JPWO2012161124A1 - Composition comprising microRNA or expression system thereof - Google Patents

Abstract

TS-miR capable of inhibiting growth of head and neck cancer cells and use thereof. 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-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, a human head and neck selected from the group consisting of 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 inhibiting the growth of cancer cells.

Description

  The present invention relates to a composition comprising microRNA or an expression system thereof and a head and neck cancer marker.

  MicroRNA (microRNA / miRNA) is a small molecule RNA consisting of 18 to 25 bases, and inhibits translation into a protein by binding to a target mRNA. To date, about 1,100 types of human miRNAs have been registered in databases (for example, miRBase (http://www.miRbase.org/)), and expression or functional abnormalities of these miRNAs are involved in various diseases. (See Non-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. Among them, miR-19 is necessary and sufficient for cell carcinogenesis, and its target One of them was shown to be a tumor suppressor gene PTEN. Thus, miRNA having an oncogenic property targeting a tumor suppressor gene is called OncomiR.

  In addition, many miRNAs have decreased expression due to cancer, and let-7, miR-15a, miR-34a, miR-143, miR-145, etc. have been reported as such miRNAs. Among them, let-7 has been shown to correlate with decreased expression and prognosis in lung cancer, and its target is known to contain the oncogene Ras. MiRNA that exerts tumor suppressor-like functions targeting oncogenes is called Tumor suppressor miR (TS-miR).

  MiR-137 and miR-193a have been suggested as TS-miR for oral cancer (Non-patent Document 2).

Special table 2008-500837 gazette

Schickel R, et al. Oncogene 27: 5959-5974, 2008. Kenichi Ozaki et al., “Comprehensive analysis of microRNA aiming at establishment of a new diagnosis and treatment system for oral cancer”, Grant-in-Aid for Scientific Research (http://kaken.nii.ac.jp), Research Project Number: 18591997

  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. In addition, if such microRNA can be used as a cancer marker in serum or saliva, it will be useful for cancer diagnosis.

  Therefore, 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. -661, hsa-miR-193b *, hsa-miR-671-5p, hsa-miR-124 *, hsa-miR-1908, hsa-miR-876-5p, hsa-miR-192 *, hsa-miR- From the group consisting of 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 selected human head and neck cancer cells.

  In another aspect, 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 *, a method for suppressing the growth of human head and neck cancer cells.

  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.

  In still another aspect, 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 cells selected from the group.

  As yet another aspect of the present invention, there is provided 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-1, hsa-miR-708 *, and hsa-miR-629 *.

  According to the present invention, for example, it can contribute to the 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, and 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, and 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 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-miR-124, hsa- selected from the group consisting of miR-760, hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR-708 *, and hsa-miR-629 *, A pharmaceutical composition for the treatment or prevention of human head and neck cancer;
[3] A method for suppressing the growth of human head and neck cancer cells, comprising contacting the cancer cells with a composition comprising microRNA or an expression system capable of expressing microRNA in the cells, 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 A method for suppressing the growth of human head and neck cancer cells selected from the group consisting of hsa-miR-708 * and hsa-miR-629 *;
[4] A method for the treatment or prevention of human head and neck cancer, comprising administering to a subject 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 a method for treating or preventing human head and neck cancer selected from the group consisting of hsa-miR-629 *;
[5] 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, wherein the microRNA comprises hsa-miR-1289 , Hsa-miR-876-5p, hsa-miR-1, hsa-miR-892b, hsa-miR-124, hsa-miR-124 *, hsa-miR-206, hsa-miR-193b *, hsa-miR -192 * and at least one selected from the group consisting of hsa-miR-192, a method for analyzing the presence or absence of head and neck cancer;
[6] hsa-miR-1289, hsa-miR-876-5p, hsa-miR-1, hsa-miR-892b, hsa-miR-124, hsa-miR-124 *, hsa-miR- in body fluid samples Use of a microRNA selected from the group consisting of 206, hsa-miR-193b *, hsa-miR-192 *, and hsa-miR-192 as a head and neck cancer marker in a body fluid sample;
[7] 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 cancer cells;
[8] 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-miR-124, hsa- selected from the group consisting of miR-760, hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR-708 *, and hsa-miR-629 *, A pharmaceutical composition for the treatment or prevention of human cancer;
[9] The composition according to [7] or [8], wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, and prostate cancer;
[10] The composition according to any one of [7] to [9], wherein the microRNA is selected from the group consisting of hsa-miR-1289, hsa-miR-892b, and hsa-miR-629 *. ;
[11] 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 hsa-miR-629 *;
[12] A method for treating or preventing human cancer, comprising administering to a subject 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 a method for treating or preventing human cancer selected from the group consisting of hsa-miR-629 *;
[13] The method according to [11] or [12], wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, and prostate cancer;
[14] The method according to any one of [11] to [13], wherein the microRNA is selected from the group consisting of hsa-miR-1289, hsa-miR-892b, and hsa-miR-629 *;
About.

