Application of ZNF750 in preparation of medicine for preventing and treating osteoporosis

Abstract

The invention discloses application of a zinc finger protein 750 (ZNF750) gene or an encoded protein thereof as a target spot in preparation of a medicine for preventing and treating osteoporosis, and belongs to the field of biological medicines. It is found for the first time that ZNF750 enhances Wnt / beta-catenin signal channel activity, promotes osteoblast differentiation and reduces adipocyte differentiation by down-regulating snail family transcription inhibition factor 1 (SNAI1) gene expression; the Znf750 gene silencing can reduce the bone mass of the mouse. Therefore, an overexpression medicine aiming at the ZNF750 coding gene or an accelerant or an activator aiming at the ZNF750 coding protein can increase osteoblasts, reduce bone marrow adipocytes and increase bone mass, and is a potential medicine for preventing and treating osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine technology and relates to the application of zinc finger protein 750 (ZNF750) in the preparation of drugs for preventing and treating osteoporosis. Background Art

[0002] Osteoporosis is a systemic bone disease characterized by low bone mass, damage to the ultrastructure of bone tissue, resulting in bone fragility and easy fracture. With the aggravation of population aging in China, the incidence of osteoporotic fractures has risen rapidly, becoming an important public health problem.

[0003] Currently, the main mechanisms of drugs for treating osteoporosis clinically include promoting bone formation and inhibiting bone resorption: there are few types of drugs for promoting bone formation, while drugs for inhibiting bone resorption will bring various adverse reactions and even increase the risk of cancer. Therefore, there is an urgent need to develop new treatment methods for osteoporosis.

[0004] Zinc finger protein 750 (ZNF750) is a transcription factor involved in terminal epidermal differentiation and cancer, with a C2H2 zinc finger structure and a nuclear localization domain. It is involved in DNA binding and protein-protein interactions. ZNF750 is involved in various biological processes such as keratinocyte and epithelial cell differentiation. ZNF750 has been identified as a potential tumor suppressor in various malignant tumors, and mutations in this gene are associated with psoriasiform dermatitis. However, there is no literature report on the study of ZNF750 regulating osteogenic and adipogenic differentiation of mesenchymal stem cells and thus regulating bone homeostasis. Currently, there is no research report in the existing technology documenting that ZNF750 has the function of regulating bone mass and bone homeostasis. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art and the actual needs, the present invention proposes the application of zinc finger protein 750 (ZNF750) in the preparation of drugs for preventing and treating osteoporosis. This drug can prevent and treat osteoporosis by targeting ZNF750. The present invention discovers for the first time that ZNF750 down-regulates the expression of the Snail family transcriptional repressor 1 (SNAI1) gene, enhances the activity of the Wnt / β-catenin signaling pathway, promotes osteoblast differentiation, and reduces adipocyte differentiation; silencing of the Znf750 gene reduces bone mass in mice. Therefore, drugs for overexpressing the gene encoding ZNF750 or promoters or activators for the protein encoded by ZNF750 can increase osteoblasts, reduce bone marrow adipocytes, and increase bone mass, and are potential drugs for preventing and treating osteoporosis.

[0006] The gene encoding ZNF750 is located on human chromosome 17 and mouse chromosome 11. The mature polypeptides of humans and mice contain 723 (human) and 703 (mouse) amino acids respectively, and the amino acid sequences are shown as SEQ NO ID.1 (human) or SEQ NO ID.2 (mouse). Existing reports show that ZNF750 has multiple functions, including participating in various biological processes such as keratinocyte and epithelial cell differentiation.

