JP6948059B2 - Promotion of osteocalcin production from osteoblasts by miR-140-3p - Google Patents

Description

The present invention provides a method for screening an osteocalcin production promoter and a bone formation promoter containing miR-140-3p as an active ingredient, an osteocalcin production promoter using miR-140-3p, and a method for ascertaining the health condition. It relates to a biomarker for determining bone formation and for determining the therapeutic effect of osteoporosis.

Bone repeatedly destroys and forms and contributes to the maintenance of morphology as a supporting tissue and calcium metabolism in the body. This bone resorption and bone formation is called bone remodeling, and osteoblasts, osteoocytes, and osteoclasts in bone tissue are involved. In addition to the direct action of osteoblasts and osteoclasts, humoral factors such as female hormones are also involved in this bone remodeling in a complex manner. When bone resorption becomes dominant, bone loss occurs, resulting in osteoporosis, which causes fragile fractures and is involved in the development of bedridden and dementia. In today's aging society, prevention of fragile fractures is considered important in preventing age-related diseases such as bedridden and dementia.

So far, bisphosphonate preparations for the purpose of inhibiting osteoclast function, parathyroid hormone preparations that act on osteoblasts, female hormone preparations, and the like have been used for the treatment of osteoporosis.

Currently, bisphosphonate preparations, which are mainly used for the treatment of osteoporosis, maintain bone mass by suppressing osteoclast function. In addition, parathyroid hormone preparations act on osteoblasts to promote bone formation, and selective estrogen receptor modulators also have an effect of improving bone quality. As described above, each therapeutic agent for osteoporosis has a characteristic action, but a bisphosphonate preparation may cause an atypical fracture of the femur by long-term administration. In addition, although parathyroid hormone preparations act directly on osteoblasts, it is stipulated that they should be administered to osteoporotic patients at high risk of fracture for a limited period of up to 24 months. As described above, conventional osteoporosis therapeutic agents have various restrictions on administration, including side effects.

It is an issue to provide a therapeutic agent for osteoporosis based on a mechanism of action different from the conventional one, which is not subject to various restrictions on such side effects and medications.

In recent years, microRNA (miRNA) has been attracting attention as a factor that regulates functions in vivo. MiRNA is a single-stranded non-coding RNA consisting of about 22 bases, which is contained in the exosomes of secretory granules, binds to the 3'UTR portion of the target gene in distant cells, and regulates its gene expression after transcription of the target gene. It is considered. MiRNAs are known to play important roles in cell defense against differentiation, development, tumorigenesis, and infection. In addition, some miRNAs are produced and secreted from osteoblasts.
In the process of osteoblast differentiation, it is miR-29, miR-208, miR-133, miR-135, miR-138 that act on the BMP pathway, and miR-29 and miR- that act on the Wnt pathway. 335 (Non-Patent Document 1). Although miR-29 acts on both pathways, the mechanism of action is considered to be independent.
More than 500 types of miRNAs present in the genome have been reported, but the functions of many miRNAs are still unknown.

The function of the miR-140-3p gene has not been specifically reported. There is a document that discloses a pharmaceutical composition containing miR-140-3p (Patent Document 1). However, Patent Document 1 does not refer to the relationship between miR-140-3p and osteocalcin, does not disclose an example using miR-140-3p, and the function of miR-140-3p is still unknown. Is. A pharmaceutical composition containing osteocalcin is also known (Patent Document 2), but there is no disclosure or suggestion regarding the relationship between miRNA or TGFβ and osteocalcin, and the target disease of the pharmaceutical composition is diabetes and the like.

Japanese Patent Application Laid-Open No. 2013-504542 Japanese Patent No. 5421109

Rahman MS et al., Bone Res. 2015 Apr14; 3: 15005

An object of the present invention is to elucidate a new mechanism of action that can treat osteoporosis in order to provide a new therapeutic agent for osteoporosis based on a mechanism of action different from the conventional one, and to produce osteocalcin based on the new mechanism of action. To provide promoters and bone formation promoters, to provide new methods for screening osteocalcin production promoters, treatments and / or preventive agents for osteoporosis, and to understand the health status of bone formation. To provide a novel biomarker for making a determination and for determining the therapeutic effect of osteoporosis.

The present inventors have a novel mechanism of action that can treat osteoporosis that miR-140-3p acts to directly link TGFβ3 and the Wnt pathway and promotes the expression of osteocalcin, which is a bone formation marker. And completed the present invention.
That is, the present invention comprises:
1. 1. An osteocalcin production promoter comprising miR-140-3p, a precursor thereof and at least one selected from the group consisting of DNA constructs encoding them as an active ingredient. 2. The agent according to item 1 above, wherein at least one selected from the group consisting of miR-140-3p, a precursor thereof and a DNA construct encoding them suppresses the gene expression of TGFβ3.
3. 3. The agent according to item 2 above, wherein the suppression of TGFβ3 gene expression is due to the binding of TGFβ3 to the 3'UTR.
4. The agent according to item 1 above, wherein at least one selected from the group consisting of miR-140-3p, a precursor thereof and a DNA construct encoding them is one in which its expression is suppressed by overexpression of Wnt3a.
5. An agent for promoting bone formation, which comprises the agent according to any one of the above items 1 to 4.
6. A method for promoting osteocalcin production by supplying the agent according to any one of the above items 1 to 4 to cells in vitro.
7. The method according to item 6 above, wherein the promotion of osteocalcin production is due to the suppression of TGFβ3 expression.
8. A method for screening a substance effective in promoting osteocalcin production, using the promotion of the expression level of miR-140-3p and / or the suppression of the expression level of TGFβ3 as a marker.
(a) The step of culturing cells in the presence of the test substance,
(b) A step of measuring the expression level of each miR-140-3p and / or TGFβ3 in the cells, and
(c) A step of selecting a substance that increases the expression level of miR-140-3p and / or a substance that suppresses the expression level of TGFβ3.
Including methods.
9. The screening method according to item 8 above, wherein the cells are osteoblasts.
10. A method for screening a substance effective for the treatment or prevention of osteoporosis, using the promotion of the expression level of miR-140-3p and / or the suppression of the expression level of TGFβ3 as a marker.
(a) The step of culturing cells in the presence of the test substance,
(b) A step of measuring the expression level of each miR-140-3p and / or TGFβ3 in the cells, and
(c) A step of selecting a substance that increases the expression level of miR-140-3p and / or a substance that suppresses the expression level of TGFβ3.
Including methods.
11. A biomarker consisting of miR-140-3p for grasping the health condition.
12. A biomarker consisting of miR-140-3p for determining bone formation.
13. A biomarker comprising miR-140-3p for determining the therapeutic effect of osteoporosis.
14. An osteoporosis preventive and therapeutic agent containing the agent according to any one of the above items 1 to 4.
15. A metabolic function improving agent containing the agent according to any one of the above items 1 to 4.
16. The agent according to item 15 above, wherein the improvement of metabolic function is the promotion of insulin production in pancreatic β cells.
17. The agent according to item 15 above, wherein the improvement of metabolic function is the promotion of insulin sensitivity in adipocytes.
18. A pharmaceutical composition comprising at least the agent according to any one of the above items 1 to 5 and 14 to 17, which comprises a pharmaceutically acceptable carrier, adjuvant, salt, diluent and / or excipient. Pharmaceutical composition.

