNA within a cell.
  • the present invention relates to a composition for suppressing the growth of human cancer cells selected from the group consisting of human lung cancer cells, human pancreatic cancer cells, and human prostate cancer cells, comprising an expression system capable of expressing RNA.
  • the present invention provides a pharmaceutical composition comprising microRNA or an expression system capable of expressing microRNA in a cell, wherein the microRNA comprises hsa-miR-1289, hsa-miR-892b, hsa-miR-661, hsa-miR-193b *, hsa-miR-671-5p, hsa-miR-124 *, hsa-miR-1908, hsa-miR-876-5p, hsa-miR-192 *, hsa -From miR-124, hsa-miR-760, hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR-708 *, and hsa-miR-629 *
  • the present invention comprises hsa-miR-1289, hsa-m
  • the human cancer to be treated or prevented is selected from the group consisting of lung cancer, pancreatic cancer, and prostate cancer.
  • the microRNA that can be included in the second pharmaceutical composition of the present invention is, in one embodiment, selected from the group consisting of hsa-miR-1289, hsa-miR-892b, and hsa-miR-629 *.
  • the second pharmaceutical composition of the present invention is a microRNA selected from the group consisting of hsa-miR-1289, hsa-miR-892b, and hsa-miR-629 * or intracellularly.
  • a pharmaceutical composition for treating or preventing human cancer selected from the group consisting of human lung cancer, human pancreatic cancer, and human prostate cancer, comprising an expression system capable of expressing microRNA.
  • the growth of human cancer cells can be suppressed. Therefore, this invention relates to the method of suppressing the growth of a human cancer cell including making the 2nd composition of this invention contact a human cancer cell in another aspect.
  • the second pharmaceutical composition of the present invention it is possible to treat or prevent human cancer. Therefore, this invention relates to the treatment or prevention method of a human cancer including administering the 2nd pharmaceutical composition of this invention to a subject as another aspect.
  • the cancer is selected from the group consisting of lung cancer, pancreatic cancer, and prostate cancer.
  • GFP stable expression strains GFP-SAS and GFP-ACCM which were isolated and established by introducing a green fluorescent protein (GFP) gene into human oral squamous cell carcinoma cell line SAS and human salivary gland carcinoma cell line ACCM, were used. These cell lines were cultured in Dulbecco's modified Eagle containing 10% fetal bovine serum (FBS, Biosource International), 100 ⁇ g / ml streptomycin, 100 U / ml penicillin, and 0.25 mg / ml amphotericin B (Invitrogen). Medium (DMEM) or RPMI1640 medium (manufactured by Sigma-Aldrich) was used as a growth culture solution, and this was performed at 37 ° C. in an incubator containing carbon dioxide gas at a rate of 5% in air.
  • FBS Dulbecco's modified Eagle containing 10% fetal bovine serum
  • FBS fetal bovine serum
  • streptomycin 100 U / ml penicillin
  • TS-miR detected only in head and neck cancer patients and detected only in healthy individuals by TS-miR expression analysis using serum derived from head and neck cancer patients and healthy individuals 2 types of TS-miR, and 5 types of TS-miR that show significant variation in the expression level between the two groups (2 types that increase in head and neck cancer patients and 3 types that increase in healthy subjects) ) was identified.
  • FIGS. 3 to 5 are examples of graphs showing the effects of TS-miR on proliferation of human cancer cells shown in Table 4, wherein FIG. 3 is a graph of hsa-miR-629 *, and FIG. 4 is a graph of hsa-miR. -1289 graph, FIG. 5 shows the hsa-miR-892b graph.
  • FIGS. 3 to 5 the growth of various human cancer cells shown in Table 4 was suppressed by hsa-miR-629 *, hsa-miR-1289 and hsa-miR-892b.
  • Hsa-miR-629 * and hsa-miR-1289 are human pancreatic cancer cells MIAPaca2, human lung squamous cell carcinoma cells EBC-1, human lung adenocarcinoma cells A549-57, and human prostate cancer cells PC-3, LNCaP shows a growth inhibition rate of 40% or more.
  • hsa-miR-629 * is 70% or more in human lung adenocarcinoma cell A549-57 and human prostate cancer cell PC-3. It showed a growth inhibition rate, and hsa-miR-1289 showed a growth inhibition rate of 70% or more in human lung squamous cell carcinoma cell EBC-1.
  • hsa-miR-892b is 30% or more in human pancreatic cancer cell MIAPaca2, human lung squamous cell carcinoma cell EBC-1, human lung adenocarcinoma cell A549-57, and human prostate cancer cell PC-3, LNCaP
  • the human pancreatic cancer cell MIAPaca2 and the human prostate cancer cell PC-3 exhibited a growth inhibition rate of 60% or more.
  • FIG. 6 is an example of a graph showing the expression of hsa-miR-1289 in human head and neck cancer tissue
  • FIG. 6A shows the expression of hsa-miR-1289 in oral squamous cell carcinoma tissue at each stage
  • FIG. 6B shows the expression of hsa-miR-1289 in salivary gland cancer tissue.
  • 6A and 6B TS-miR expression is shown as a relative value with RNU6B expression as an internal control.
  • hsa-miR-1289 tends to clearly decrease in oral squamous cell carcinoma tissue and salivary gland cancer tissue regardless of stage as compared with normal oral mucosal tissue.
  • hsa-miR-1289 replacement therapy may be effective in cases where hsa-miR-1289 expression is decreased.
  • FIG. 7 is an example of a graph showing the expression of hsa-miR-892b in human head and neck cancer tissue
  • FIG. 7A shows the expression of hsa-miR-892b in oral squamous cell carcinoma tissue at each stage
  • FIG. 7B shows hsa-miR-892b expression in salivary gland cancer tissue.
  • 7A and 7B the expression of TS-miR is shown as a relative value with the expression of RNU6B as an internal control.
  • FIGS. 7A and B there were cases where the expression of hsa-miR-892b was clearly reduced in oral squamous cell carcinoma tissue and salivary gland cancer tissue compared to normal oral mucosal tissue.
  • the expression of hsa-miR-892b was markedly decreased in all cases except one case regardless of the stage.
  • the present invention is useful in drug development related to head and neck cancer, medical field of head and neck cancer, research field of head and neck cancer, and the like.
  • SEQ ID NO: 1-18 Sequence of mature miRNA registered in miRBase (Release 17: April 2011)