[0007] SEQ NO ID.1:

[0008] MSLLKERKPKKPHYIPRPPGKPFKYKCFQCPFTCNEKSHLFNHMKYGLCKNSITLVSEQDRVPKCPKSNSLDPKQTNQPDATAKPASSKSVANGLSAFDSKLQHSSAREDIKENLELQARGTHRCLGQKPALHRASPCKSPAPEAALGAQPALEGAARPSAFVPVGEHRLKGPDNAEAPETLALHNPTAKAVSFHTKSAFHTPGYPWKAGSPFLPPEFPHKISSTKGLGAISPYMHPTIPEYPPHFYTEHGLATIYSPYLLAGSSPECDAPLLSVYGTQDPRHFLPHPGPIPKHLAPSPATYDHYRFFQQYPSNLPIPYGFYRPESAFSSYGLRLPPVTGLTRDQSSHLLEEATLVYPASSPSRLNPSDPNRKHVEFESPIPEAKDSSKAGQRDTEGSKMSPRAGSAATGSPGRPSPTDFMQTSQTCEGLYDLSNKAASSALGRLYPPEQSLTAFRPVKKSTECLPAQAAETTAESPVSLNVVNGDPPAPTGSASLVSEAAPSSPDDSSGMGPLNLSKKSEINLAATHEPTYQGSPQAETASFSELQDLPLNLSVKDPCNTQAPRPAFPGRPRAAEPAAAVPQKTGTEGSEDGPSHPETKPGSLDGDGAPPTGPGEEAPDACAVDSSEEQKQTAAVALCQLAAYSPRNIRVGDGDAAAPEPACRQDTPTLSSMESQEAQCDLRPKGQKRTSLRDAGKSQQGAKKAKLQDTARVFTLRRRARVS

[0009] SEQ NO ID.2:

[0010] MSLLKERKPKKPHYIPRPPGKPFKYKCFQCPFTCNEKSHLFNHMKYGLCKNSITLVSEQDRIPKCPKSSSLDPKQTHQPEPTSKPATSKSLLNGLSSFDPKSQQGSAKEDAKENLEMQARGAHKGPQKPALQKEMAPEAILSTQPCLDSGVRHSAFVPVGEHRLRGPEDTEATEVLANSTTKASSFHAKSAFHTPGYPWKAGSPFLPPDFPHKISSTKGFGAISPYMHPAIPEYPHPFYAEHGLAAIYSPYLLTGNTPECETTLLSVYGTQDQRHFLSPAGPIPKHLNTSPSTYDHYRFFQQYHSNLPIPYGFYRPESAFPSYSLRLPSVTGITRDQSSRLLEDATLAYPASSPSELNLSSSHRKHTECEKGSPVPEAKDPSKDGQRDAEEAKMSPRAGSAATGSPGRPSPTNFTQTSQTFEGLCDLSNKAASSGTLERLQQAEQSPTAFKPVQRGSESPHSQPPANRTESPKSLQAMNGDPPAQTGSSNSFITEAPPSSPEDHSRIGPLNLSKKLETNPAATYGPMYASNAQADTLQDLPLNLSVKDLCNAWAPRPALPGPPQGAEPAATPKTETKGSEDRTSRVETPQDKAHSRTTPDVHTEDSSDEQKQTAAVALCQLAAYSPGNVRVADEEGTVQEPTRQDVPTLSATENLEAQCDLRPKGQKRTSQRDTGKSQQGTKKPKLNDPVPRVLTLRRRTRVS

[0011] According to the present invention, the ZNF750 can promote the osteogenic differentiation of mesenchymal stem / progenitor cells, while inhibiting the adipogenic differentiation of mesenchymal stem / progenitor cells, thereby increasing bone mass.

[0012] According to the present invention, ZNF750 enhances the activity of the Wnt / β-catenin signaling pathway by downregulating the expression of the Snail family transcriptional repressor 1 (SNAI1) gene, increases the expression of osteogenic differentiation marker genes Runx2, Osx, Alp, and Opn in mesenchymal cells, and inhibits the expression of adipogenic differentiation marker genes Pparγ, C / ebpα, Fabp4, and adipsin, thereby increasing the number of osteoblasts, reducing the number of adipocytes, and promoting bone mass increase.