According to the present invention, it is possible to obtain an osteocalcin production-promoting agent and a bone formation-promoting agent whose mechanism of action is to promote the expression level of miR-140-3p and / or suppress the expression level of TGFβ3.

The outline of the Wnt3a overexpression experiment in mouse-derived osteoblasts is shown. (Example 1) The expression of phosphorylated β-catenin and β-catenin in mouse-derived osteoblasts due to overexpression of Wnt3a was investigated. (Example 1) It shows the ratio of alkaline phosphatase (ALP) -positive cells and the change in alkaline phosphatase (ALP) expression in mouse-derived osteoblasts due to overexpression of Wnt3a. (Example 1) The expression of type I collagen (Col1), RUNX2 and osteocalcin (OCN) in mouse-derived osteoblasts due to overexpression of Wnt3a was confirmed. (Example 1) It shows the result of miRNA microarray analysis (Wnt3a vs eGFP). It shows that 14 types of miRNAs whose expression was increased more than 2-fold due to overexpression of Wnt3a and 21 types of miRNAs whose expression was decreased were identified, and miR-140-3p was focused on among them. (Example 1) The expression of miR-140-3p in mouse-derived osteoblasts due to overexpression of Wnt3a was confirmed. (Example 1) It shows the change in TGFβ3 expression in mouse-derived osteoblasts due to overexpression of Wnt3a. (Example 1) It shows the change in the expression of TGFβ3 by the introduction of miR-140-3p mimic into mouse-derived osteoblasts. (Example 2) It shows the result of performing luciferase using 3'UTR of TGFβ3. (Example 2) It outlines the transfection of miR-140-3p mimic into mouse-derived osteoblasts. (Example 3) It is shown that the introduction of miR-140-3p mimic into mouse-derived osteoblasts promoted osteocalcin (OCN) expression. (Example 3) It shows the outline which treated the mouse-derived osteoblast with rTGFβ3. (Example 3) It is shown that the expression of osteocalcin (OCN) in mouse-derived osteoblasts was suppressed by the administration of TGFβ3. A. As a result of overexpression of Wnt3a in mouse-derived osteoblasts, (1) the activity of alkaline phosphatase (ALP) is promoted, (2) the expression of miR-140-3p is suppressed, and (3) the gene and protein of TGFβ3 It indicates that the expression was increased. B. It is shown that the expression of miR-140-3p in mouse-derived osteoblasts promotes the expression of osteocalcin, and the suppression of the expression of TGFβ3 promotes the expression of osteocalcin. It describes the effects of osteocalcin on the whole body.

Wnt is a secretory glycoprotein that has undergone lipid modification with palmitic acid having a molecular weight of about 40,000. Two types of Wnt receptors have been reported: Frizzled, which is a 7-transmembrane receptor, and LRP5 / 6, which is a 1-transmembrane receptor. Traditionally, Wnt signaling has been thought to promote bone formation (Rawadi, G. & Roman-Roman, S., Expert Opin TherTargets. 2005 Oct; 9 (5): 1063-77).

Osteoblastic osteoblasts are derived from pluripotent mesenchymal stem cells (MSCs). Wnt-β-catenin signaling is required for mesenchymal stem cells to differentiate into osteoblast lineages and suppresses cell fate to adipocytes and chondrocytes. Once the cell fate is determined, classical Wnt signaling is essential for the proliferation and differentiation of osteoblast progenitor cells. Wnt-β-catenin signaling, but not in all cases, is also involved in the downregulation of osteoblast apoptosis. In addition, Wnt-β-catenin signaling inhibits osteoclastic bone resorption in the osteoblast lineage, which is the final differentiated cell and contains the most abundant osteoocytes. In fact, Wnt-β-catenin signaling is required for the expression of the anti-osteoclast factor OPG, a decoy receptor for RANKL in osteoblasts and osteoocytes (Roland Baron & Michaela Kneissel, NatMed. 2013 Feb; 19). (2): 179-92).

Transforming growth factor β (TGFβ) is thought to be associated with a wide range of biological activities, including cell proliferation, cell death, cell differentiation, inflammation, and immune response, by regulating the expression of specific combinations of target genes. There are three subtypes of TGFβ, TGFβ1, TGFβ2 and TGFβ3. All three subtypes bind to type I and type II TGFβ receptors. These receptors belong to the serine / threonine kinase family and activate signal transduction systems to target genes via transcription factors of the Smad family. It is generally believed that the functions of growth factors belonging to the TGFβ family vary depending on the state of the cell and the type of cell.