  As used herein, “microRNA / miRNA” refers to a kind of low-molecular non-coding RNA, and has a normal meaning used in the art. In the present specification, the microRNA referred to using an ID can refer to a database (for example, miRBase (http://www.miRbase.org/)). In this specification, microRNA refers to microRNA consisting of mature 18-25 bases unless otherwise specified.

  In the present specification, “a 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. Examples of the “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”. Examples of the “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.

  As used herein, “suppression of cell growth” includes inhibition of cell proliferation and cell death, and includes suppression of cell growth in vivo, in vitro, and ex vivo. As used herein, “cell” can include human cells and non-human cells, with human cells being preferred.

  As used herein, “head and neck cancer” refers to cancer that can be formed from the neck, excluding the brain and eyes. Generally, oral cancer, nasal sinus cancer, lip cancer, and pharyngeal cancer. Including laryngeal cancer, cervical tumor, ear cancer. In the present specification, 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. In the present specification, “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.

[Composition for inhibiting growth of human head and neck cancer cells]
In one aspect, the present invention is a composition for suppressing the growth of human head and neck cancer cells, 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- And a composition selected from the group consisting of 708 * and hsa-miR-629 * (hereinafter also referred to as “the composition of the present invention”). In one embodiment of the composition of the invention, 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 (miRBase, Release 17: April 2011) are shown in SEQ ID NOS: 1 to 17 in Table 1 below, respectively.

  When 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. When 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.

  When the 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. As the expression vector for linking the RNA precursor or a similar sequence thereof, a known microRNA expression vector may be used.

  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 in vivo, in vitro, or ex vivo.

[Pharmaceutical composition for treatment or prevention of human head and neck cancer]
In another aspect, 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 a pharmaceutical composition selected from the group consisting of -708 * and hsa-miR-629 * (hereinafter also referred to as “the pharmaceutical composition of the present invention”). As used herein, prevention of cancer includes prevention of recurrence of cancer.

  The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier. The pharmaceutical carrier is not particularly limited. For example, 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. As the dosage form of the pharmaceutical composition of the present invention, known dosage forms of microRNA and / or siRNA supplements can be used.

[Method of inhibiting growth of human head and neck cancer cells]
According to 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. Moreover, 10-25 nM is mentioned as a dosage amount to the cell of the expression system which can express microRNA in the said microRNA or a cell in the lipofection method and other introduction methods, for example.

[Method of treating and / or preventing human head and neck cancer]
According to the pharmaceutical 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 dosage is 0.01 μg to 1 g / kg body weight, preferably 0.01 to 100 mg / kg body weight, more preferably 1 to 10 mg / kg body weight. As the administration frequency, it can be administered once or more daily, weekly, monthly or yearly, 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.

[Head and neck cancer marker]
As another aspect, 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. Therefore, 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.

  Examples of the “body fluid sample” include a blood sample and / or a saliva sample. Examples of the “blood sample” include whole blood, plasma, and serum. However, 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. Therefore, in still another aspect, 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.

  Examples of the measurement value include measurement values of the amount or concentration of the microRNA in the body fluid sample to be analyzed, 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. As a reference, a measurement value obtained by measuring the amount or concentration of each microRNA of a healthy person and / or head and neck cancer patient in the same manner as the analysis target can be used.

  In the head and neck cancer marker of the present invention, 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. For this type of marker, 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.

  In the head and neck cancer marker of the present invention, 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. 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.

  In the head and neck cancer marker of the present invention, 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.

  Among the markers, hsa-miR-892b and hsa-miR-124 are markers whose measured values increase 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. Alternatively, if 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.

  The measured values of hsa-miR-193b *, hsa-miR-192 *, and hsa-miR-192 are increased in the 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. Alternatively, if 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.

[Determination or diagnosis kit for head and neck cancer]
As another aspect, 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. In addition, 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, and more preferably 10 to 40 mer. In addition, said polynucleotide or its one part can be obtained by chemically synthesizing using a well-known method in the said technical field.

  Although it does not restrict | limit especially as said array which fix | immobilizes said polynucleotide or its part, A glass substrate, a silicon substrate, etc. can be illustrated suitably, A glass substrate can be illustrated more suitably. 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. Although not limited, 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) that can amplify the sequence of microRNA 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. And 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) And 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. As a specific 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. In addition, 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.

