JP5850702B2 - Differentiation regulator of mesenchymal cells, medicament using the same, and screening method for substances having differentiation regulating action on mesenchymal cells - Google Patents

Description

  The present invention relates to a differentiation regulator (differentiation promoter / differentiation inhibitor) for mesenchymal cells, a drug using the same, and a method for screening a substance having a differentiation regulating action on mesenchymal cells.

  Recently, various proteins that regulate the differentiation of osteoblasts, a type of mesenchymal cell, have been identified, but they act on various tissues such as bone morphogenetic protein (BMP). Or a transcriptional regulator that acts directly on DNA. For example, BMP has a strong osteoinductive effect, but also ossifies soft tissues such as muscles, making it difficult to use as a medicine. Also. In addition to cbfa1, homeobox genes such as Msx and Dlx are known as transcriptional regulatory factors that regulate osteoblast differentiation. Among these, Dlx5 is known to be specifically expressed in bone tissue during the development of vertebrate individuals, and the expression level of Dlx5 gene increases as osteoblasts mature, and Dlx5 protein is expressed in osteoblasts. It has been reported that the phenotypic traits of the gene are controlled at the gene level. Furthermore, it has been reported that the Dlx5 gene is specifically induced in osteoblasts by stimulation of BMP, and that differentiation is remarkably enhanced in osteoblasts overexpressing this Dlx5.

  Here, conventionally, the possibility that miRNA (microRNA, microRNA) is involved in osteoblast differentiation has been reported. miRNA is a non-coding RNA of about 20 to 25 bases that is endogenous to cells. miRNA is first transcribed from a miRNA gene on genomic DNA as a primary transcript (pri-miRNA) having a length of about several hundred to several thousand bases. Then, it is processed to become a pre-miRNA having a hairpin structure of about 60 to 70 bases. Thereafter, the miRNA moves from the nucleus into the cytoplasm and is further processed to become a mature miRNA composed of a dimer (guide strand and passenger strand) of about 20 to 25 bases. In mature miRNA, the guide strand (antisense strand) of these forms a complex with a protein called RISC (RNA-Induced Silencing Complex) and acts on the mRNA of the target gene, thereby inhibiting the translation of the target gene. It is known to do.

  Specifically, for example, in Patent Document 1, miR-125b suppresses differentiation of mesenchymal cells, and the function inhibitor of miR-125b promotes differentiation of mesenchymal cells. It is disclosed that it can be used as a medicine for treating and preventing diseases such as osteoporosis.

  In addition, in the Patent Document 2, the present inventors also inhibited miRNA-141 and miRNA-200a, which are other miRNAs, from inhibiting the differentiation of mesenchymal cells through binding to the 3 ′ untranslated region of the Dlx5 gene. MiRNA-208 targets the Ets1 gene and suppresses differentiation, and these miRNA inhibitors promote differentiation of mesenchymal cells to prevent / treat diseases such as osteoporosis It is disclosed that it can be used as a medicine.

JP 2008-184450 A International Publication No. 2010/058824 Pamphlet

  As described above, several techniques for regulating the differentiation of mesenchymal cells using miRNA have been proposed so far, but those that are decisively effective as preventive / therapeutic agents for various diseases remain. The current situation is that it has not been developed. In addition, from the viewpoint of enhancing the lineup of preventive and therapeutic agents according to the type of disease and its severity, many differentiation regulators, drugs using them, and substances that can be candidates for such drugs There is great significance in developing a screening method.

  Therefore, an object of the present invention is to provide a novel agent for regulating the differentiation of mesenchymal cells, a drug using the same, and a screening method for substances that can be candidates for such drugs.

  The present inventors have conducted intensive studies in view of the above problems. As a result, it was found that miRNA-294 has an action of promoting the differentiation of mesenchymal cells, and the present invention has been completed.

