Application of PRMT6 inhibitor in preparation of anti-bone resorption product

Abstract

The invention relates to the field of biological medicine, in particular to application of a PRMT6 inhibitor in preparation of a bone resorption resisting product. The PRMT6 inhibitor disclosed by the invention is any one or more of EPZ020411, antisense oligonucleotide, double-stranded RNA (Ribonucleic Acid), short hairpin RNA, a nucleic acid construct or a nucleic acid composition. According to the invention, PRMT6 is determined as a new metabolic checkpoint, metabolic switching from fatty acid oxidation to glycolysis is mediated, and an RANK signal is converted into metabolic switching in an osteoclast differentiation process. The PRMT6 induces asymmetric double methylation of H3R2 on loci of PPARD, Acox3 and Cpt1a, and chromatin accessibility of genes related to fatty acid beta-oxidation is reduced, so that fatty acid oxidation is inhibited. The PRMT6 inhibition effectively inhibits osteoclast differentiation induced by RANKL and bone loss of ovariectomized mice, and a promising target is provided for anti-absorption treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of PRMT6 inhibitors in the preparation of anti-bone resorption products. Background Art

[0002] Excessive osteoclastogenesis is a key shared factor in the progression of diseases such as osteoporosis, periodontitis, rheumatoid arthritis, and metastatic cancer. Specifically, in postmenopausal osteoporosis, estrogen deficiency abolishes the inhibition of osteoclast differentiation and increases levels of receptor activator of nuclear factor κB ligand (RANKL), leading to excessive osteoclast activity and increased bone resorption. Antiresorptive therapy is the mainstay of treatment for these conditions, and a thorough investigation of the mechanisms of osteoclastogenesis is needed to uncover new therapeutic targets. Osteoclasts are bone-specific multinuclear cells derived from bone marrow mononuclear cells / macrophages (BMMs). Their differentiation and formation depend on two essential cytokines: macrophage colony-stimulating factor (M-CSF) and RANKL. Activation of RANK and its ligand triggers a series of downstream signaling cascades, including NF-κB, JNK, p38, ERK, and Src, which converge to initiate osteoclast differentiation and activation. Concurrently, cells undergo metabolic reprogramming in response to RANKL stimulation. Activation of RANK signaling in BMMs increased glycolysis, whereas genetic knockout of Ldha or Glut1 inhibited osteoclast differentiation.

[0003] Protein arginine methyltransferase 6 (PRMT6) is a type 1 prnmt that asymmetrically dimethylates arginine residues on certain proteins, including histones, transcription factors, and core regulatory factors. Epigenetic regulation plays a crucial role in cellular life, and histone modification is a key form of this regulation. Within the context of histone modifications, the catalytic sites of PRMT6 are located at arginine 2 (H3R2me2a), arginine 17 (H3R17me2a), and arginine 42 (H3R42me2a) of histone 3, as well as at arginine 26 (H2AR26me2a) of histone H2A19. H3R2me2a is considered a repressive histone modification that reduces chromatin accessibility by reciprocally repelling H3K4me3. Current research has revealed the crucial roles of PRMT6 in tumorigenesis, viral diseases, neurodegenerative diseases, and numerous physiological functions, including mitosis, DNA repair, aging, development, and differentiation. However, the role and mechanism of how PRMT6 regulates osteoclastogenesis and metabolic reprogramming remain unclear. Summary of the Invention

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a use of a PRMT6 inhibitor in the preparation of an anti-bone resorption product to solve the problems in the prior art.

[0005] To achieve the above objectives and other related objectives, the present invention provides the use of a PRMT6 inhibitor in the preparation of an anti-bone resorption product.

[0006] Preferably, the PRMT6 inhibitor is any one or more of EPZ020411, antisense oligonucleotide, double-stranded RNA, short hairpin RNA, nucleic acid construct or nucleic acid composition; wherein the nucleic acid construct comprises a nucleic acid fragment encoding a Cas protein and a guide RNA; or, the nucleic acid composition comprises a Cas protein or a ribonucleic acid molecule encoding a Cas protein, and a guide RNA or a ribonucleic acid molecule encoding a guide RNA.

[0007] The present invention also provides the use of a PRMT6 inhibitor in the preparation of an epigenetic regulation product, wherein the epigenetic regulation product is a product that regulates chromatin accessibility.

[0008] As described above, the use of the PRMT6 inhibitor of the present invention in the preparation of an anti-bone resorption product has the following beneficial effects:

[0009] This study identifies PRMT6 as a novel metabolic checkpoint that mediates a metabolic switch from fatty acid oxidation to glycolysis, translating RANK signaling into a metabolic switch during osteoclast differentiation. Mechanistically, PRMT6 induces asymmetric dimethylation of H3R2 at the PPARD, Acox3, and Cpt1a loci, reducing chromatin accessibility at genes involved in fatty acid β-oxidation, thereby inhibiting fatty acid oxidation. PRMT6 inhibition effectively suppresses RANKL-induced osteoclast differentiation and bone loss in ovariectomized mice, providing a promising target for antiresorptive therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shown is a schematic diagram of the results of the present invention showing the upregulation of PRMT6 in response to RANK signal activation.

[0011] Figure 2 Schematic diagram showing the results of PRMT6 promoting RANKL-induced osteoclastogenesis in vitro in the present invention.

[0012] Figure 3 Graph showing the results of the present invention showing that PRMT6 deficiency alleviates ovariectomy-induced bone loss.

[0013] Figure 4 Schematic diagram showing the results of the present invention showing that PRMT6 deficiency reduces the HIF-1 signaling pathway in RANKL-induced BMMs.

[0014] Figure 5 Schematic diagram showing the results of the present invention showing that PRMT6 deficiency leads to RANKL-induced BMMs metabolism shift from glycolysis to fatty acid oxidation.

[0015] Figure 6 Schematic diagram showing the results of the present invention showing that PRMT6 reprograms the metabolic transition of RANKL-induced BMMs by regulating chromatin accessibility.

[0016] Figure 7 Shown is a schematic diagram of the results of the present invention showing that inhibition of PRMT6 alleviates ovariectomy-induced osteoporosis.

[0017] Figure 8 Schematic diagram showing the results of the effects of PRMT6 inhibition on alkaline phosphatase or bone mineralization in the present invention.

[0018] Figure 9 It is shown that PRMT6 in the present invention - / - Cells and PRMT6 + / + Schematic representation of the results showing cell accessibility to the osteoclast marker Mmp9.