[0013] The present invention provides a potential drug for preventing and treating osteoporosis. The drug targets the gene encoding ZNF750 or its protein, regulates the expression of genes or proteins related to osteoporosis, and achieves the purpose of preventing and treating osteoporosis.

[0014] 1. Use of zinc finger protein 750 (ZNF750) in the preparation of a drug for preventing and treating osteoporosis. It is characterized in that: ZNF750 is used to regulate the osteogenic / adipogenic differentiation of mesenchymal stem / progenitor cells, by down-regulating the expression of the SNAI1 (Snail family transcriptional repressor 1) gene, enhancing the activity of the Wnt / β-catenin signaling pathway, increasing the expression of osteogenic differentiation marker genes Runx2, Osx, Alp, and Opn, and inhibiting the expression of adipogenic differentiation marker genes Pparγ, C / ebpα, Fabp4, and adipsin, thereby increasing the number of osteoblasts, reducing the number of adipocytes, and promoting bone mass increase.

[0015] 2. The use according to claim 1, wherein the drug is a ZNF750 regulator, which is used to increase the number and activity of osteoblasts, and thus prevent and treat the osteoporosis.

[0016] 3. The use according to claim 1, wherein the drug is a ZNF750 regulator, which is used to reduce the number of adipocytes, and thus prevent and treat the osteoporosis.

[0017] 4. The use according to claim 1, wherein the drug is a ZNF750 regulator, which inhibits the expression of the SNAI1 gene, enhances the activity of the Wnt / β-catenin signaling pathway, and thus prevents and treats the osteoporosis.

[0018] 5. The use according to any one of claims 1-4, wherein the drug comprises at least one of an overexpression drug targeting the gene encoding ZNF750 or a promoter or activator drug targeting the protein encoded by ZNF750.

[0019] 6. The use according to claim 5, wherein the overexpression drug targeting the gene encoding ZNF750 comprises an overexpression vector targeting the gene encoding ZNF750, cells overexpressing ZNF750, or recombinant proteins.

[0020] 7. The use according to claim 5, wherein the promoter or activator drug targeting the ZNF750 protein comprises any one of small molecule compounds, nucleic acids, amino acids, and polypeptides.

[0021] 8. The application according to claim 5, wherein the dosage form of the drug includes oral liquid, injection, tablet, pill, dispersant, capsule, drop, granule, suspending agent or emulsion.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention discovers for the first time that ZNF750 can promote osteoblast differentiation and inhibit adipocyte differentiation, which helps to increase bone mass. In addition, the present invention discovers for the first time that ZNF750 enhances the activity of the Wnt / β-catenin signaling pathway by downregulating the expression of the SNAI1 gene, increases the expression of osteogenic differentiation marker genes Runx2, Osx, Alp, and Opn of mesenchymal stem / progenitor cells, and inhibits the expression of adipogenic differentiation marker genes Pparγ, C / ebpα, Fabp4, and adipsin, thereby increasing the number of osteoblasts, reducing the number of adipocytes, and promoting bone mass increase. ZNF750 has the potential to prevent and treat osteoporosis. Description of the Drawings

[0024] Figure 1 It is the result of ZNF750 promoting osteogenic differentiation of mouse stromal cell line ST2. A is the Znf750 mRNA expression level detected by qRT-PCR after transfection of Znf750 overexpression plasmid in ST2 cells; B is the ALP staining after osteogenic induction for 14 days; C is the mRNA expression of osteogenic differentiation key factors Runx2, Osterix, Alp, and Opn detected by qRT-PCR after osteogenic induction for 3 days; D is the protein expression of Runx2, Osterix, and ALP detected by Western blotting after osteogenic induction for 3 days.