  That is, according to one aspect of the present invention, miRNA-294, its precursor, and one or more selected from the group consisting of DNA constructs encoding them are contained as active ingredients, and An agent for regulating differentiation of mesenchymal cells that promotes differentiation is provided.

  Moreover, according to the other form of this invention, the differentiation regulator of a mesenchymal cell which contains the inhibitor of miRNA-294 as an active ingredient, and suppresses the differentiation of a mesenchymal cell is provided.

  In the differentiation regulator described above, the mesenchymal cell is preferably an osteoblast.

  According to still another aspect of the present invention, a medicament for preventing or treating a disease caused by abnormal differentiation of mesenchymal cells is also provided. The medicament contains the differentiation regulator described above.

  In the medicament described above, the disease is preferably a disease caused by a disorder of mesenchymal cell differentiation.

  In the above-described medicament, preferably, the mesenchymal cell is an osteoblast, and the disease is one or more selected from the group consisting of osteoporosis, osteogenesis dysfunction, and growth inhibition in the developmental stage.

  According to still another aspect of the present invention, there is also provided a method for screening a substance having an action of regulating differentiation into mesenchymal cells. The method comprises a step of supplying a test substance to a mesenchymal stem cell or mesenchymal progenitor cell or non-human animal expressing miRNA-294, and the mesenchymal stem cell or mesenchymal progenitor cell or non-human animal. The expression or function of miRNA-294 in a human animal is compared with the expression or function of miRNA-294 when the test substance is not supplied, and based on the comparison result, differentiation of the test substance into mesenchymal cells is promoted And the step of evaluating the action of inhibiting the differentiation.

  According to the present invention, there are provided a novel agent for regulating differentiation of mesenchymal cells, a drug using the same, and a screening method for substances that can be candidates for such drugs.

It is a schematic diagram of mouse pre-miRNA-294. In Example 1, it is a photograph which shows the result of having investigated the influence on BMP-2 induction osteoblast differentiation in miRNA-294 introduction | transduction MC3T3-E1 cell. In Example 2, it is a photograph which shows the result of having investigated the influence on the differentiation by the presence or absence of BMP-2 induction | guidance | derivation of miRNA-294 introduction | transduction MC3T3-E1 cell. In Example 3, it is a photograph which shows the result of having investigated the influence on differentiation by the presence or absence of BMP-2 induction | guidance | derivation of miRNA-294 introduction | transduction human osteoblast cell line HFO-SV-40.

  The disclosure herein relates to miRNA-294 and uses thereof. The disclosure of the present specification is based on the discovery that miRNA-294 has an action of promoting differentiation of mesenchymal cells. That is, according to the disclosure of the present specification, miRNA-294, a precursor thereof, and a DNA construct encoding them can be used as a differentiation regulator that promotes differentiation of mesenchymal cells. In addition, miRNA-294 inhibitors can be used as differentiation regulators that suppress the differentiation of mesenchymal cells. Furthermore, according to the disclosure of the present specification, a medicine using such a mesenchymal cell differentiation regulator and a method for screening a substance having a differentiation regulating action on mesenchymal cells are also provided. Hereinafter, embodiments of the present disclosure will be described in more detail.

  In the following description, in miRNA, pri-miRNA, and pre-miRNA, an antisense strand (or an RNA region containing the antisense strand) against the mRNA that is finally paired with mRNA of a target gene is used. Expressed as “guide chain”. On the other hand, a sense strand (or an RNA region containing the sense strand) with respect to the antisense strand is expressed as a “passenger strand”.