[Composition for inhibiting the growth of human cancer cells]
In still another embodiment, 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 cells selected from the group (hereinafter also referred to as “the second composition of the present invention”).

  In one embodiment, the cancer to be inhibited from growth is a human cancer selected from the group consisting of lung cancer, pancreatic cancer, and prostate cancer. In other embodiments, 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 *. In yet another embodiment, 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.

[Pharmaceutical composition for treatment or prevention of human cancer]
In still another embodiment, 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 relates to a pharmaceutical composition (hereinafter, also referred to as “second pharmaceutical composition of the present invention”) for treatment or prevention of human cancer selected from the group consisting of

  In one embodiment, 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 *. In yet another embodiment, 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.

[Methods for inhibiting proliferation of human cancer cells, methods for treating and preventing human cancer cells]
According to the second composition of the present invention, 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. In addition, according to 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. In one embodiment of the methods of the present invention of these aspects, the cancer is selected from the group consisting of lung cancer, pancreatic cancer, and prostate cancer.

1. Identification of TS-miR that inhibits the growth of human head and neck cancer cells About the inhibition of the growth of human head and neck cancer cells by introducing microRNA into the following two types of human head and neck cancer cell lines using the synthetic miRNA library Exhaustive analysis was performed. Specifically, the following conditions were used.

[Cell lines used]
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 μg / 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.

[Comprehensive functional analysis using synthetic miRNA library]
In each well of a 96-well plastic plate (trade name: BD Falcon, manufactured by BD), 1000 μl of 10% FBS-containing DMEM medium containing 2 × 10 ³ human head and neck cancer cell lines GFP-SAS or GFP-ACCM 4 μmol each of human synthetic microRNA (trade name: Synthetic microRNA Library-Human v13.0, manufactured by Hokkaido System Science) and 40 μl of Opti-MEM (manufactured by Invitrogen) containing 0.4 μl of RNAiMAX (Invitrogen) Added. After culturing for 80 hours, the GFP fluorescence intensity of each well was measured with Wallac ARVO MX 1420 Multilabel Counter (manufactured by PerkinElmer), and then the number of cells was quantified using Cell Counting Kit-8 (manufactured by Dojindo).

  By comprehensive functional analysis using the synthetic miRNA library described above, 16 types exhibiting a growth inhibition rate of 70% or more in human oral squamous cell carcinoma cell GFP-SAS, and 50% or more in human salivary gland cancer cell GFP-ACCM Two types of TS-miR showing a growth inhibition rate were identified. The results are shown in FIG. Note that has-miR-193a-3p in FIG. 1 and Table 2 is a microRNA suggested in Non-Patent Document 2 to be TS-miR of head and neck cancer.

[Analysis of TS-miR expression level in human serum]
Serum 0.5 ml was collected from 10 untreated head and neck cancer patients and 10 healthy subjects, miRNA was extracted from each sample, and microarray analysis was performed on the expression level of TS-miR in Table 2 above. . The results are shown in Table 3 below. In Table 3 below, the numerical values indicate relative signal intensity.

  As shown in Table 3 above, three types of 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.

2. Confirmation of growth inhibition for various cancer cells by introduction of TS-miR Introducing hsa-miR-629 *, hsa-miR-1289 or hsa-miR-892b into 8 types of human cancer cell lines shown in Table 4 below Then, the growth inhibitory effect of each human cancer cell was examined. The cell lines are the same as those in 1. above except that the 8 cell lines shown in Table 4 below were used. A cell line was prepared in the same manner as [Used cell line]. In addition, the analysis of human cancer cell proliferation was performed in the above 1. except that the above three types of TS-miR and cell lines were used. [Analysis of proliferation of human cancer cells]. The results are shown in 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. As shown in 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. In particular, 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 In particular, the human pancreatic cancer cell MIAPaca2 and the human prostate cancer cell PC-3 exhibited a growth inhibition rate of 60% or more.

3. Analysis of hsa-miR-1289 expression in human head and neck cancer tissue Each cancer tissue was collected from untreated oral squamous cell carcinoma patients and salivary gland cancer patients, miRNA was extracted from each specimen, and hsa -Microarray analysis was performed on the expression level of miR-1289. Oral squamous cell carcinoma tissues were collected from patients with stage I, II, III, and IV oral squamous cell carcinomas (total of 11 patients), and salivary gland cancer tissues were collected from 5 salivary gland cancer patients. In addition, oral mucosal tissues were collected from healthy subjects and similarly microarray analysis was performed for the expression of hsa-miR-1289. The result is shown in FIG. FIG. 6 is an example of a graph showing the expression of hsa-miR-1289 in human head and neck cancer tissue, and 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.