Osteocalcin (OCN) is used as a bone formation marker along with alkaline phosphatase (ALP). These markers are osteoblast-derived proteins that increase in concentration as osteoblasts proliferate. In particular, osteocalcin is a protein specifically produced from osteoblasts, and an increase in serum osteocalcin level in a living body is regarded as an index of bone formation. In addition, these markers are also usually used as bone metabolism markers in osteoporosis bone metabolism diseases.

The terms used herein have the following definitions:

In this specification, the abbreviations of nucleotides, polynucleotides, DNA, RNA, etc. are referred to as "Guidelines for preparing specifications including base sequences or amino acid sequences" (Japan), which is a guideline common to the Trilateral Offices. (Edited by the Japan Patent Office) and the conventions in this technical field shall be followed.

As used herein, the term "nucleic acid" is used to include RNA, DNA, and modified polynucleotides thereof. The DNA includes any of cDNA, genomic DNA, and synthetic DNA. Further, the RNA includes any of total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, shRNA, non-coding RNA, pri-miRNA, pre-miRNA, and synthetic RNA. Further, the modified polynucleotide includes a polynucleotide obtained by converting a natural sugar portion, base portion or phosphodiester bond of a nucleic acid into an unnatural structure, for example, LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), phosphorothioate. Polynucleotides having a bond, artificial polynucleotides having a 2'-O-alkylribose group, and the like are included. Also, herein, nucleic acids are used interchangeably with polynucleotides.

As used herein, the term "gene" includes not only RNA and double-stranded DNA, but also single-stranded DNA such as the positive strand (or sense strand) or complementary strand (or antisense strand) that compose it. It is used with the intention of doing so. Moreover, the length is not particularly limited.

Unless otherwise specified, the term "gene" as used herein refers to double-stranded DNA containing human genomic DNA, single-stranded DNA containing cDNA (positive strand), and single-stranded DNA having a sequence complementary to the positive strand (single-stranded DNA). Complementary strands), and fragments thereof, and any of the human genomes. Further, the "gene" is not only a "gene" represented by a specific base sequence (or SEQ ID NO:), but also an RNA having a biological function equivalent to that of RNA encoded by these, for example, a homologue (that is, homologue). , Mutants such as gene polymorphisms, and "genes" encoding derivatives are included. Specific examples of the "gene" encoding such a homologue, mutant, or derivative include the conditions described in molecular cloning-A LABORATORY MANUAL THIRD EDITION (Sambrook et al., Cold Spring Harbor Laboratory Press). In (), a "gene" having a base sequence that hybridizes with the complementary sequence of any specific base sequence shown in SEQ ID NO: 1 or 2 can be mentioned. The "gene" does not ask whether the functional region is different, and may include, for example, an expression control region, a coding region, an exon, or an intron. In addition, the "gene" may be contained in a cell, may be released extracellularly and exist alone, or may be contained in a vesicle surrounded by a lipid bilayer called an exosome. There may be.

As used herein, the term "transcript" refers to RNA synthesized using the DNA sequence of a gene as a template. RNA is synthesized in such a way that RNA polymerase binds to a site called a promoter upstream of a gene and ribonucleotides are bound to the 3'end so as to be complementary to the base sequence of DNA. This RNA includes not only the gene itself, but also the entire sequence from the transcription start site to the end of the poly A sequence, including the expression control region, coding region, exon or intron.

Unless otherwise stated, "microRNA (miRNA)" herein is transcribed as an RNA precursor with a hairpin-like structure, cleaved by dsRNA cleaving enzyme with RNase III cleaving activity, and incorporated into a protein complex called RISC. It is intended to use 15-25 bases of RNA that are involved in the suppression of mRNA translation. Further, the "miRNA" contains not only the "miRNA" represented by a specific base sequence (or SEQ ID NO:) but also the precursors (pre-miRNA, tri-miRNA) of the "miRNA", and is encoded by these. Also included are miRNAs that are biologically equivalent to miRNAs, such as "miRNAs" that encode homologues (ie, homologues), variants such as gene polymorphisms, and derivatives. The "miRNA" encoding such a precursor, homologue, mutant or derivative can be identified by miRBase (http://www.mirbase.org/) and has a specific base sequence under stringent conditions. Can be mentioned as "miRNA" having a base sequence that hybridizes with the complementary sequence of.

As used herein, the term "variant" means, in the case of a nucleic acid, a natural variant due to polymorphism, mutation, selective splicing during transcription, etc., or the nucleotide sequence shown by SEQ ID NOs: 1 and 2 or a nucleotide sequence thereof. A mutant containing a deletion, substitution, addition or insertion, preferably substitution of one or two nucleotides in a complementary base sequence, or about 90% or more, about 95% or more, about with the base sequence or a partial sequence thereof. It means a mutant showing 97% or more, about 98% or more, about 99% or more% identity, or a nucleic acid that hybridizes with a polynucleotide or an oligonucleotide containing the base sequence or a partial sequence thereof under stringent conditions. ..

As used herein, the term "miR-140-3p" refers to miRNA (miRbase ID: hsa-miR-140-3p, miRbase Accession No. MIMAT0004597) consisting of the RNA sequence "UACCACAGGGUAGAACCACGG" shown in SEQ ID NO: 1. Other species such as homologs are included. In the present specification, "miR-140-3p" refers to RNA consisting of the RNA sequence shown in SEQ ID NO: 1 or other biological homologues (for example, miRbase ID: mmu-miR-140-3p, miRbase Accession No. which are mouse homologues). MIMAT0000152) may be used, and it may be expressed from RNA encoding the RNA sequence shown in SEQ ID NO: 1 or from DNA encoding the RNA sequence shown in SEQ ID NO: 2 described below. Later, it may have been processed.