[0019] Figure 10 Schematic diagram showing the results of the effects of glycolysis inhibition on osteoclast differentiation and PRMT6 deficiency on fatty acid (β-) oxidation in the present invention.

[0020] Figure 11 Schematic diagram showing the effects of RANKL stimulation on glycolysis and fatty acid oxidation during normal osteoclastogenesis in the present invention.

[0021] Figure 12 Schematic diagram showing the effects of ovariectomy on the uterine weight and body weight of mice in the present invention.

[0022] Figure 13 Shown is a schematic diagram of the results of genotyping of PRMT6-deficient mice in the present invention. DETAILED DESCRIPTION

[0023] The present invention provides use of a PRMT6 inhibitor in preparing an anti-bone resorption product.

[0024] In the present invention, the PRMT6 inhibitor can be a PRMT6 protein activity inhibitor or a PRMT6 gene inhibitor. Wherein, the gene inhibitor refers to a molecule that has an inhibitory effect on a gene. The inhibitory effect on a gene includes, but is not limited to, inhibiting the expression or activity of the gene. Taking the PRMT6 gene as an example, a PRMT6 gene inhibitor refers to a molecule that has an inhibitory effect on PRMT6, that is, the target gene of the PRMT6 gene inhibitor is PRMT6. The inhibitory effect on PRMT6 includes, but is not limited to, inhibiting the expression or activity of PRMT6.

[0025] In some specific embodiments, the nucleotide sequence of the PRMT6 gene can be obtained through Gene ID: 55170 (Mus musculus) or Gene ID: 55170 (Homo) in combination with the public database NCBI.

[0026] Inhibiting gene activity means reducing gene activity and thus reducing the biological function of the gene. Preferably, the gene activity is reduced by at least 10%, such as at least 30%, 50%, 70% or 90%, compared to before inhibition.

[0027] Inhibiting gene expression may be inhibiting gene transcription or translation. Specifically, it may mean preventing gene transcription or reducing gene transcription activity, preventing gene translation or reducing gene translation level.

[0028] Those skilled in the art can use conventional methods to regulate gene expression, such as gene knockout, homologous recombination, interfering RNA, etc.

[0029] The inhibition of gene expression can be verified by detecting the expression level through PCR and Western Blot.

[0030] Preferably, gene expression is reduced by at least 10%, more preferably by at least 30%, even more preferably by at least 50%, even more preferably by at least 70%, even more preferably by at least 90%, and most preferably, the gene is not expressed at all compared to the wild type.

[0031] The anti-bone resorption product must include a protein activity inhibitor or a gene inhibitor, and use the protein activity inhibitor or the gene inhibitor as an effective ingredient for the aforementioned efficacy.

[0032] In the product, the active ingredient that exerts the aforementioned function may be only a protein activity inhibitor or a gene inhibitor, or may contain other molecules that can exert the aforementioned function.

[0033] That is, the protein activity inhibitor or gene inhibitor is the only active ingredient or one of the active ingredients of the product.

[0034] The product can be a single-component substance or a multi-component substance.

[0035] The product is mainly targeted at mammals. The mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0036] The protein activity inhibitor or gene inhibitor can be selected from one or more of nucleic acid molecules, polypeptides, proteins, small molecules or viruses.

[0037] In some specific embodiments, the protein activity inhibitor may be EPZ020411, whose CAS number is 1700663-41-7, and whose chemical structure is as follows:

[0038]

[0039] In some embodiments, the nucleic acid molecule can be any one or more of an antisense oligonucleotide, a double-stranded RNA (dsRNA), a short hairpin RNA (shRNA), a nucleic acid construct, or a nucleic acid composition. The nucleic acid construct comprises a nucleic acid fragment encoding a Cas protein and a guide RNA; or the nucleic acid composition comprises a Cas protein or an RNA molecule encoding a Cas protein, and a guide RNA or an RNA molecule encoding a guide RNA.

[0040] Furthermore, the nucleotide sequence encoding the guide RNA is shown as any one or more of SEQ ID No. 1: ACG AAT CCC AGC AGGCCC CG or SEQ ID No. 2: GAG ATC GCC TAT GCA AGT TG.

[0041] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the Cas protein is shown in SEQ ID No. 3.

[0042] In some embodiments, the anti-bone resorption product is a product that inhibits activation of HIF-1α and HIF-1 signaling pathways and / or a product that promotes fatty acid (β-) oxidation.

[0043] Furthermore, the product that inhibits activation of HIF-1α and HIF-1 signaling pathway is selected from one or more of the following products:

[0044] 1) Products that reduce HIF-1α expression;

[0045] 2) Products that reduce Pkm expression;

[0046] 3) Products that reduce Ldha expression;

[0047] 4) Products that reduce Pdk1 expression;

[0048] 5) a product that decreases Hk1 expression;

[0049] 6) a product that decreases Pfkp expression;

[0050] 7) a product that decreases Pfkm expression. Wherein the decrease is a decrease in mRNA expression level relative to healthy bone marrow monocyte / macrophage cells.

[0051] Further, the product that promotes fatty acid (beta-) oxidation is selected from one or more of the following:

[0052] 8) a product that increases Ppard expression;

[0053] 9) a product that increases Cpt1a expression;

[0054] 10) a product that increases Acox expression;

[0055] 11) a product that increases Hadha expression;

[0056] 12) a product that increases Ivd expression;

[0057] 13) a product that increases Abcd2 expression;

[0058] 14) a product that increases Scp2 expression. Wherein the increase is an increase in mRNA expression level relative to healthy bone marrow monocyte / macrophage cells.