<< Mesenchymal cell differentiation regulator >>
The differentiation regulator disclosed in the present specification regulates differentiation into mesenchymal cells. Therefore, the application target of the differentiation regulator is a cell having the ability to differentiate into mesenchymal cells. There is no restriction | limiting in particular as a kind of cell which has the differentiation ability to a mesenchymal cell, According to the objective, it can select suitably, For example, a mesenchymal stem cell and a mesenchymal progenitor cell are mentioned. The method for obtaining mesenchymal stem cells and mesenchymal progenitor cells is not particularly limited and can be appropriately selected according to the purpose.For example, it can be obtained by isolation from the bone marrow of an individual, It can also be obtained from various organizations as already mesenchymal stem cells that have been cloned. Such already cloned mesenchymal stem cells are not particularly limited and may be appropriately selected depending on the purpose. For example, examples of mouse-derived mesenchymal stem cells include MC3T3-E1, which is a skull-derived progenitor osteoblast, ST2 cells, which are bone marrow-derived preadipocytes, and NRG cells (each of which is a RIKEN BioResource Center). (Available from (BRC)). In addition, even cells other than mesenchymal stem cells can be used as an application target of a differentiation regulator as long as they are cells capable of differentiating into mesenchymal cells.

  Moreover, there is no restriction | limiting in particular also about the kind of mesenchymal cell, According to the objective, it can select suitably, For example, an osteoblast, an adipocyte, a muscle cell, a chondrocyte etc. are mentioned. Of these, osteoblasts are particularly preferable.

  The differentiation regulator for mesenchymal cells according to one aspect of the present invention contains, as an active ingredient, one or more selected from the group consisting of miRNA-294, a precursor thereof, and a DNA construct encoding them. . In this case, the differentiation regulator has an action of promoting the differentiation of mesenchymal cells.

(MiRNA-294)
The mature sequence (guide strand sequence) of mouse miRNA-294 that the above-mentioned differentiation regulator can contain as an active ingredient is as shown below.

  The presence of human miRNA-294 has been suggested in literature (Gurman Singh Pall et al., Nucleic Acids Research, 2007, Vol. 35, No. 8, e60). Although the base sequence of human miRNA-294 is not described in this document, since the mouse miRNA-294 shows the mesenchymal cell differentiation-inducing action in Examples described later, the base sequence of human miRNA-294 Is considered to be almost identical to that of mouse miRNA-294.

  Such miRNA-294 is based on its base sequence and is isolated from natural products, chemically synthesized, or genetically engineered in vivo or in vitro using a conventionally known method. Can be produced. The miRNA-294 may be an analog synthesized so as to mimic an endogenous mature miRNA. Such analogs are available from Ambion, for example. Such miRNA-294 may be single-stranded or double-stranded with a complementary strand.

(Precursor of miRNA-294)
Examples of the precursor of miRNA-294 include pri-miRNA and pre-miRNA. Specific examples thereof include pre-miRNA-294 shown below for mice. Here, FIG. 1 shows a schematic diagram of mouse pre-miRNA-294. In addition, it is considered that the base sequences of human pri-miRNA-294 and pre-miRNA-294 are highly conserved with respect to those of mice.

  Such miRNA-294 precursors can also be produced by isolation from natural products, chemical synthesis, or in vivo or in vitro genetic engineering using conventionally known methods. Can do. Furthermore, as the precursor of miRNA-294, an analog of the precursor of miRNA-294 synthesized so as to mimic the precursor of endogenous miRNA-294 can also be used. The precursor of miRNA-294 may be single-stranded or double-stranded.

(DNA constructs such as miRNA)
The above-described DNA construct capable of transcribing miRNA-294 or its precursor-encoding DNA is a DNA construct encoding such miRNA-294 or its precursor in a transcribable manner in vivo or in vitro. For example, a construct capable of transcribing miRNA-294 or a precursor thereof in vitro includes a vector in which a DNA encoding miRNA-294 or a precursor thereof is linked under the control of a viral promoter. Examples of the construct capable of transcribing miRNA-294 or a precursor thereof in vivo include a vector in which a DNA encoding miRNA-294 or a precursor thereof is linked under the control of a promoter effective in mammalian cells. Such vectors for in vivo or in vitro transcription are commercially available, and such vectors can be easily constructed based on the base sequence of miRNA-294 or its precursor according to the protocol.