[0025] Figure 2 It is the result of ZNF750 inhibiting adipogenic differentiation of mouse mesenchymal cell line C3H10T1 / 2. A is the Znf750 mRNA expression level detected by qRT-PCR after transfection of Znf750 overexpression plasmid in C3H10T1 / 2 cells; B is the Oil Red O staining after adipogenic induction for 5 days; C is the result of extracting Oil Red O from the stained cells with 100% isopropanol and then measuring OD520; D is the mRNA expression of adipogenic differentiation key factors Pparγ, C / ebpα, Fabp4, and adipsin detected by qRT-PCR after adipogenic induction for 2 days; E is the protein expression of adipogenic differentiation key factors PPARγ, C / EBPα, and FABP4 detected by Western blotting after adipogenic induction for 3 days.

[0026] Figure 3It is the result of Znf750 gene silencing inhibiting the osteogenic differentiation of primary bone marrow stromal cells (BMSCs) and promoting their adipogenic differentiation. A shows the knockdown of Znf750 gene expression in BMSCs infected with Znf750 shRNA detected by qRT-PCR; B shows the ALP staining after osteogenic induction for 14 days; C shows the mRNA expression of key osteogenic differentiation factors Runx2, Osterix, Alp, and Opn detected by qRT-PCR after osteogenic induction for 3 days; D shows the protein expression of key osteogenic differentiation factors Runx2, Osterix, and ALP detected by Western blotting after osteogenic induction for 3 days; E shows the Oil Red O staining after adipogenic induction for 5 days; F shows the result of extracting Oil Red O from the stained cells with 100% isopropanol and then measuring OD520; G shows the mRNA expression of key adipogenic differentiation factors Pparγ, C / ebpα, Fabp4, and adipsin detected by qRT-PCR after adipogenic induction for 2 days; H shows the protein expression of key adipogenic differentiation factors PPARγ, C / EBPα, and FABP4 detected by Western blotting after adipogenic induction for 3 days.

[0027] Figure 4 It is the result of ZNF750 transcriptionally inhibiting Snai1 gene expression and enhancing the activity of the Wnt / β-catenin signaling pathway. A shows the mRNA expression of Snai1 detected by qRT-PCR after overexpressing ZNF750 in the stromal cell line ST2; B shows the predicted binding sequence of ZNF750 and the Snai1 promoter by bioinformatics; C shows the verification by dual-luciferase reporter gene assay that ZNF750 transcriptionally inhibits the Snai1 promoter, and the transcriptional inhibitory effect disappears after mutation of the predicted binding site; D shows the changes in the proteins of the Wnt / β-catenin signaling pathway detected by Western

[0028] blotting after overexpressing ZNF750; E shows the changes in the proteins of the Wnt / β-catenin signaling pathway detected by Western blotting after overexpressing Snai1.

[0029] Figure 5Overexpression of Snai1 alleviates the dysregulation of mesenchymal stem / progenitor cell differentiation induced by ZNF750. Znf750 and Snai1 overexpression plasmids were co-transfected into ST2 cells (A-D) or C3H10T1 / 2 cells (E-H). A Detection of changes in Wnt / β-catenin signaling pathway proteins after co-transfection of Znf750 and Snai1 in ST2 cells by Western blotting; B ALP staining of ST2 cells after osteogenic induction for 14 d; C Detection of mRNA expression of key osteogenic differentiation factors Runx2, Osterix, Alp, and Opn by qRT-PCR after osteogenic induction for 3 d; D Detection of protein expression of key osteogenic differentiation factors Runx2, Osterix, and ALP by Western blotting after osteogenic induction for 3 d; E Oil Red O staining of C3H10T1 / 2 cells after adipogenic induction for 5 d; F Results of extracting Oil Red O from stained cells with 100% isopropanol and then measuring OD520; G Detection of mRNA expression of adipogenic differentiation factors Pparγ, C / ebpα, Fabp4, and adipsin by qRT-PCR after adipogenic induction for 2 d; H Detection of protein expression of adipogenic differentiation factors PPARγ, C / EBPα, and FABP4 by Western blotting after adipogenic induction for 3 d.