[0059] In some embodiments, the nucleotide sequence of HIF-1α can be obtained by Gene ID: 15251 in conjunction with the public database NCBI; or, the nucleotide sequence of Pkm can be obtained by Gene ID: 18746 in conjunction with the public database NCBI; or, the nucleotide sequence of Ldha can be obtained by Gene ID: 16828 in conjunction with the public database NCBI; or, the nucleotide sequence of Pdk1 can be obtained by Gene ID: 228026 in conjunction with the public database NCBI; or, the nucleotide sequence of Hk1 can be obtained by Gene ID: 15275 in conjunction with the public database NCBI; or, the nucleotide sequence of Pfkp can be obtained by Gene ID: 56421 in conjunction with the public database NCBI; or, the nucleotide sequence of Pfkm can be obtained by Gene ID: 18642 in conjunction with the public database NCBI; or, the nucleotide sequence of Ppard can be obtained by Gene ID: 19015 in conjunction with the public database NCBI; or, the nucleotide sequence of Cpt1a can be obtained by Gene ID: 19015 in conjunction with the public database NCBI. The nucleotide sequence of Acox3 can be obtained through Gene ID: 12894 in conjunction with the public NCBI database; or, the nucleotide sequence of Hadha can be obtained through Gene ID: 97212 in conjunction with the public NCBI database; or, the nucleotide sequence of Ivd can be obtained through Gene ID: 56357 in conjunction with the public NCBI database; or, the nucleotide sequence of Abcd2 can be obtained through Gene ID: 26874 in conjunction with the public NCBI database; or, the nucleotide sequence of Scp2 can be obtained through Gene ID: 20280 in conjunction with the public NCBI database. In some embodiments, the anti-bone resorption product is an osteoporosis treatment drug. The product is a pharmaceutical. The dosage form of the pharmaceutical is not specifically limited, for example, an oral preparation or an injection. The pharmaceutical can be a sustained-release preparation.

[0060] Furthermore, the osteoporosis may be primary osteoporosis or secondary osteoporosis. Specifically, the primary osteoporosis is selected from one or more of postmenopausal osteoporosis, senile osteoporosis, or idiopathic osteoporosis. Preferably, the osteoporosis is postmenopausal osteoporosis.

[0061] The present invention also provides the use of a PRMT6 inhibitor in the preparation of an epigenetic regulation product, wherein the epigenetic regulation product is a product that regulates chromatin accessibility.

[0062] In some embodiments, the epigenetic regulation product is selected from one or more of the following products:

[0063] 15) Products that increase the expression of the open histone mark H3K27ac;

[0064] 16) Products that increase the expression of the open histone mark H3k56ac;

[0065] 17) Products that increase the expression of the open histone mark H3K4me3;

[0066] 18) Products that reduce the expression of the repressive histone mark H3k27me3;

[0067] 19) Products that reduce the expression of the repressive histone mark H3K9me3;

[0068] 20) A product that reduces the expression of the histone marker H3R2me2a, wherein the increase / decrease refers to an increase / decrease in protein expression relative to healthy bone marrow monocytes / macrophages.

[0069] In this application, the term "anti-resorptive" generally refers to the process of inhibiting or slowing down the breakdown and resorption of bone by osteoclasts.

[0070] In this application, the term "accessible histone mark" generally refers to histone modifications associated with the open state of chromatin. Chromatin openness refers to the looseness of the chromatin structure, which determines whether transcription factors and other regulatory proteins can enter and interact with chromatin, thereby affecting gene transcriptional activity.

[0071] In this application, the term "repressive histone mark" generally refers to histone modifications associated with the closed state of chromatin. Modifications associated with the closed state of chromatin change the structure of chromatin, making it more compact, thereby limiting the binding of transcription factors and other regulatory proteins, leading to downregulation or shutdown of gene expression.

[0072] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0073] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0074] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0075] The sequence information used in this application is as follows:

[0076] Cas9 RNA (wherein T is U in the RNA) SEQ ID No. 3:

[0077]

[0078] The materials used in the examples of this application are as follows:

[0079]

[0080]

[0081] **Zhang,H.,Han,C.,Li,T.,Li,N.,and Cao,X.(2019).The methyltransferasePRMT6attenuates antiviral innate immunity by blocking TBK1-IRF3signaling.Cell Mol Immunol 16,800-809.

[0082] The experimental methods used in the examples of this application are as follows:

[0083] Experimental animal preparation

[0084] PRMT6-deficient mice were prepared using the CRISPR-Cas9 gene editing system. Specifically, two guide RNAs (5′-ACG AAT CCC AGC AGG CCC CG-3′ and 5′-GAG ATC GCC TAT GCA AGT TG-3′) were first designed to target a single exon encoding PRMT6 located on chromosome 3. Cas9 mRNA and the above-mentioned gRNA were then microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. These sequence-verified F0 generation mice were further mated with C57BL / 6J mice to generate positive F1 generation heterozygous mice, which were then bred to obtain viable homozygous mice. Mice with the expected genotype were identified by tail genomic DNA PCR using the following primers: 5′-TTTCGCCGTCTGGTTTCA-3′ and 5′-GGTCAGGGATGCTCACTTTT-3′ for wild type; 5′-AGGCTACCCATACGTTCT-3′ and 5′-CCTTTCTCCCAGTTTCAT-3′ for PRMT6 knockout type. Figure 13 All mice were bred and maintained under specific pathogen-free (SPF) conditions. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Animals and approved by the Scientific Investigation Committee of the Naval Medical University.

[0085] Cell culture

[0086] Mouse bone marrow cells (BMCs) were isolated from 4-6 week old littermate mice by flushing the bone marrow from the tibia and femur. Primary MSCs were obtained by culturing freshly isolated BMCs in α-MEM (Hyclone, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin and streptomycin (Gibco, USA). The culture medium was changed every 3 days, and the first-generation MSCs were treated with osteogenic induction medium containing 50 μg / ml ascorbic acid, 5 mM β-glycerophosphate, and 100 nM dexamethasone (Cyagen, USA). To induce osteoclastogenesis in vitro, BMCs were first induced with 50 ng / ml M-CSF (Preprotech, USA) for 3 days to form bone marrow-derived monocytes / macrophages (BMMs), followed by stimulation with an additional 100 ng / ml RANKL (Peprotech, USA) for 4-6 days.

[0087] CCK-8 assay

[0088] 10 μl of Cell Counting Kit-8 reagent (NCMBiotech, China) was added to each well of a 96-well plate containing 100 μl of culture medium, followed by incubation for an additional 3 hours at 37°C under 5% CO 2 . The absorbance was measured at 450 nm using a microplate reader (Bio-Tek, USA).

[0089] Quantitative RT-PCR

[0090] Total RNA from cultured cells was extracted using TRIzol (Invitrogen, USA) according to the manufacturer's instructions and quantified using a NanoDrop 2000 (Thermo Fisher Scientific, USA). RNA was then reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara, Japan). qRT-PCR was performed using SYBR Premix ExTaq II (Takara, Japan) on a QuantStudio™3 RT-PCR system. Relative gene expression was determined by 2- ΔΔ Determined by Ct method.