As used herein, the term "miR-140" or "precursor of miR-140-3p" consists of the RNA sequence "UGUGUCUCUCUCUGUGUCCUGCCAGUGGUUUUCCUAUGGUAGGUUACGUCAUGCUGUUCUACCACAGGGUAGAACCACGGACAGGAUACCGGGGCACC" shown in SEQ ID NO: 2. , MiRbase Accession No. MI0000456) and other species homologues (eg, miRbase ID: mmu-mir-140, miRbase Accession No. MI0000165, which are mouse homologues) are included. As used herein, "miR-140" refers to "a precursor of miR-140-3p" and includes the RNA sequence of miR-140-3p.

When the RNA molecule is a single polynucleotide, there is a linker region between the miRNA region and the complementary region. In some embodiments, the single polynucleotide is capable of forming a hairpin loop structure as a result of binding between the miRNA region and the complementary region. The linker constitutes the hairpin loop. In some embodiments, the linker region is at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 in length. , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 residues, or there It is intended to be any range that can be derived from. In certain embodiments, the linker is 3 to 30 residues in length (including the numbers at both ends).

In addition to having miRNA and complementary regions, there can be additional flanking sequences at either the 5'or 3'end of the region. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides or more, or any deriving there, adjacent to one or both sides of these regions. There is a range.

In one aspect of the invention, the osteocalcin production promoter or bone formation promoter is at least one selected from the group consisting of miR-140-3p, miR-140-3p precursors, and DNA constructs encoding them. May be an agent containing as an active ingredient. miR-140-3p is an RNA or other species homolog consisting of the RNA sequence set forth in SEQ ID NO: 1. The precursor of miR-140-3p comprises the RNA sequence of miR-140-3p and is an RNA consisting of the RNA sequence shown in SEQ ID NO: 2 or a homologue of other species. The DNA construct may include a DNA sequence expressing miR-140. Examples of the DNA construct include a vector expressing miR-140-3p.

In one aspect of the invention, the osteocalcin production promoter is based on suppressing the gene expression of TGFβ3. Here, "suppressing gene expression" means suppressing gene expression at the mRNA level or the protein level. For example, the mRNA level may be suppressed by degrading the mRNA after the gene to be expressed by the gene is expressed. In one aspect of the invention, suppression of TGFβ3 expression may result from miR-140-3p binding to the 3'UTR of TGFβ3.

In one aspect of the present invention, the 3'UTR of TGFβ3 is not particularly limited as long as it is an untranslated region that is not translated into a protein located on the 3'side of the coding region of TGFβ3 mRNA. The 3'UTR of TGFβ3 preferably contains an RNA sequence consisting of "GCUUGAGAAUCACUGUGGUA" and an RNA sequence consisting of "ACAUGGAAAUUCUGUGGUG".

In one aspect of the present invention, miR-140-3p is suppressed by overexpression of Wnt3a.

In one aspect of the invention, the method of promoting osteocalcin production comprises at least one selected from the group consisting of miR-140-3p, miR-140-3p precursors, and DNA constructs encoding them as active ingredients. This is done by supplying the cell with an agent containing as. Preferably, the supply is done by introducing into cells. Feeding the cells may be done in vitro. Preferably, the cell is an osteoblast. In addition, promotion of osteocalcin production may be caused by suppression of TGFβ3 expression.

In one aspect of the present invention, the cell to be targeted for promoting the production of osteocalcin may be any cell that produces osteocalcin, and examples thereof include osteoblasts and precursor cells of osteoblasts. .. Examples of osteoblast progenitor cells include bone marrow stromal cells and cell lines derived from them.

The degree of osteoblast differentiation can be assessed, for example, by measuring the intracellular alkaline phosphatase activity. For the method of evaluating the degree of osteoblast differentiation, for example, J. Biol. Chem. 2006, 281 (26), 18015-24 and the like can be referred to.

In one aspect of the present invention, recombinant cells can be mentioned as target cells for promoting the production of osteocalcin. The recombinant cell is, for example, a vector expressing miR-140-3p, such as cultured osteoblast cells (mouse osteoblast progenitor cells, rat femoral / cervical bone marrow-derived mesenchymal stem cells, human bone marrow-derived stem cells, which will be described below). It can be prepared by introducing it into bone marrow mononuclear cells).

For mouse osteoblast progenitor cells, cut the cranial crown of a 18.5 day embryonic mouse fetus or newborn, cut it into small pieces with scissors, add collagen gel (cell matrix type IA), and then after the gel has hardened. , ΑMEM is added and cultured for 10 to 14 days. Then, the cells are treated with 0.2% collagenase, centrifuged to collect the cells, the cells are resuspended in αMEM (containing 10% FBS), and the cells are seeded and cultured on a culture plate.

For rat femoral / cervical bone marrow-derived mesenchymal stem cells, after removing the femoral bone / cervical bone, flush out the bone marrow with a culture medium, centrifuge after pipetting, discard the supernatant, and use αMEM (including 10% FBS) to remove the cells. It can be prepared by resuspending, seeding and culturing cells on a culture plate coated with laminin, and culturing by adding bFGF at a final concentration of 3 ng / ml on the 9th day after seeding.

Human bone marrow mononuclear cell fractions can be obtained, for example, from ALLCELLS or LONZA.

In one aspect of the invention, the nucleic acid is introduced into cells by calcium phosphate transfection, lipid transfection, electroporation, microinjection, or injection. In addition, the cells may be in a subject that can be a patient or animal model. In this case, the nucleic acid can be administered to the subject or patient using a mode of administration well known to those of skill in the art, especially for therapeutic applications. It is specifically intended that the patient is a human or any other mammal or animal with miRNA.