(Inhibitor of miRNA-294)
The inhibitor of miRNA-294 may be any substance that reduces the function of miRNA-294, and its specific form is not particularly limited. Examples of such an inhibitor include a first RNA strand (so-called “antisense miRNA”) which is a complementary strand of at least a part of the guide strand of miRNA-294 or a precursor thereof. The complementary strand in this first RNA strand does not necessarily have to be the entire complementary strand of miRNA-294 or its precursor, and may be a partial complementary strand as long as it has an action of reducing its function. Good. Moreover, it is not necessary to be a complete complementary strand, and an incomplete complementary strand may be used as long as it has an action of reducing the functions of miRNA-294 and its precursor.

  The first RNA strand is preferably one that has increased complementarity compared to the passenger strand for the guide strand in endogenous miRNA-294 or its precursor. That is, in the case of endogenous miRNA-294, when there is a mismatch between the guide strand and the passenger strand, the number of such mismatches is preferably reduced in the first RNA strand. How much the mismatch should be reduced is not particularly limited, but as long as the first RNA strand functions as an inhibitor, it is not necessary to eliminate all mismatches, and only a part of the mismatches are eliminated It may be. The position of the mismatch (position in the guide strand of endogenous miRNA-294) that is eliminated in the complementary strand as an inhibitor is not particularly limited, but the inhibitor that has been eliminated in the mismatch at the 3 ′ end side is an inhibitor. preferable.

  The inhibitor of miRNA-294 may comprise a first RNA strand and a second RNA strand that is a guide strand of miRNA-294 or a precursor thereof. Even in such a double-stranded state, it is effective as an inhibitor.

  The first RNA strand and the second RNA strand described above can be produced by chemical synthesis or genetic engineering, and can also be obtained commercially from Ambion or the like. The first RNA strand and the second RNA strand may be appropriately modified in their bases, sugars, etc. from the viewpoint of ensuring stability in the cell or the like. For example, PNA (for example) is a point which can form a duplex more strongly with miRNA-294 or its precursor, and can inhibit (reduce) the function of miRNA-294 or its precursor more effectively. Peptide nucleic acid and LNA (Locked Nucleic Acid) are preferred. As a complementary strand having a modification such as LNA (Locked Nucleic Acid), for example, miRCURY Knockdown Probes (Exiqon) can be used (for example, Biomedecine & Pharmacotherapy, Volume 60, Issue 9, November 2006, Pages). 633-638). In addition, the complementary strand can be present more stably by preventing degradation in the cell, and preferably has a modification such as 2′-O-methyl (for example, Nucleic Acids Research, 2003, Vol. 31, No. 11, 2705-2716).

  The inhibitor may be a DNA construct encoding the first RNA strand against miRNA-294 or a precursor thereof, or a DNA construct that holds the DNA encoding the first RNA strand and the second RNA strand in a transcribable manner. May be. Such a DNA construct can be easily obtained by using a vector for in vivo or in vitro transcription.

  The active ingredient of the differentiation regulator disclosed in the present specification is selected according to the purpose. That is, when a differentiation regulator is used for the purpose of promoting the differentiation of mesenchymal cells, one or more selected from the group consisting of miRNA-294, a precursor thereof, and a DNA construct encoding them are used. It can be contained as an active ingredient. Moreover, when using a differentiation regulator for the purpose of suppressing the differentiation of mesenchymal cells, the above-described inhibitor of miRNA-294 can be contained as an active ingredient. As the active ingredient, one active ingredient contributing to the same purpose may be used alone, or two or more active ingredients may be used in combination. Moreover, there is no restriction | limiting in particular about content of the active ingredient in the differentiation regulator of a mesenchymal cell, According to the objective, it can set suitably.