[0091] Western Blot

[0092] The cells were first washed with ice-cold PBS and then lysed in RIPA buffer containing protease and phosphatase inhibitors (Epizyme, China). The lysate was sonicated and centrifuged at 14,000 rpm for 15 minutes at 4°C. The total protein concentration of a small amount of lysate was determined using a BCA protein assay kit (Epizyme, China), and most of the remaining lysate was diluted with 5× SDS loading buffer and heated at 95°C for 10 minutes. Then, equal amounts of total protein from each sample were separated on 10% or 12.5% ​​SDS-polyacrylamide gels and subsequently transferred to PVDF membranes using a wet transfer apparatus. The membranes were blocked with protein-free rapid blocking solution (Epizyme, China) for 30 minutes at room temperature and then incubated with primary antibodies overnight at 4°C. After incubation with goat anti-rabbit or anti-mouse IgG secondary antibodies conjugated to HRP for two hours at room temperature, the immunoreactive bands on the membranes were visualized using enhanced chemiluminescence (Epizyme, China).

[0093] Chromatin immunoprecipitation (ChIP)

[0094] use ChIP experiments were performed using an enzymatic chromatin IP kit (CST, USA) according to the manufacturer's instructions. Briefly, cells were treated with 1% formaldehyde for 10 minutes at room temperature for chromatin cross-linking. After the reaction was terminated by adding glycine for 5 minutes, the nuclei were isolated by incubation in cold buffer containing DTT and a protease inhibitor cocktail for 10 minutes. The chromatin was then digested with Micrococcal Nuclease at 37°C for 20 minutes and released by ultrasonic sonication. The chromatin fragments were confirmed to be 150-900bp by agarose gel electrophoresis. Subsequently, ChIP was performed using primary antibodies against H3 (provided in the kit), H3R2me2a (Abcam, UK) and IgG (provided in the kit). The precipitated DNA samples were quantified by qRT-PCR, and the data were expressed as a percentage of the input DNA.

[0095] ALP and Alizarin Red Staining

[0096] After 15 minutes of fixation of MSCs for 5 days of osteogenic induction, they were fixed with 4% paraformaldehyde at room temperature and then stained with an ALP assay kit (Solarbio, China) by azo coupling method in the dark for 15 minutes to detect alkaline phosphatase (ALP) activity. ALP activity was determined using an ALP microplate test kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. Cells stained with alizarin red underwent 14 days of osteogenic induction. After fixation, they were stained with 1% alizarin red solution (Sigma-Aldrich, USA) at 37°C for 20 minutes and washed with PBS to remove nonspecific staining. Subsequently, the mineralized nodules were dissolved with 10% pyridinium chloride (Sigma, USA) for semi-quantitative analysis using an absorbance of 562 nm.

[0097] TRAP staining and bone resorption assay

[0098] TRAP staining was performed when mature osteoclasts were successfully generated in the control group. The cells were first fixed with 4% formaldehyde for 15 minutes, treated with 0.1% Triton-100 for 30 minutes, and then incubated with TRAP staining solution (Servicebio, China) for 1 hour in the dark at 37°C for observation. For bone resorption assays, BMMs were seeded on the surface of bovine cortical bone slices (JoyTech, China) and induced to differentiate into osteoclasts with 50ng / ml M-CSF and 100ng / ml RANKL. After osteoclast maturation, they were further cultured for two days, and then the surface of the slices were carefully cleaned and bone resorption pits were observed using SEM (ZEISS, Germany).

[0099] Lactate determination

[0100] The cell culture supernatants were collected at 0, 6, 12, and 24 h after RANKL induction, and the lactate concentration was measured using a lactate assay kit (Sangon Biotech, China) according to the manufacturer's instructions.

[0101] Seahorse

[0102] The extracellular acidification rate (ECAR) was measured using an XF-96 Extracellular Flux Analyser (Seahorse Bioscience, Agilent Technologies, USA). Baseline values ​​of ECAR were measured after the addition of 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxy-D-glucose (Sigma, USA).

[0103] FAOBlue

[0104] Cells were incubated with 5 μM FAOBlue in serum-free medium for 40 minutes at 37°C. After washing twice, blue fluorescence (Ex. 405 nm / Em. 430-480 nm) was observed using a fluorescence microscope (LEICA, Germany) and quantified using Image J software (NIH, USA).

[0105] ATAC-seq

[0106] Given that changes in chromatin structure precede gene transcription and protein expression, ATAC-seq was performed at 0 and 12 hours after RANKL stimulation, as well as RNA-seq and proteomic analysis at 0 and 24 hours after RANKL stimulation. ATAC-seq was performed according to the method described in Buenrostro, JD, Giresi, PG, Zaba, LC, Chang, HY, and Greenleaf, WJ (2013). Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10, 1213-1218. Briefly, nuclei were first extracted from the sample, and the nuclear pellet was resuspended in Tn5 transposase reaction mix. The transposition reaction was incubated at 37°C for 30 minutes. After transposition, equimolar amounts of Adapter1 and Adapter2 were added, and the library was subsequently amplified by PCR. After PCR reaction, the library was purified by AMPure beads and the library quality was assessed using Qubit. Index-encoded samples were clustered using the TruSeqPECluster Kit v3-cBot-HS (Illumina) on the cBot Cluster Generation System according to the manufacturer's instructions. After cluster generation, library preparation was performed on the Illumina platform for sequencing to generate 150bp paired-end reads. The raw fastq format data was first processed using fastp (version 0.20.0) to remove reads containing adapters, poly-N, and low quality. At the same time, the Q20, Q30, and GC content of the clean data were calculated. The clean reads were aligned to the reference genome using BWA mem. Reads derived from mitochondrial DNA and chloroplast DNA, incorrect pairing, and PCR duplicates were discarded to obtain high-quality data (MAPQ ≥ 13). All peak calls were performed using MACS2 (version 2.1.0), and ChIPseeker was used to retrieve the nearest genes around the peak and annotate the genomic region of the peak. Peak-associated genes were identified using ChIPseeker, and Gene Ontology (GO) enrichment analysis was performed using the GOseq R package to determine functional enrichment results. KOBAS software was used to test for statistical enrichment of peak-associated genes within Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Peaks from different groups were merged using 'bedtools merge'. The mean RPM of each group in the merged peaks was calculated. Only peaks with an RPM fold change of more than 2 were considered differential peaks.