In one aspect of the invention, it comprises introducing into the cell a test substance that affects the expression of miR-140-3p in the cell in an amount effective to achieve the desired physiological result. In one aspect of the invention, an increase or decrease in the level of expression of miR-140-3p in cells correlates with the disease state as compared to the level of expression of miR-140-3p in normal cells. This correlation allows the following screening and diagnostic methods to be performed when the expression level of miR-140-3p is measured in the assessed biological sample and then compared to the expression level of normal cells. ..

In one aspect of the present invention, a substance effective in promoting osteocalcin production can be screened using the promotion of the expression level of miR-140-3p as a marker. More specifically, the following steps:
(a) The step of culturing cells in the presence of the test substance,
(b) A step of measuring the expression level of each miR-140-3p in the cells, and
(c) Screening can be performed by a method including the step of selecting a substance that increases the expression level of miR-140-3p.

In one aspect of the present invention, a substance effective in promoting osteocalcin production can be screened using the suppression of the expression level of TGFβ3 as a marker. More specifically, the following steps:
(a) The step of culturing cells in the presence of the test substance,
(b) A step of measuring the expression level of TGFβ3 in the cells, and
Screening can be performed by a method including (c) a step of selecting a substance that reduces the expression level of TGFβ3.

In one aspect of the present invention, a substance effective for the treatment or prevention of osteoporosis can be screened using the promotion of the expression level of miR-140-3p and / or the suppression of the expression level of TGFβ3 as markers. More specifically, the following steps:
(a) The step of culturing cells in the presence of the test substance,
(b) A step of measuring the expression level of miR-140-3p and / or the expression level of TGFβ3 in the cells, and
(c) Screening can be performed by a method including a step of selecting a substance that increases the expression level of miR-140-3p and / or a substance that decreases the expression level of TGFβ3.

In one aspect of the present invention, in the screening method of the present invention, the expression level of miR-140-3p can be measured by any method known to those skilled in the art. For example, the expression level of miR-140-3p can be measured using a primer or probe that specifically hybridizes to miR-140-3p. Such a primer or probe can be appropriately designed by a person skilled in the art based on the base sequence of miR-140-3p with reference to the information in the database and the like.

Northern blotting is a classical method as a measurement method using such a primer or probe. There are reports of miRNA microarrays (Liu et al, 2004; Lim et al, 2005), improved invader methods (Allawi et al, 2004), and bead-based flow cytometer methods (Luetal, 2005). In addition, there is real-time PCR as a quantitative method (Chen et al, 2005).

The miRNA microarray is a method capable of quantifying mature miRNA, and a commonly used method may be used. For example, a microRNA chip at the following URL (https://www.m-chemical.co.jp/genome/products/micro_rna.html) can be used.

The base sequence of the primer or probe preferably has an appropriate number of bases that enables specific binding with the template, for example, about several tens of bp and 10 to 30 bp. In addition, the base sequence may have a hairpin structure within the primer in the miRNA microarray probe design. For example, commercially available primer design software such as Oligo ™ (National Bioscience Inc.) can also be used.

In one aspect of the invention, the agent of the invention comprises at least one selected from the group consisting of miR-140-3p, a precursor thereof and a DNA construct encoding them as an active ingredient.

In one aspect of the present invention, the agent of the present invention is useful as an osteocalcin production promoter, a bone formation promoter, an osteoblast differentiation promoter, or a preventive and therapeutic agent for osteoporosis. Osteoporosis includes primary osteoporosis (degenerative osteoporosis (postmenopausal osteoporosis, senile osteoporosis), idiopathic osteoporosis), secondary osteoporosis (drug-induced, rheumatoid arthritis, diabetes, hyperthyroidism, sexual dysfunction, immobility, etc.) (Nutrition, congenital diseases), etc., but are not limited to these.

The subject of administration of the agent of the present invention is usually a mammal. Mammals include, for example, rodents such as mice, rats, hamsters and guinea pigs, experimental animals such as rabbits, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, and humans. Primates such as monkeys, crab monkeys, red-tailed monkeys, guinea pigs, oran wootans, and chimpanzees can be mentioned, but are not limited thereto. Mammals are preferably primates (such as humans) or rodents (such as mice).

The dose of the agent of the present invention should be appropriately determined according to the age, sex, body weight, symptoms, administration route, etc. of the patient, and is not limited to the following, but is, for example, about 0.1 μg / kg per adult day. It is in the range of ~ 100 mg / kg, preferably in the range of about 1 μg / kg to 10 mg / kg. The pharmaceutical product may be administered daily during the treatment, every day for 4 weeks, or at intervals of several days, 2 weeks, or 1 month at equal time intervals.

Osteocalcin is known to promote insulin secretion from the pancreas and insulin sensitivity in adipose tissue, and administration of miR-140-3p may have a positive effect on the whole body (Fig. 15). From this, miR-140-3p is useful not only as a mere bone formation promoting agent and an osteoporosis preventive and therapeutic agent, but also as a metabolic function improving agent or a preventive agent for improving the entire metabolic system in the living body, and is useful for extending healthy life expectancy. It is thought to contribute.

In one aspect of the present invention, a diagnostic means using miR-140-3p as a marker is provided. For example, by measuring miR-140-3p in the body, it is considered to be useful as a biomarker capable of grasping the health condition. For the measurement of miR-140-3p in the body, a sample obtained from the measurement target can be used. In the present specification, the sample obtained from the subject refers to a biological tissue in which the expression of miR-140-3p of the present invention changes, a substance derived from the biological tissue, and a substance in contact with the biological tissue, and examples thereof include a blood sample. The method of extracting total RNA from a sample and measuring the target miR-140-3p from the sample can be measured by any method known to those skilled in the art as described above. In addition, when miR-140-3p is used as a biomarker, it is considered useful because the effect of treatment for bone formation and osteoporosis can be determined earlier than the measurement of osteocalcin as a biomarker.