  As shown in FIGS. 6A and B, the expression of 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. Was recognized. Therefore, it was suggested that hsa-miR-1289 replacement therapy may be effective in cases where hsa-miR-1289 expression is decreased.

4. Analysis of the expression of hsa-miR-892b in human head and neck cancer tissues Each cancer tissue was collected from untreated oral squamous cell carcinoma patients and salivary gland cancer patients, miRNA was extracted from each specimen, hsa Microarray analysis was performed for the expression of -miR-892b. Oral squamous cell carcinoma tissues were collected from patients with stage I, II, III, and IV oral squamous cell carcinomas (total of 11 patients), and salivary gland cancer tissues were collected from 5 salivary gland cancer patients. In addition, oral mucosal tissues were collected from healthy subjects, and microarray analysis was similarly performed for the expression of hsa-miR-892b. The result is shown in FIG. FIG. 7 is an example of a graph showing the expression of hsa-miR-892b in human head and neck cancer tissue, and 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.

  As shown in 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 mucosa tissue. In particular, in the oral squamous cell carcinoma tissue, the expression of hsa-miR-892b was markedly decreased in all cases except one case regardless of the stage. These results suggest that hsa-miR-892b replacement therapy may be effective in patients with decreased expression of hsa-miR-892b.

  The present invention is useful in pharmaceutical development relating to head and neck cancer, the medical field of head and neck cancer, the research field of head and neck cancer, and the like.

SEQ ID NO: 1-18: Mature miRNA sequence registered in miRBase (Release 17: April 2011)

Claims (14)

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-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, a human head and neck selected from the group consisting of hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR-708 *, and hsa-miR-629 * A composition for inhibiting the growth of cancer cells. 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-miR-124, hsa-miR-760 Human head and neck selected from the group consisting of hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR-708 *, and hsa-miR-629 * A pharmaceutical composition for the treatment or prevention of cancer. A method for inhibiting the growth of human head and neck cancer cells, comprising contacting the cancer cells with microRNA or a composition comprising an expression system capable of expressing microRNA in the cells, 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- A method for suppressing the growth of human head and neck cancer cells selected from the group consisting of miR-708 * and hsa-miR-629 *. A method of treating or preventing human head and neck cancer, comprising administering to a subject 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 a method for treating or preventing human head and neck cancer selected from the group consisting of hsa-miR-629 *. A method for analyzing the presence or absence of head and neck cancer,
Detecting microRNA in a body fluid sample and comparing the measured value to a reference value;
The microRNA is hsa-miR-1289, hsa-miR-876-5p, hsa-miR-1, hsa-miR-892b, hsa-miR-124, hsa-miR-124 *, hsa-miR-206, A method for analyzing the presence or absence of head and neck cancer, which is at least one selected from the group consisting of hsa-miR-193b *, hsa-miR-192 *, and hsa-miR-192. Hsa-miR-1289, hsa-miR-876-5p, hsa-miR-1, hsa-miR-892b, hsa-miR-124, hsa-miR-124 *, hsa-miR-206, hsa in body fluid samples -Use of a microRNA selected from the group consisting of miR-193b *, hsa-miR-192 *, and hsa-miR-192 as a head and neck cancer marker in a body fluid sample. 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-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, A human cancer cell selected from the group consisting of hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR-708 *, and hsa-miR-629 * A composition for inhibiting the growth of potato. 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-miR-124, hsa-miR-760 Selected from the group consisting of: hsa-miR-192, hsa-miR-206, hsa-miR-629, hsa-miR-1, hsa-miR-708 *, and hsa-miR-629 * A pharmaceutical composition for the treatment or prevention of cancer. The composition according to claim 7 or 8, wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, and prostate cancer. The composition according to any one of claims 7 to 9, wherein the microRNA is selected from the group consisting of hsa-miR-1289, hsa-miR-892b, and hsa-miR-629 *. A method for inhibiting 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, 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- A method for inhibiting the growth of human cancer cells, selected from the group consisting of 708 * and hsa-miR-629 *. A method of treating or preventing human cancer comprising administering to a subject a composition comprising microRNA or an expression system capable of expressing microRNA in a cell, wherein said 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 A method for treating or preventing human cancer selected from the group consisting of hsa-miR-629 *. The method according to claim 11 or 12, wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, and prostate cancer. The method according to any of claims 11 to 13, wherein the microRNA is selected from the group consisting of hsa-miR-1289, hsa-miR-892b, and hsa-miR-629 *.

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