  The differentiation regulator disclosed in the present specification may contain only the above-mentioned active ingredient, but may contain other ingredients as necessary. There is no restriction | limiting in particular as another component, According to the objective, it can select suitably. Examples thereof include water and various buffer solutions for diluting the above active ingredient to a desired concentration. In addition, as the other component, a reagent for introduction for introducing an active ingredient into a cell (target cell; for example, mesenchymal stem cell such as osteoblast) that wants to suppress or promote differentiation into mesenchymal cells And so on. The introduction reagent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include Lipofectamine 2000 (Invitrogen).

  The differentiation regulator disclosed in the present specification can promote or suppress differentiation into mesenchymal cells such as mesenchymal stem cells or mesenchymal progenitor cells. Therefore, the differentiation regulator can be used as a medicament for preventing or treating a disease associated with differentiation into mesenchymal cells. Moreover, it can be preferably used for evaluation and screening of research reagents and drugs related to differentiation into mesenchymal cells.

≪Pharmaceuticals for preventing or treating diseases caused by abnormal differentiation of mesenchymal cells≫
The medicament disclosed in the present specification is a medicament for preventing or treating a disease caused by abnormal differentiation of mesenchymal cells, and contains the above-described differentiation regulator as an active ingredient. At this time, the differentiation regulator is selected from the various differentiation regulators already disclosed in the present specification according to the type of disease targeted by the medicine. When the medicament disclosed in the present specification is a disease caused by impaired differentiation of mesenchymal cells, one or two selected from the group consisting of miRNA-294, a precursor thereof, and a DNA construct encoding them The above can be used as an active ingredient of a medicine. On the other hand, when the medicament disclosed in the present specification is a disease caused by excessive differentiation of mesenchymal cells, the aforementioned miRNA-294 inhibitor can be used as an active ingredient of the medicament.

  There is no restriction | limiting in particular about content of the differentiation regulator in the pharmaceutical disclosed by this specification, According to the objective, it can select suitably. Moreover, the medicament disclosed in the present specification may be composed of only active ingredients, but may contain other ingredients as necessary. There is no restriction | limiting in particular as another component, According to the objective, it can select suitably. Typically, a pharmaceutically acceptable carrier and the like can be mentioned. There is no restriction | limiting in particular as said carrier, For example, it can select suitably according to a pharmaceutical dosage form etc. Moreover, there is no restriction | limiting in particular as content of the other component in a pharmaceutical, According to the objective, it can select suitably.

  The medicament disclosed in the present specification can take various conventionally known preparation forms. It can be appropriately selected according to the desired administration method as will be described later. For example, oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), oral liquid preparations (internal solutions, syrups, elixirs) Etc.), injections (solutions, suspensions, solid agents for dissolution at the time of use), ointments, patches, gels, creams, powders for external use, sprays, inhaled powders and the like. Additives that are appropriately required in these various preparations are well known to those skilled in the art, and those skilled in the art can produce various preparations according to the purpose.

  The medicament disclosed in the present specification preferably takes a dosage form suitable for transfection of a medicament into a cell with respect to an administration subject or a tissue containing cells collected from the administration subject. For example, it can take the form of an injection preparation (subcutaneous injection, intravenous injection, intramuscular injection, bone marrow injection, dental pulp injection, intraperitoneal injection, etc.) according to the application site. Injections include, for example, active ingredients, pH regulators, buffers, stabilizers, isotonic agents, local anesthetics, etc., and are used for subcutaneous, intramuscular, intravenous, etc. by conventional methods. An injection can be produced. Examples of the pH adjuster and buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

  The administration method of the medicine disclosed in the present specification is not particularly limited, but is appropriately selected according to the dosage form of the medicine. As described above, the medicament disclosed in the present specification can be administered, for example, by introducing an active ingredient into cells or tissues in a patient. The cells and tissues are preferably locations where mesenchymal stem cells and mesenchymal progenitor cells are present. For example, in a disease caused by abnormal differentiation into osteoblasts, it is preferable to administer a site where stem cells or progenitor cells to osteoblasts are present, typically bone marrow.