In one aspect of the present invention, the vector expressing miR-140-3p is used for the production of miR-140-3p, which is the active ingredient of the pharmaceutical composition of the present invention, or the vector itself is formulated to form a pharmaceutical. It can be used as a composition.

Vectors include, for example, plasmids, phages, viruses and the like. Examples of plasmids include, but are not limited to, E. coli-derived plasmids (eg, pRSET, pTZ19R, pBR322, pBR325, pUC118, pUC119, etc.), bacteriophage-derived plasmids (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YEp24, etc.). YCp50 etc.), Ti plasmid, etc., examples of phages include λ phages, etc. Further, examples of virus vectors include animal virus vectors such as retrovirus, vaccinia virus, lentivirus, adenovirus, adeno-associated virus, etc. , Insect virus vectors such as bacteriophage.

The vector may include a polylinker or multicloning site for incorporating the DNA of interest, and may also include several regulatory elements to express the DNA encoding the miR-140-3p gene. Control elements include, for example, promoters, enhancers, poly-A addition signals, replication origins, selectable markers, ribosome binding sequences, terminators and the like.

Examples of selection markers are drug resistance genes (eg neomycin resistance gene, ampicillin resistance gene, canamycin resistance gene, puromycin resistance gene, etc.), nutritional requirement complementary genes (eg dihydrofolate reductase (DHFR) gene, HIS3 gene, LEU2 gene, etc.) , URA3 gene, etc.).

Examples of host cells include, but are not limited to, Escherichia genus such as Escherichia coli, Bacillus genus such as Bacillus subtilis, Pseudomonas genus such as Pseudomonas petita, Saccharomyces cerevisiae, Saccharomyces genus Pombe and the like. , Yeasts such as Candida and Pikia, animal cells such as CHO, COS, HEK293, NIH3T3 and NS0, insect cells such as Sf9 and Sf21, plant cells and the like.

In one aspect of the invention, the pharmaceutical composition of the invention is at least one selected from the group consisting of pharmaceutically effective amounts of miR-140-3p of the invention, precursors thereof and DNA constructs encoding them. As an active ingredient, it may be in the form of a sterile aqueous or non-aqueous solution, suspension, or emulsion. Further, it may contain a pharmacologically acceptable carrier such as a salt, a buffer, an adjuvant, an additive and the like.

As the pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used. Specific examples include excipients, lubricants, binders, disintegrants in solid formulations, solvents in liquid formulations, solubilizers, suspending agents, tonicity agents, buffers, soothing agents and the like. Be done. At the time of formulation, if necessary, formulation additives such as preservatives, antioxidants, colorants, and sweeteners may be used.

Pharmaceutically acceptable additives include, for example, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, poly. Excipients such as propylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, starch, carmellose, hydroxypropyl starch, sodium-glycol-starch, disintegrants such as sodium hydrogen carbonate, calcium phosphate, calcium citrate, stearic acid Lubricants such as magnesium, talc, sodium lauryl sulfate, citric acid, menthol, diammonium glycyrrhizinate, glycine, orange oil and other flavoring agents, sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben and other preservatives, citrate Stabilizers such as acid, sodium citrate, acetic acid, suspending agents such as methylcellulose, povidone, aluminum stearate, dispersants such as surfactants, diluents such as water, physiological saline, orange juice, cacao butter, Examples thereof include base waxes such as polyethylene glycol and white kerosene, but the present invention is not limited thereto.

The agent of the present invention is one or more carriers that are pharmacologically acceptable for at least one selected from the group consisting of the active ingredient miR-140-3p, a precursor thereof, and a DNA construct encoding them. It can be mixed with and manufactured by any method well known in the technical field of pharmaceutics. The agents of the present invention may further comprise any active ingredient for other treatments for bone disease. Such therapeutic agents include, but are not limited to, calcium preparations (calcium L-aspartate, calcium gluconate, calcium lactate, etc.), active vitamin D3 preparations (alphacalcidol, calcitriol, etc.), etc. Female hormone drugs (estriol, bound estrogen, etc.), calcium preparations (sake calcitonin, elcatonin, etc.), vitamin K preparations (menatetrenone, etc.), bisphosphonate preparations (disodium etidronate, sodium alendronate hydrate, sodium lysedronate, etc.) Hydrate, etc.), selective estrogen receptor modulators (laroxyphenic acid hydrochloride, etc.), ipriflavon, anti-RANKL antibody, etc. are included.

It is desirable to use the most effective route of administration for treatment, and it is usually administered parenterally or orally, such as transdermally or intravenously. The parenteral preparation includes, but is not limited to, an intravenous preparation, an intramuscular preparation, an intraperitoneal preparation, a subcutaneous preparation, a topical preparation and the like. Topical administration includes direct administration to injured, fractured, or damaged bones such as the skull, femur, sternum, vertebrae, ribs, etc., and contains the active ingredient in an artificial bone component such as hydroxyapatite. It may be administered as a transplanted preparation in the form of a fracture. Parenteral-administered preparations include, for example, injections, infusions, suppositories, transdermal absorbents, liposomes, nanoparticle-encapsulated preparations, and the like. Oral preparations include, for example, tablets, pills, granules, capsules, powders, solutions, suspensions, delayed release preparations, enteric-coated preparations and the like.

Osteoporosis model non-human mammals are well known to those skilled in the art and include, for example, ovariectomized non-human mammals (Am.J.Pathol.2007, 170 (4), 1277-1290, etc.), arthritis non-human mammals (Biochemical Pharmacol). .2005,70 (12), 1744-1755, etc.), gravity reduction due to tail suspension Non-human mammals (Exp.Cell.Res.2006,312 (16), 3075-3083, etc.), etc. Not limited to these.

The evaluation of the symptoms of osteoporosis is performed based on parameters well known to those skilled in the art, such as bone density, bone mass, and blood alkaline phosphatase activity.