[0107] RNA-seq

[0108] After total RNA was extracted using Trizol reagent, mRNA was enriched using Oligo(dT) beads and fragmented into short fragments. These mRNAs were then reverse transcribed into cDNA using random primers. The cDNA fragments were then purified using the QiaQuick PCR Extraction Kit, end-repaired, poly(A) added, and ligated with Illumina sequencing adapters. The ligation products were size-selected by agarose gel electrophoresis, PCR amplified, and sequenced using an Illumina HiSeq™ 2500. To obtain high-quality data, the raw reads were further filtered to remove reads containing adapters, reads containing more than 10% unknown nucleotides, and reads containing more than 50% low-quality (Q value <= 20) bases. Reads that aligned to rRNA were then removed using the short-read alignment tool Bowtie2. The remaining reads were then aligned to the reference genome using TopHat2 (version 2.0.3.12). Gene abundance was quantified using RSEM software and normalized using the FPKM (fragments per kilobase of transcript per million mapped reads) method.

[0109] Proteome

[0110] The cells were transferred to a 1.5 ml tube and lysed with DB lysis buffer (8 M urea, 100 mM triethylamine bicarbonate, pH 8.5), followed by sonication on ice for 5 minutes. The lysate was centrifuged at 12,000 g for 5 minutes at 4 ° C, and the supernatant was incubated at 56 ° C for 1 hour with 10 mM DL-dithiothreitol, followed by alkylation with sufficient iodoacetamide for 1 hour at room temperature in the dark. Each protein sample was taken, supplemented to 100 μL with DB lysis buffer, and then digested with trypsin and 100 mM triethylamine bicarbonate for 4 hours at 37 ° C. Then, trypsin and CaCl2 were added for overnight digestion. The digested sample was mixed with formic acid, adjusted to a pH below 3, and centrifuged at 12,000 g for 5 minutes at room temperature. The supernatant was slowly loaded onto a C18 desalting column, washed three times with wash buffer (0.1% formic acid, 3% acetonitrile), and then elution buffer (0.1% formic acid, 70% acetonitrile) was added. The eluate of each sample was collected and freeze-dried. The freeze-dried powder was then dissolved and separated using a C18 column (Waters BEH C18, 4.6×250 mm, 5 μm) on a Rigol L3000 HPLC system. UHPLC-MS / MS analysis was performed using a nanoElute UHPLC system (Bruker, Germany) coupled to a tims TOF pro2 mass spectrometer (Bruker, Germany). All obtained spectra were searched in the UniProt database using the search engine MaxQuant (Bruker, Tims) and further used to filter the search results. Protein quantification results were statistically analyzed by T-test.

[0111] Ovarian removal

[0112] PRMT6 + / + (8) and PRMT6 - / -(8) 12-week-old female littermates were randomly assigned to either ovariectomy (4 per group) or sham surgery (4 per group). All experimental mice (16 in total) were anesthetized with isoflurane before the surgical procedure. The ovaries were exposed through a small dorsal incision and then carefully removed after ligation of the accompanying blood vessels and fallopian tubes. For the sham surgery group, the exposed ovaries were placed back into the abdominal cavity. After wound closure, the mice were placed on an electric blanket at 37°C until they recovered from anesthesia. After 5 weeks, the mice were euthanized and blood samples were collected for serum separation. Intact femurs were dissected and fixed in 4% formaldehyde for 2 days and then stored in 70% ethanol. For inhibitor administration, 10-week-old female C57BL / 6J mice (20) were randomly divided into four groups: sham surgery (5), OVX (5), OVX+EPZ (LD) (5), and OVX+EPZ (HD) (5). Mice were treated with EPZ via intraperitoneal injection for 5 weeks at a dose of 5 or 10 mg per mouse per day, corresponding to the OVX+EPZ (LD) and OVX+EPZ (HD) groups. Mice in the sham and OVX groups were injected with an equal volume of saline.

[0113] Micro-CT

[0114] The distal femur was scanned using a SkyScan 1076 (Bruker, Germany) microCT scanner at a resolution of 9 μm per pixel. To analyze cancellous microarchitecture, 200 sections beneath the growth plate were examined, and parameters such as bone mineral density (BMD), bone volume to total volume ratio (BV / TV), cancellous trabecular number (Tb.N), cancellous trabecular thickness (Tb.Th), and cancellous trabecular spacing (Tb.Sp) were calculated. Femoral cortical thickness (Ct.Th) was measured in 100 sections of the midshaft.

[0115] Histology and immunohistochemistry

[0116] Femurs were decalcified in 10% EDTA, dehydrated through a series of ethanol treatments (70%–100%), embedded in paraffin, and cut into 5-μm-thick sections. These sections were subsequently stained with hematoxylin and eosin (HE) and tautomer (TRAP). For immunohistochemistry, sections were antigen-retrieved by immersion in citric acid buffer at 99°C for 20 minutes, then incubated overnight at 4°C with an anti-PRMT6 primary antibody (Santa Cruz, USA), followed by incubation with an HRP-conjugated secondary antibody. Sections were then developed using a DAB kit (Beyotime Biotech, USA).

[0117] ELISA

[0118] The CTX-1 ELISA kit (CST, USA) was used to detect the level of CTX-1 in serum according to the manufacturer's instructions.

[0119] Statistical analysis

[0120] All data were analyzed using SPSS 24.0 (IBM, USA). Statistical differences were analyzed using unpaired two-tailed Student's t-test or one-way analysis of variance (ANOVA) followed by least significant difference test. A p-value less than 0.05 was considered statistically significant.

[0121] Example 1 Omics analysis reveals that PRMT6 is upregulated in response to RANK signaling activation

[0122] To identify potential regulators of metabolic reprogramming following RANK signaling activation, we used RANKL to stimulate bone marrow monocytes / macrophages (BMMs) for RNA sequencing. Principal component analysis revealed that clusters before and after stimulation were separated 24 hours after induction ( Figure 1 A), indicating that there are significant differences in overall gene expression between samples before and after RANKL stimulation. This example identified 2454 differentially expressed genes (DEGs) (FC≥2, p<0.05), of which 2284 genes were upregulated and 170 genes were downregulated ( Figure 1 B). Gene ontology enrichment analysis of these DEGs highlighted ten significantly enriched GO biological processes related to cellular metabolism and molecular mechanisms ( Figure 1 C). These biological processes were analyzed by Venn diagram ( Figure 1 D), sixteen overlapping DEGs were revealed, among which PRMT6 ( Figure 1 E) and Auts2( Figure 1 F) was significantly upregulated. However, the remaining fourteen DEGs have been previously reported in osteoclastogenesis (Table 1). Considering that PRMT6 has a higher expression level than Auts2, PRMT6 is likely to be a candidate regulator that mediates the conversion of RANK signaling activation to metabolic reprogramming. Further ATAC-seq data analysis also showed that after 12 hours of RANKL stimulation, the genomic accessibility of the gene loci of PRMT6 and osteoclast marker Mmp9 increased ( Figure 1 G). Subsequently, we verified that PRMT6 was expressed in RANKL-induced BMMs at the gene ( Figure 1 H-1J) and protein levels ( Figure 1 In addition, in ovariectomized bone tissue, which usually indicates overactive RANK signaling in vivo, PRMT6 expression was also increased compared with the Sham group ( Figure 1 L). Thus, activation of RANK signaling upregulates PRMT6 expression, making it a possible regulator of metabolic reprogramming.