  In addition, it can be administered by introducing it into mesenchymal stem cells or mesenchymal progenitor cells collected from a patient or the like or a tissue containing these cells and transplanting the cells into the patient. The introduction method is not particularly limited, and for example, a conventionally known method such as lipofection or electroporation can be appropriately selected and used. There is no particular limitation on the method for transplanting the cells transfected in vitro to the patient, and for example, conventionally known cell transplantation methods can be appropriately used. For example, systemic administration or local administration may be performed by embedding in liposomes or other nanoparticles outside the body.

  In addition, there is no restriction | limiting in particular as dosage of the medicine disclosed by this specification, According to the age of a patient who is an administration object, a body weight, the grade of a desired effect, etc., it can select suitably. The number of administrations is not particularly limited, and can be appropriately selected according to the age, weight, desired degree of effect, etc. of the patient to be administered. Furthermore, there is no restriction | limiting in particular as the administration time, According to the objective, it can select suitably, For example, it administers prophylactically with respect to the disease resulting from the differentiation abnormality (suppression or excess) of a mesenchymal cell. May be administered therapeutically. The mammal to be administered with the medicament disclosed in the present specification is not particularly limited and can be appropriately selected according to the purpose. For example, in addition to humans, mice, rats, cows, pigs, monkeys, dogs And non-human mammals such as cats.

  The disease targeted for prevention or treatment by the medicament disclosed in the present specification is a disease caused by abnormal differentiation (suppression or excess) of mesenchymal cells. Examples of diseases caused by suppression of differentiation (insufficient differentiation) of mesenchymal cells include osteoporosis (postmenopausal osteoporosis, senile osteoporosis, osteoporosis due to steroid treatment, diabetic osteoporosis) caused by suppression of differentiation or insufficient differentiation of osteoblasts. Bone dysplasia, growth inhibition during development, and the like. Examples of the diseases caused by excessive differentiation of mesenchymal cells include osteoblast type osteosarcoma caused by excessive differentiation of osteoblasts.

≪Method for evaluating differentiation regulator and screening method for substances having differentiation regulating action on mesenchymal cells≫
According to the disclosure of the present specification, a method for evaluating a differentiation regulator of mesenchymal cells is also provided. In the evaluation method, an increase or decrease in the expression and / or function of miRNA-294 can be used as an index. According to this evaluation method, a differentiation regulator capable of regulating (promoting or inhibiting) the differentiation of mesenchymal cells can be obtained.

  The evaluation method typically includes a step of supplying a test substance to a mesenchymal stem cell or a mesenchymal progenitor cell or a non-human animal expressing miRNA-294 (a supply step), and the mesenchyme The expression or function of miRNA-294 in a stem cell or mesenchymal progenitor cell or a non-human animal is compared with the expression or function of miRNA-294 when the test substance is not supplied, and the test is performed based on the comparison result. And a step (evaluation step) for evaluating differentiation promoting action or differentiation inhibiting action on mesenchymal cells of the substance. The evaluation method may include a step of selecting a test substance that promotes or suppresses differentiation of mesenchymal stem cells after the above-described “evaluating step”. In other words, it can be said that the evaluation method is a screening method for a substance having an action of regulating differentiation into mesenchymal cells.

  Examples of the test substance include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. It may be a well-known one.

  As described above, a conventionally known transfection method used when administering the differentiation regulator or medicine disclosed in the present specification to cells or patients can be adopted for the above-described supplying step.

  In the evaluation step described above, the expression or function of miRNA-294 in a mesenchymal stem cell or mesenchymal progenitor cell or a non-human animal is compared with the expression or function of miRNA-294 when the test substance is not supplied. Based on the comparison result, the differentiation promoting action or differentiation inhibiting action on mesenchymal cells of the test substance is evaluated. There is no restriction | limiting in particular about the specific method of evaluation, According to the objective, it can select suitably. For example, the expression level of endogenous miRNA-294 in the presence (at the time of supply) and absence (at the time of non-supply) of the test substance can be measured and used as an evaluation index for expression. In addition, when mesenchymal stem cells or mesenchymal progenitor cells for osteoblasts are used as cells, differentiation indexes to various osteoblasts such as alkaline phosphatase activity can also be used as evaluation indexes.