If the symptoms of osteoporosis in non-human mammals treated with the test substance are significantly reduced compared to the symptoms of osteoporosis in control non-human mammals not treated with the test substance, the test substance is used. , Can be obtained as a substance having an activity of preventing and / or treating osteoporosis, an activity of promoting bone formation, and an activity of promoting osteoblast differentiation.

Hereinafter, the present invention will be described in detail by way of examples, but the technical scope of the present invention is not construed as being limited by the description of the following examples. One of ordinary skill in the art can make many modifications and modifications based on the description herein without departing from the technical scope of the invention. Unless otherwise specified, the following examples include, for example, Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FMetal., Current Protocols in Molecular Biology, John. It can be performed according to standard genetic engineering and molecular biology techniques known to those skilled in the art as described in Wiley & Sons, New York, NY, 1995, etc. When using the kit, follow the instruction manual of the kit. The contents of the documents cited in the present specification constitute the disclosure of the present specification and a part of the contents.

In this example, the expression of miR-140-3p was suppressed by overexpression of Wnt3a, and the expression of miR-140-3p suppressed the expression of TGFβ3 and promoted the expression of osteocalcin (OCN). show.
1. 1. Overexpressing Wnt3a Mouse-derived osteoblasts were cultured in each well of a 4-well plate (5 ml) with 2.5x10 ⁴ cells and in each well of a 6-well plate (2 ml) with 1x10 ^{5 cells.} The medium in each well consists of one containing phenol red-free α minimum essential medium, 10% fetal bovine serum, penicillin, streptomycin. Vector (control) expressing Wnt3a (NM-009522, 1275 bp from start codon) or eGFP (L29345, 820 bp from start codon) in cultured mouse-derived osteoblasts (MOI: 200), RNA is extracted from cells 1, 3, and 7 days after infection, and miRNA microarray, alkaline phosphatase (ALP) staining, and Western blot are used from cells 3 days after infection. Was done.
RNA extraction was performed with miRN easy Mini kit or TRIzol, and anti-phosphorylated β-catenin antibody (Cell Signaling Technology), anti-TGFβ3 antibody (Abcam), and anti-RUNX2 antibody (Cell Signaling Technology) were used for Western blotting. The outline is shown in FIG.

(1) Effect of overexpression of Wnt3a on osteoblast differentiation As shown in FIG. 1, analysis of overexpression of Wnt3a in mouse-derived osteoblasts revealed that the osteoblasts were in a phosphorylated (Ser675) state. High expression of β-catenin was observed (Fig. 2). Phosphorylated β-catenin is thought to translocate into the nucleus and form a complex with other transcription factors to promote the expression of the target gene, alkaline phosphatase (ALP).

Therefore, the expression of alkaline phosphatase (ALP) in mouse-derived osteoblasts overexpressing the control eGFP and mouse-derived osteoblasts overexpressing Wnt3a was investigated. Alkaline phosphatase (ALP) staining was performed by a conventional method.
As a result, it was confirmed that the overexpression of Wnt3a promoted the activity of alkaline phosphatase (ALP) in mouse-derived osteoblasts (Fig. 3).

Similarly, the expression of type I collagen and osteocalcin (OCN) in mouse-derived osteoblasts due to overexpression of Wnt3a was examined. As can be seen from the results of type I collagen and comparison, the expression of osteocalcin (OCN) was significantly suppressed by the overexpression of Wnt3a (Fig. 4).

(2) MiRNA microarray analysis Wnt3a and eGFP were introduced into mouse-derived osteoblasts, respectively, and miRNA was purified using the miRNeasy Mini kit. Microarray analysis was performed using 3D-Gene miRNA Microarray Platform (Toray).
A scatter plot was obtained with the global normalization of the fluorescence intensity of Wnt3a on the vertical axis and the global normalization of the fluorescence intensity of the control eGFP on the horizontal axis (FIG. 5).
As shown in FIG. 5, among the miRNAs that fluctuated more than twice due to overexpression of Wnt3a, 14 types of miRNAs with increased expression and 21 types of miRNAs with decreased expression were obtained.
The spot indicated by the arrow in FIG. 5 is miR-140-3p, which was obtained as a miRNA having a large variation in expression due to overexpression of Wnt3a and a high expression level.

(3) Expression analysis of miR-140-3p As shown in FIG. 1, when the expression analysis of mouse-derived osteoblasts overexpressing Wnt3a was performed, suppression of the expression of miR-140-3p was observed. (Fig. 6).

(4) Effect of overexpression of Wnt3a on TGFβ3 expression The target gene of miR-140-3p obtained as a miRNA gene related to the expression of Wnt3a is stored in a known database [miRBase (http://www.mirbase.org). /), miRDB (http://www.mirdb.org/), microRNA. Forecast using org (http://www.microrna.org/microrna/home.do)]. As a result, TGFβ3 was nominated as a target gene for miR-140-3p. Therefore, as shown in FIG. 1, the effect of overexpression of Wnt3a on the expression of TGFβ3 was investigated.
As a result, it was found that the overexpression of Wnt3a increased the expression of TGFβ3 in mouse-derived osteoblasts (Fig. 7).

Summarizing the above results, as a result of overexpression of Wnt3a in mouse-derived osteoblasts, (1) the activity of alkaline phosphatase (ALP) was promoted, (2) the expression of miR-140-3p was suppressed, and (3) ) Increased expression of TGFβ3 gene and protein (Fig. 14A).
Alkaline phosphatase is an early differentiation marker for osteoblasts, and osteocalcin is a late differentiation marker for osteoblasts. At the stage of early differentiation, only weak expression of miR-140-3p was observed, but as the differentiation progressed, the expression of miR-140-3p and osteocalcin was promoted.