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Abstract

L'invention concerne un miR suppresseur de tumeur (TS-miR) qui peut inhiber la croissance des cellules du cancer de la tête et du cou, et un procédé d'utilisation du TS-miR. La présente invention concerne une composition pour l'inhibition de la croissance des cellules du cancer de la tête et du cou humain qui contient un micro-ARN ou un système d'expression apte à exprimer un micro-ARN dans des cellules, le micro-ARN étant choisi dans le groupe consistant en hsa-miR-1289, hsa-miR-892b, hsa-miR-661, hsa-miR-193b*, hsa-miR-671-5p, hsa-miR-124*, hsa-miR-1908, hsa-miR-876-5p, hsa-miR-192*, hsa-miR-124, hsa-miR-760, hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR-708* et hsa-miR-629*.
PCT/JP2012/062825 2011-05-20 2012-05-18 Composition contenant un micro arn ou système d'expression associé Ceased WO2012161124A1 (fr)

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JPWO2015099122A1 (ja) * 2013-12-26 2017-03-23 学校法人東京医科大学 遺伝子発現制御のための人工ミミックｍｉＲＮＡおよびその用途
US10934542B2 (en) 2013-12-27 2021-03-02 Bonac Corporation Artificial match-type miRNA for controlling gene expression and use therefor
US11027023B2 (en) 2014-12-27 2021-06-08 Bonac Corporation Natural type miRNA for controlling gene expression, and use of same
US11142769B2 (en) 2015-03-27 2021-10-12 Bonac Corporation Single-stranded nucleic acid molecule having delivery function and gene expression regulating ability
WO2019117270A1 (fr) 2017-12-13 2019-06-20 国立大学法人広島大学 Procédé d'aide à la détection du cancer de la tête et du cou
JPWO2019117270A1 (ja) * 2017-12-13 2020-12-24 国立大学法人広島大学 頭頸部がんの検出を補助する方法
JP7390002B2 (ja) 2017-12-13 2023-12-01 国立大学法人広島大学 頭頸部がんの検出を補助する方法
JP2021513508A (ja) * 2018-02-12 2021-05-27 インテアールエヌエー テクノロジーズ ビー．ヴイ．ＩｎｔｅＲＮＡ Ｔｅｃｈｎｏｌｏｇｉｅｓ Ｂ．Ｖ． 抗がんマイクロｒｎａ及びその脂質製剤
EP3976058A4 (fr) * 2019-06-03 2023-07-19 Carmel-Haifa University Economic Corporation Ltd. Traitement du cancer de la tête et du cou humain

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