  In the evaluation step, for example, when the expression level of miRNA-294 or the like increases in the presence of the test substance (during supply) compared to the absence of the test substance (during supply), the test substance is endogenous. It can be evaluated that the action of miRNA-294 is increased. That is, the test substance can be evaluated as a substance having an action of promoting differentiation into mesenchymal cells. On the other hand, when the expression level of miRNA-294 or the like decreases in the presence of the test substance, it can be evaluated that the test substance decreases the action of endogenous miRNA-294. That is, the test substance can be evaluated as a substance having an action of suppressing differentiation into mesenchymal cells.

  In addition, any of the above supply steps or evaluation steps, or between these steps, may be appropriately subjected to a treatment for inducing differentiation into mesenchymal cells of mesenchymal stem cells or mesenchymal progenitor cells. Good. As the differentiation induction process, for example, BMP-2 or the like can be used for the induction process to osteoblasts. By evaluating the action of the test substance under such differentiation induction treatment, it is possible to more clearly detect the differentiation inhibiting ability and differentiation promoting ability of the test substance.

  Moreover, as described above, the evaluation method (screening method) according to the present invention includes a step (selection step) of selecting a test substance that promotes or suppresses differentiation of mesenchymal stem cells after the above-described evaluation step. Can do. The selection step is a step of selecting a test substance evaluated as a substance having an action of promoting or suppressing differentiation into mesenchymal cells as a regulator of differentiation into mesenchymal cells. The selected test substance is useful as, or as a candidate for, the active ingredient of the differentiation regulators and medicaments disclosed herein.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

[Example 1: Effect on BMP-2-induced osteoblast differentiation in miRNA-294-introduced MC3T3-E1 cells]
The effect of miRNA-294 introduction on the differentiation of BMC-2 induced mouse MC3T3-E1 cells (skull-derived progenitor osteoblasts) into osteoblasts was examined by the following procedure. Mouse MC3T3-E1 cells are one type of mesenchymal stem cells.

  Specifically, miRNA-294 synthesized to mimic mouse endogenous miRNA molecules, its antisense inhibitor Anti-miRNA-294, and miRNA-Negative Control (all from Applied Biosciences) ) Were introduced into MC3T3-E1 cells to a final concentration of 40 nM by the lipofection method using Lipofectamime (registered trademark) RNAiMAX (manufactured by Invitrogen).

Subsequently, each of the introduced cells described above and a control cell into which nothing has been introduced (with or without BMP-2 added) were cultured in an OPTI-MEM serum-free medium (Invitrogen) for 18 hours, and then MEM-α ( 10% FBS, 100 U / ml penicillin, containing 100 μg / ml streptomycin), and cultured for 72 hours after the medium was changed. Thereafter, cells adjusted to 1 × 10 ⁵ cells / ml are seeded in a 96-well multiplate at 500 μl / well, and BMP-2 (Becton Dickinson) is added to a final concentration of 200 ng / ml 3 hours after seeding. In addition, differentiation was induced and cultured for 72 hours.

  Thereafter, the cells were washed twice with PBS (−), fixed with ethanol, and stained with alkaline phosphatase (ALP), a differentiation index, using TRACP & ALP double-staining Kit (manufactured by Takara Bio Inc.). The degree of cell differentiation was evaluated. The results are shown in FIG.