[0030] Figure 6 μCT results of the tibias of mice transplanted with BMSCs with silenced Znf750 gene. A 3D reconstruction diagram of the tibial metaphysis; B Trabecular bone volume fraction; C Trabecular bone thickness; D Trabecular bone mineral density; E Structure model index.

[0031] Figure 7 Histological staining results of the tibial metaphysis of mice transplanted with BMSCs with silenced Znf750 gene. A Results of ALP immunohistochemical staining; B Results of counting ALP-positive osteoblasts; C HE

[0032] staining results; D Results of counting adipocytes; E Percentage of adipocyte area in the total bone marrow area. Detailed implementation manners

[0033] To further elaborate on the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with examples and drawings. It can be understood that the specific implementation manners described herein are only used to explain the present invention, rather than limiting the present invention.

[0034] For those technical details or conditions not specified in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through regular commercial channels.

[0035] The materials and methods involved in the embodiments of the present invention are described as follows.

[0036] (1) Cell culture and induced differentiation

[0037] The mouse stromal cell line ST2 was cultured in α-MEM medium containing 10% fetal bovine serum (FBS), and the mouse mesenchymal cell line C3H10T1 / 2 was cultured in D-MEM medium containing 10% fetal bovine serum (FBS).

[0038] Isolation of primary bone marrow stromal cells (BMSCs): Primary bone marrow stromal cells were isolated by the whole bone marrow adherent culture method. After sacrificing 4-6-week-old mice, the bilateral femurs and tibias of the hind limbs were taken out under sterile conditions, the epiphyses were removed, the bone marrow cavities were exposed, and the bone marrow cavities were repeatedly rinsed with 5 mL of syringe containing DMEM medium to collect cells, and then inoculated into α-MEM containing 10% fetal bovine serum. The medium was changed for the first time after 3 days of culture, the non-adherent cells were removed, and the fresh culture medium was replaced after washing with PBS. The culture medium was changed every 3 days. Passage was carried out when the cell confluence reached 80%. The third-generation cells were used for subsequent experiments.

[0039] Osteogenic induction of BMSCs and ST2 cells: When the cell confluence reached about 80%, the α-MEM complete medium containing osteogenic inducer (10% FBS, 50 μg / ml ascorbic acid, 5 mmol / L β-glycerophosphate) was replaced. The above medium was changed every 3 days. mRNA and protein were collected after 72 hours of osteogenic induction, and alkaline phosphatase (ALP) staining was performed after 14 days of induction.

[0040] Adipogenic induction of BMSCs and C3H10T1 / 2: When the cell confluence reached 100%, the α-MEM complete medium containing adipogenic inducer (5 μg / mL insulin, 0.25 mM methylisobutylxanthine, 0.5 μM dexamethasone and 50 μM indomethacin) was replaced for cell differentiation. The medium was updated after 3 days of differentiation, and further differentiated in α-MEM containing 5 μg / mL insulin for 2 days, followed by Oil Red O staining. Oil Red O in the cells was extracted with 100% isopropanol, and the absorbance at 520 nm was measured. In addition, mRNA was extracted from the cells differentiated for 2 days, and protein was extracted from the cells differentiated for 3 days.

[0041] (2) Cell transfection

[0042] The expression construct of mouse Znf750 was from Origene (MR224624, Rockville, MD, USA). PCR primers were designed for the coding sequence (CDS) of Snai1, namely Forward Primer: TTGGTACCGAGCTCGGATCCGCCACCATGCCGCGCTCCTTCCTGGTC; Reverse Primer: GCTGGATATCTGCAGAATTCTCAGCGAGGGCCTCCGGAGC. Using mouse cDNA as a template, PCR amplification was carried out. The obtained CDS amplification fragment was ligated into the pcDNA3.1(+) vector from the BamHI / EcoRI site using a seamless cloning kit (TransGen Biotech, Beijing, China) to construct the Snai1 overexpression plasmid (Snai1 Construct).