[0123] Table 1. Figure 1 There are 16 overlapping DEGs in the Venn diagram of D.

[0124]

[0125]

[0126] Among them, asterisks indicate genes previously reported to be related to osteoclasts, while hash marks indicate genes that have not been reported in osteoclast studies.

[0127] Example 2 PRMT6 promotes RANKL-induced osteoclastogenesis in vitro

[0128] To evaluate the role of PRMT6 in RANKL-induced osteoclastogenesis, a selective inhibitor of PRMT6 (EPZ020411) was used in this example to interfere with osteoclast differentiation. Notably, PRMT6 inhibitors inhibited the formation of TRAP-positive multinuclear cells (TRAP+ / MNCs) in a dose-dependent manner ( Figure 2 A-2C, and Figure 8 A) and reduces osteoclast-induced bone erosion ( Figure 2 A and 2D). Then, in this example, PRMT6 was compared. + / + and PRMT6 - / - Osteoclastogenesis between BMMs. + / + Mature osteoclasts differentiated from BMMs exhibited strong bone resorption capacity. - / - The cells showed a significant decrease in the number and area of ​​TRAP+ / MNCs ( Figure 2 E-2G), and fewer absorption pits ( Figure 2 E and 2H). In addition, PRMT6 deficiency reduced Acp5 ( Figure 2 I), Ctsk( Figure 2 J) and Mmp9 ( Figure 2 K) gene expression, and protein expression at 4 days ( Figure 2 L). Considering the known roles of activated NF-κB and MAPK signaling in RANKL-induced osteoclastogenesis, this example examined the effect of PRMT6 deficiency on their activation. + / + In cells, RANKL stimulation rapidly activated the phosphorylation of p65, erk, jnk, and p38. - / - The activation of p65 and ERK in cells was significantly reduced, while the activation of p38 and JNK was basically unaffected ( Figure 2M and 2N). In addition, this example evaluates the effect of PRMT6 on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In this example, there are also the following results: PRMT6 inhibition ( Figure 8 B, 8C, 8E and 8F) or defective ( Figure 8 D, 8G, and 8H) had no significant effect on alkaline phosphatase (ALP) or bone mineral formation. These results together support the role of PRMT6 in promoting RANKL-induced osteoclastogenesis in vitro without significant effect on osteogenesis.

[0129] Example 3 PRMT6 deficiency alleviates ovariectomy-induced bone loss

[0130] Given that ovariectomized mice are often accompanied by high levels of RANKL and over-activated osteoclast formation, this example studies the effect of PRMT6 on ovariectomy-induced bone loss. In the sham group, Micr-CT-based parameters, including trabecular bone mineral density (Tb.BMD), bone volume ratio (BV / TV), trabecular number (Tb.N) and trabecular separation (Tb.Sp), were significantly increased in the sham group (P<0.05). - / - and PRMT6 + / + There were no significant differences between the mice ( Figure 3 A-3B). However, trabecular thickness (Tb.Th) was slightly increased in PRMT6- / - mice ( Figure 3 B), while the cortical thickness (Ct.Th) was slightly reduced ( Figure 3 B) Five weeks after ovariectomy, expression of PRMT6 + / + Compared with mice, PRMT6 - / - Mice showed significant improvement in these parameters ( Figure 3 A-3B), but there was no significant difference in Ct.Th ( Figure 3 B). Histomorphological analysis showed a similar trend. In the sham group, PRMT6 - / - and PRMT6 + / + There were no significant differences in the number of osteoclasts per unit bone perimeter (Oc.N / B.Pm) and the area of ​​osteoclasts per unit bone area (Oc.S / BS) between the mice ( Figure 3 C-3D). However, in femoral sections of OVX mice, PRMT6 - / - Oc.N / B.Pm and Oc.S / BS in mice and PRMT6+ / + Compared with mice, the Figure 3 C-3D). In addition, PRMT6 - / - The serum CTX-1 level in OVX mice was also significantly lower than that in PRMT6 + / + OVX mice, showing that PRMT6 deficiency inhibits bone resorption and osteoclast activity ( Figure 3 E) These findings indicate that PRMT6 supports osteoclastogenesis and promotes bone loss in ovariectomized mice.