2. Effect of miR-140-3p expression on TGFβ3 expression miR-140-3p mimic or control miR-NC (mirVana ™ miRNA mimic or mimic negative) having a sequence similar to miR-140-3p in mouse-derived osteoblasts After introducing control, Ambion), mouse-derived osteoblasts were cultured for 3 days, and the gene expression and protein expression of TGFβ3 were examined.
TGFβ3 expression was reduced at both mRNA and protein levels in miR-140-3p mimic-introduced cells as compared to miR-NC-introduced cells (FIG. 8).

A reporter expression vector construct was prepared by ligating the 3'UTR portion of the luciferase gene and the wild-type TGFβ3 gene downstream of the PGK promoter, and the reporter expression vector construct was derived from mice together with miR-NC or miR-140-3p mimic. It was co-introduced into osteoblasts and cultured, and a luciferase assay was performed.
The 3'UTR region of TGFβ3 and miR-140-3p have complementary sequences (Fig. 9). The expression of luciferase was suppressed in the cells expressing miR-140-3p (Fig. 9). Therefore, it was shown that miR-140-3p suppresses the transcriptional activity of TGFβ3 via the 3'UTR region of TGFβ3.

Summarizing the above results, introduction of miR-140-3p mimic into osteoblasts reduced TGFβ3 expression, and the reduction in gene expression was via the 3'UTR of TGFβ3 linked downstream of the luciferase gene. From what was done, it was shown that miR-140-3p acts on the 3'UTR of TGFβ3 and suppresses the gene expression of TGFβ3.

3. 3. Effect of miR-140-3p expression on genes related to bone metabolism α
Mouse-derived osteoblasts were cultured in ^{3x10 4} cells in each well of a 24-well plate (1 ml). The medium in each well consists of one containing phenol red-free α minimum essential medium, 10% fetal bovine serum, penicillin, streptomycin. Cultured mouse-derived osteoblasts were transfected with miR-140-3p mimic or negative control (NC) miRNA, and medium exchange (MC) was performed 1 day later to obtain RNA from the cells 2 days after transfection. Extraction was performed and cells 3 days after transfection were used for Western blot. The outline is shown in FIG.

As shown in FIG. 10, miR-NC or miR-140-3p mimic was introduced into mouse-derived osteoblasts, and alkaline phosphatase (ALP) known as a bone formation marker and type I collagen (Col1) known as a bone resorption marker were introduced. ), The transcription factor Runx2 that functions in osteoblast differentiation, and osteocalcin (OCN), which is known as a bone formation marker, were examined for gene expression and protein expression.
As a result, the expression of osteocalcin was promoted by introducing miR-140-3p mimic into mouse-derived osteoblasts (Fig. 11).

4. Effect of TGFβ3 administration on osteocalcin (OCN) expression 24 hours before administration of recombinant TGFβ3 (rTGFβ3, purchased from Cell Signaling Technology), 4x10 ^{4 mouse-derived osteoblasts were placed in each well of a 24-well plate (1 ml).} It was cultured. The medium in each well consists of phenol red-free α minimum essential medium, 10% fetal bovineserum, penicillin, streptomycin. rTGFβ3 was administered to the medium at a concentration of 5 ng / ml, and RNA was extracted 2, 5, 24, and 72 hours after the administration of rTGFβ3. The outline is shown in FIG.

The expression of osteocalcin was suppressed in the mouse-derived osteoblasts to which TGFβ3 was administered as compared with the mouse-derived osteoblasts (control) to which TGFβ3 was not administered (FIG. 13).

As shown in this example, it was revealed that miR-140-3p is involved in the Wnt3a / TGFβ3 signaling pathway and regulates osteoblast differentiation. In addition, miR-140-3p can be used as an osteocalcin (OCN) promoter because it suppresses the TGFβ3 signaling pathway and promotes the expression of osteocalcin (OCN) (FIG. 14).

The present inventors have found that miR-140-3p acts to directly link TGFβ3 and the Wnt3a pathway. Gene transfer of miR-140-3p into osteoblasts promoted the expression of osteocalcin, a bone formation marker. This mechanism of action is completely different from conventional osteoporosis therapeutic agents, and is considered to be effective for bone formation. In addition, miRNA exists in a physiological state, and a therapeutic agent containing miR-140-3p as an active ingredient is expected to have fewer side effects than conventional therapeutic agents.
Considering the progress of the aging society in the future, prevention of the onset of osteoporosis is considered to play an important role in extending healthy life expectancy. It is essential to be diagnosed with osteoporosis in the use of conventional therapeutic agents for osteoporosis, and there is concern about the occurrence of side effects. It is considered that miR-140-3p according to the present invention can be used not only for treatment but also for prevention, and contributes to promotion of prevention of illness and long-term care and creation of a health industry that does not depend on public insurance, and creates a health industry. I think I can.

Claims (7)

An osteocalcin production promoter in osteoblasts containing a DNA construct encoding miR-140-3p as an active ingredient. The agent according to claim 1, wherein the DNA construct encoding miR-140-3p suppresses the gene expression of TGFβ3. The agent according to claim 2 , wherein the suppression of TGFβ3 gene expression is due to the binding of TGFβ3 to the 3'UTR. The agent according to claim 1, wherein the DNA construct encoding miR-140-3p is one in which its expression is suppressed by overexpression of Wnt3a. A method for promoting osteocalcin production by supplying the agent according to any one of claims 1 to 4 to osteoblasts in vitro. The method of claim 5 , wherein the promotion of osteocalcin production is due to suppression of TGFβ3 expression. A method for screening a substance effective in promoting osteocalcin production using the promotion of the expression level of miR-140-3p as a marker.
(a) Step of culturing osteoblasts in the presence of the test substance,
(b) measuring the expression level of miR-140-3p that put in the cell and,
(c) A step of selecting a substance that increases the expression level of miR-140-3p,
Including methods.

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