  As shown in FIG. 2, when BMP-2 is added, differentiation of MC3T3-E1 cells into osteoblasts is induced, but when miRNA-294 is introduced, bones of MC3T3-E1 cells induced by BMP-2 are induced. It was confirmed that differentiation into blast cells was further enhanced. On the other hand, when anti-miRNA-294, an antisense inhibitor of miRNA-294, was introduced, it was confirmed that differentiation of MC3T3-E1 cells induced by BMP-2 into osteoblasts was attenuated. Introducing miRNA-Negative Control did not affect the differentiation of MC3T3-E1 cells induced by BMP-2 into osteoblasts.

  From the above, it was shown that miRNA-294 has an action of promoting the differentiation of mesenchymal stem cells into osteoblasts. It was also shown that Anti-miRNA-294 has an action of suppressing the differentiation of mesenchymal stem cells into osteoblasts.

[Example 2: Effect of differentiation on presence or absence of BMP-2 induction in miRNA-294-introduced MC3T3-E1 cells]
Except that BMP-2 was not added, the effect of miRNA-294 introduction on the differentiation of mouse MC3T3-E1 cells into osteoblasts was examined by the same method as in Example 1 described above. The results are shown in FIG. 3 in comparison with the case where BMP-2 was added. In FIG. 3, “BMP-2 (−)” indicates the result of the group to which BMP-2 was not added, and “BMP-2 (+)” indicates the result of the group to which BMP-2 was added.

  As shown in “BMP-2 (+)” in FIG. 3, differentiation of MC3T3-E1 cells is induced by the addition of BMP-2, and the differentiation potential of MC3T3-E1 cells introduced with miRNA-294 is further enhanced. (Confirmation of Example 1 described above). On the other hand, as shown in “BMP-2 (−)” of FIG. 3, MC3T3-E1 cells into which miRNA-294 has been introduced, without adding BMP-2 (without inducing differentiation), Its differentiation ability increased.

  From the above, it was suggested that miRNA-294 exhibits differentiation promoting action even when used alone and introduced into cells.

[Example 3: Effect of differentiation on presence or absence of BMP-2 induction in miRNA-294-introduced human osteoblast cell line]
An experiment similar to Example 2 described above was performed except that HFO-SV-40 cells, which are human osteoblast cell lines, were used instead of MC3T3-E1 cells. The results are shown in FIG. In FIG. 4, “BMP-2 (−)” indicates the result of the group to which BMP-2 was not added, and “BMP-2 (+)” indicates the result of the group to which BMP-2 was added.

  As shown in FIG. 4, miRNA-294 also showed an action of promoting osteoblast differentiation, although the action was weaker than that of the same experiment using MC3T3-E1 cells (Example 2, FIG. 3).

  From the above, it was suggested that miRNA-294 shows the mesenchymal cell differentiation promoting effect even across species.

[SEQ ID NO: 1]
It is the RNA sequence of the mature sequence of mouse miRNA-294 (guide strand sequence).
[SEQ ID NO: 2]
It is the RNA sequence of mouse pre-miRNA-294.

Claims (4)

miRNA-294, contain one or more members selected from the group consisting of DNA constructs encoding the precursor and these as active ingredients, promotes the differentiation of osteoblasts, osteoblast differentiation-regulating agent . Antisense inhibitors of miRNA-294 contains as an active ingredient, inhibits the differentiation of osteoblasts, differentiation-regulating agent of osteoblast. A pharmaceutical agent for preventing or treating one or more diseases selected from the group consisting of osteoporosis, bone dysplasia, and growth inhibition in the developmental stage, wherein the differentiation regulator according to claim 1 or 2 Containing a medicament. supplying a test substance to a mesenchymal stem cell or mesenchymal progenitor cell or a non-human animal expressing miRNA-294;
The expression or function of miRNA-294 in the mesenchymal stem cell or mesenchymal progenitor cell or non-human animal is compared with the expression or function of miRNA-294 when the test substance is not supplied, and based on the comparison result A step of evaluating the differentiation promoting action or differentiation inhibiting action of the test substance on osteoblasts ,
A screening method for a substance having an osteoblast differentiation-regulating action.