[0131] Example 4 PRMT6 deficiency reduces RANKL-induced HIF-1 signaling in BMMs

[0132] To elucidate the potential mechanism of PRMT6 in RANKL-induced osteoclastogenesis, this example used PRMT6 - / - and PRMT6 + / + BMMs were subjected to ATAC-seq, mRNA-seq, and quantitative LC / LM proteomics analysis after and without RANKL stimulation. - / - Cells and PRMT6 + / + cells showed lower levels of accessibility to the osteoclast marker Mmp9 ( Figure 9 By comparing RANKL-induced PRMT6 - / - and PRMT6 + / + ATAC-seq data of the samples found that both groups had 54,080 overlapping accessible regions, of which 27,001 were specific to PRMT6. - / - Samples, 7903 of which are specific to PRMT6 + / + sample( Figure 4 A) RANKL-stimulated and PRMT6 + / + The genome-wide KEGG analysis of accessible regions in the cells revealed significant enrichment of the “HIF-1 signaling pathway” and the “glycolysis / gluconeogenesis signaling pathway”, with the highest enrichment factors ( Figure 4 B) To understand the transcriptional status of these signaling pathways, this example analyzed PRMT6 - / - and PRMT6 + / + RNA-seq data of BMMs 24 hours after RANKL induction ( Figure 4 C). KEGG enrichment analysis showed that "HIF-1 signaling pathway" and "glycolysis / gluconeogenesis signaling pathway" were among the top 12 significantly enriched pathways ( Figure 4 D) Further investigation of PRMT6 - / -KEGG enrichment analysis was performed on the significantly downregulated DEGs in the samples. The results showed that the HIF-1 signaling pathway ranked first in the KEGG gene set signal transduction category, and the glycolysis / gluconeogenesis signaling pathway was also significantly enriched ( Figure 4 E) Some key genes in the HIF-1 signaling pathway are expressed in PRMT6 - / - The expression level in cells was significantly lower than that of PRMT6 + / + cell( Figure 4 F). These findings were validated by qPCR, confirming the significant downregulation of genes associated with the HIF-1 signaling pathway (HIF-1α, Ldha, and Pdk1), as well as other important glycolysis key genes (Pkm, Hk1, Pfkp, and Pfkm) ( Figure 4 G). Considering PRMT6 - / - The transcription of HIF-1α was significantly reduced in the samples and its key role in the HIF-1 signaling pathway was evaluated in this example. + / + and PRMT6 - / - Protein expression in BMMs during osteoclastogenesis. The results showed that PRMT6 was expressed under RANKL stimulation. + / + The expression of HIF-1α in cells increased, while PRMT6 - / - The cells were never activated ( Figure 4 H). Furthermore, the addition of alfa-KG (α-KG), which is known to increase PHD activity and deplete HIF-1α, adversely affected osteoclastogenesis in vitro ( Figure 4 These data highlight PRMT6 as a key mediator of RANK signaling-induced HIF-1α expression and activation, supporting RANKL-induced osteoclastogenesis.

[0133] Example 5 PRMT6 deficiency causes RANKL-induced BMMs metabolism to shift from glycolysis to fatty acid oxidation

[0134] Given the well-known role of HIF-1α as a promoter of glycolysis and the significant enrichment of glycolysis / gluconeogenesis as described in Example 3, this example investigated whether PRMT6 deficiency attenuated glycolysis during osteoclastogenesis. Proteomic analysis showed that PRMT6 was downregulated after RANKL induction. + / + Glycolytic enzymes were widely upregulated in BMMs, and PRMT6 deficiency significantly weakened this upregulation effect ( Figure 5A and 5B). Key enzymes of glycolysis, including HK1, PFKM / L, PKM, and LDHA, showed similar patterns as verified by Western Blot, and PRMT6 deficiency prevented their upregulation at 12 and 24 hours after RANKL induction ( Figure 5 C). In addition, PRMT6 - / - Lactate produced by BMMs during osteoclast differentiation is higher than that produced by PRMT6 + / + Fewer cells ( Figure 5 D) To explore the role of PRMT6 in glycolysis in the early stages of osteoclast differentiation, the extracellular acidification rate (ECAR), which reflects the glycolytic capacity, was measured in this example. - / - The ECAR of BMMs was significantly lower than that of PRMT6 + / + BMMs( Figure 5 E and 5F). This example further inhibited glycolysis by using 2-deoxyglucose (2-DG), and found that osteoclast differentiation was significantly inhibited ( Figure 10 A-10C). These results indicate that in the absence of PRMT6, the activation of glycolysis is significantly inhibited during RANKL-induced osteoclast differentiation. In order to comprehensively evaluate the changes in metabolic patterns, this example used RNA-seq data to analyze the expression of PRMT6 induced by RANKL. - / - and PRMT6 + / + The main metabolic pathways between cells were compared. GSEA analysis showed that PRMT6 - / - Cellular glycolysis was significantly reduced ( Figure 5 G). and PRMT6 + / + Compared with the group, PRMT6 - / - Fatty acid oxidation was significantly increased in the group ( Figure 5 HI). In addition, genes related to the fatty acid oxidation pathway were widely upregulated in PRMT6- / - samples, including Ppard, Cpt1a, Acox3, Hadha, Ivd, Abcd2, and Scp2 ( Figure 5 J) In addition, PRMT6 was expressed 24 hours after RANKL induction. - / - The FAOBlue fluorescence intensity of BMMs was higher than that of PRMT6 + / + cells, indicating enhanced fatty acid oxidation ( Figure 5 K and 5L). These findings suggest that PRMT6 deficiency leads to a metabolic shift from glycolysis to fatty acid oxidation when RANK signaling is activated. However, during normal osteoclastogenesis, RANKL stimulation induces an increase in glycolysis and a significant decrease in fatty acid oxidation ( Figure 11A and 11B-11C). PRMT6-deficient cells exhibit a markedly different metabolic pattern in response to RANKL stimulation than wild-type cells, highlighting the critical role of PRMT6 in promoting the metabolic switch between glycolysis and fatty acid oxidation.

[0135] Example 6 PRMT6 reprograms RANKL-induced metabolic transition in BMMs by regulating chromatin accessibility

[0136] Considering that PRMT6 catalyzes asymmetric dimethylation of H3R2 (H3R2me2a), which antagonizes trimethylation of H3K4 (H3K4me3), this example explored whether PRMT6 regulates the metabolic shift from fatty acid oxidation to glycolysis in BMMs induced by RANKL by regulating chromatin accessibility. In this example, ATAC-seq data were used to compare the expression of PRMT6 after RANKL stimulation. + / + and PRMT6 - / - Global chromatin accessibility in BMMs and discovery of PRMT6 - / - The overall accessibility of the sample is higher ( Figure 6 A). Differential accessibility analysis showed that PRMT6 - / - 26.3% of the peaks in the sample had RPM values ​​of PRMT6 + / + samples>1.3 times, while in PRMT6 + / + Only 11.4% of the peaks in the group had RPM values ​​of PRMT6 - / - Group>1.3 times ( Figure 6 B). Given the close relationship between chromatin accessibility and histone modification, in this example, GESA analysis was performed on RNA-seq data, and it was observed that PRMT6 deficiency had a significant effect on histone binding ( Figure 6 C) To investigate the relationship between histone modification and chromatin accessibility, this example involved PRMT6. - / - and PRMT6 + / + The expression levels of open histone marks (H3K27ac, H3k56ac, and H3K4me3), repressive marks (H3k27me3, H3K9me3), and the PRMT6 target H3R2me2a were examined in BMMs during RANKL-induced osteoclast differentiation. This example found that PRMT6 deficiency inhibited the increase in asymmetric dimethylation of H3R2, enhanced the expression of open histone marks, and reduced the expression of repressive marks ( Figure 6D). These results indicate that PRMT6 reduces chromatin accessibility by regulating H3R2 methylation (H3R2me2a). Considering the enhanced fatty acid oxidation in PRMT6-deficient cells, this example subsequently analyzed ATAC-seq data to compare the expression of fatty acid oxidation-related genes in PRMT6-deficient cells stimulated by RANKL. - / - and PRMT6 + / + Chromatin accessibility between BMMs. In this example, among the peaks corresponding to fatty acid oxidation-related genes, PRMT6 - / - The accessibility of the group was significantly higher than that of PRMT6 + / + Group( Figure 6 E). This suggests that increased chromatin accessibility due to PRMT6 deficiency may contribute to enhanced fatty acid β-oxidation. Subsequently, in this example, Venn analysis was performed on genes related to fatty acid oxidation ( Figure 6 F), including PRMT6 in ATAC-seq - / - Genes upregulated in chromatin accessibility, PRMT6 in RNA-seq - / - The mRNA expression of the genes in the group was upregulated and the mRNA expression of the genes in wild-type cells induced by RANKL was downregulated. This analysis identified 7 common genes, including Ppard, Akt2, Cpt1a, Hadha, Acox3, Irs2 and Abcd2, which may be potential targets of PRMT6 in regulating osteoclast differentiation ( Figure 6 F) Given the key roles of PPARD, Cpt1a, and Acox3 in fatty acid oxidation, this example performed ChIP-qPCR analysis and observed that PRMT6 + / + The enrichment of H3R2me2a at these gene loci in cells was significantly higher than that of PRMT6 - / - cell( Figure 6 G). In conclusion, PRMT6 plays a key role in promoting the metabolic switch between glycolysis and fatty acid oxidation through its epigenetic regulation of chromatin accessibility.

[0137] Example 7 Inhibition of PRMT6 Alleviates Ovariectomy-Induced Osteoporosis

[0138] Given the role of PRMT6 in promoting osteoclast differentiation in vivo and in vitro, this example investigated whether inhibition of PRMT6 could alleviate ovariectomy-induced osteoporosis. Five weeks after ovariectomy, mice showed significant uterine atrophy ( Figure 12 A). Injection of a selective PRMT6 inhibitor (EPZ020411) had no significant effect on the body weight of mice ( Figure 12 B), Micro-CT images show that mice injected with PRMT6 inhibitors had reduced trabecular bone loss compared with mice injected with saline ( Figure 7A). In addition, Micro-CT-based parameters, including Tb.BMD, BV / TV, Tb.N, Tb.Th, and Tb.Sp, showed dose-dependent improvements in mice injected with PRMT6 inhibitors compared with mice injected with saline ( Figure 7 B). However, PRMT6 inhibition failed to prevent the loss of cortical bone thickness ( Figure 7 A and 7B). Histological analysis showed that PRMT6 inhibition led to a significant decrease in the number of TRAP-positive osteoclasts ( Figure 7 C). This finding was further supported by quantitative analysis of histomorphometric parameters, including Oc.N / B.Pm and Oc.S / BS, which were significantly reduced after PRMT6 inhibitor treatment ( Figure 7 D). In addition, the serum CTX-1 level was significantly reduced after PRMT6 inhibitor injection compared with saline injection ( Figure 7 E). Taken together, these results demonstrate that PRMT6 inhibitors can attenuate ovariectomy-induced bone resorption and trabecular bone loss, highlighting its potential as a drug target for antiresorptive therapy.

[0139] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of PRMT6 inhibitors in the preparation of anti-bone resorption products.

2. The use according to claim 1, characterized in that The PRMT6 inhibitor is selected from one or more of nucleic acid molecules, polypeptides, proteins, small molecules or viruses.

3. The use according to claim 2, characterized in that The PRMT6 inhibitor is any one or more of EPZ020411, antisense oligonucleotide, double-stranded RNA, short hairpin RNA, nucleic acid construct or nucleic acid composition; wherein the nucleic acid construct comprises a nucleic acid fragment encoding a Cas protein and a guide RNA; or, the nucleic acid composition comprises a Cas protein or a ribonucleic acid molecule encoding a Cas protein, and a guide RNA or a ribonucleic acid molecule encoding a guide RNA.

4. The use according to claim 3, characterized in that The nucleotide sequence encoding the guide RNA is shown in any one or more of SEQ ID No. 1 or SEQ ID No.

2.

5. The use according to claim 1, characterized in that The anti-bone resorption product is a product that inhibits the activation of HIF-1α and HIF-1 signaling pathways and / or a product that promotes fatty acid (β-) oxidation.

6. The use according to claim 5, characterized in that The product that inhibits the activation of HIF-1α and HIF-1 signaling pathway is selected from one or more of the following products: 1) Products that reduce HIF-1α expression; 2) Products that reduce Pkm expression; 3) Products that reduce Ldha expression; 4) Products that reduce Pdk1 expression; 5) Products that reduce Hk1 expression; 6) Products that reduce Pfkp expression; 7) Products that reduce Pfkm expression.

7. The use according to claim 5, characterized in that The product that promotes fatty acid (β-) oxidation is selected from one or more of the following products: 8) Products that increase Ppard expression; 9) Products that increase Cpt1a expression; 10) Products that increase Acox expression; 11) Products that increase Hadha expression; 12) Products that increase Ivd expression; 13) Products that increase Abcd2 expression; 14) Products that increase Scp2 expression.

8. The use according to claim 1, characterized in that The anti-bone resorption product is an osteoporosis treatment drug; preferably, the osteoporosis is primary osteoporosis or secondary osteoporosis; more preferably, the primary osteoporosis is selected from one or more of postmenopausal osteoporosis, senile osteoporosis or idiopathic osteoporosis; more preferably, the osteoporosis is postmenopausal osteoporosis.

9. Use of a PRMT6 inhibitor in the preparation of an epigenetic regulatory product, wherein the epigenetic regulatory product is a product that regulates chromatin accessibility.

10. The use according to claim 9, characterized in that The epigenetic regulation product is selected from one or more of the following products: 15) Products that increase the expression of the open histone mark H3K27ac; 16) Products that increase the expression of the open histone mark H3k56ac; 17) Products that increase the expression of the open histone mark H3K4me3; 18) Products that reduce the expression of the repressive histone mark H3k27me3; 19) Products that reduce the expression of the repressive histone mark H3K9me3; 20) Products that reduce the expression of the histone mark H3R2me2a.

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