[0043] Plasmid transfection: When the cell density reached 70%, transfection was carried out using Jet PRIME transfection reagent (Polyplus, Illkirch, France). Taking a 24-well plate as an example, 0.5 μg of plasmid DNA was taken into a 1.5 mL EP tube, 50 μL of Jet PRIME Buffer was added and mixed well for 10 s, then 1 μL of Jet PRIME was added and gently mixed. After standing for 10 minutes, the transfection reagent-nucleic acid complex was formed and added to each well of the cells, gently mixed, and placed in a CO2 cell culture incubator for culture. The medium was changed 4 hours after transfection.

[0044] (3) Quantitative real-time polymerase chain reaction (qRT-PCR)

[0045] Total cellular RNA was extracted using a total RNA extraction kit (Omega, USA). Reverse transcription was carried out using a first-strand cDNA synthesis kit (PerfectStart Uni RT&qPCR Kit, TransGen, Beijing, China). Real-time fluorescence quantitative PCR was performed using the SYBR Green fluorescence PCR method. Using β-actin as an internal reference, the relative expression levels of each target gene were calculated using the 2 －ΔΔCt calculation method: ΔCt = Ct value of the target gene - Ct value of the internal reference gene; ΔΔCt = ΔCt of the experimental group - ΔCt of the control group. All experiments were repeated 3 times.

[0046] (4) Western Blotting

[0047] Total cellular proteins were extracted using RIPA lysis buffer, separated by SDS-PAGE gel electrophoresis, transferred to a nitrocellulose membrane, blocked with 5% non-fat milk at room temperature for 1 h, incubated with the primary antibody overnight at 4 °C, then incubated with the horseradish peroxidase (HRP)-labeled secondary antibody at room temperature, and the expression of the target protein was detected using an ECL chemiluminescence kit (ABclonal, Wuhan, China).

[0048] (5) Lentivirus packaging and infection

[0049] The annealed oligonucleotide pair of ZNF750 shRNA (Sense strand:

[0050] GATCCGTTACAGAGACCGTACTTCATGTTCAAGAGACATGAAGTACGGTCTCTGTAATTTTTTACGCGTG; Anti-sense strand:

[0051] AATTCACGCGTAAAAAATTACAGAGACCGTACTTCATGTCTCTTGAACATGAAGTACGGTCTCTGTAACG) was inserted into the pLVX-shRNA2 vector at the BamHI / EcoRI site to construct the lentiviral expression plasmid of ZNF750 shRNA. The ZNF750 shRNA lentiviral plasmid was transfected into 293T cells using a lentivirus packaging kit (GeneCopoeia, Shanghai, China), and the supernatant was collected 48 h after transfection. The virus particles were purified using a virus purification reagent (Beyotime, Shanghai, China). Primary BMSCs were infected at a multiplicity of infection (MOI) of 20.

[0052] (6) Alkaline phosphatase staining

[0053] Alkaline phosphatase staining was performed 14 days after osteogenic induction differentiation. The cells were washed with 1×PBS buffer, fixed with 4% paraformaldehyde for 15 min, and incubated with the BCIP / NBT staining working solution (Beyotime, Shanghai, China) at room temperature in the dark for 15 min.

[0054] (7) Oil Red O staining

[0055] Oil Red O staining was performed 5 days after adipogenic induction differentiation. The cells were washed with 1×PBS buffer, fixed with 4% paraformaldehyde for 15 min, then the culture was rinsed with 60% isopropanol, and subsequently incubated in a 0.24% (w / v) Oil Red O solution for 5 min. To evaluate the staining intensity, the dye was extracted from the cells using 100% isopropanol and the optical density was measured at a wavelength of 520 nm.

[0056] (8) Promoter study

[0057] Primers were designed for the Snai1 promoter fragment (-1507 / +12 nt): Forward Primer: TCGCTAGCCTCGAGGATATCCATGCCAGCCAGCATCCATC; Reverse Primer: AGTACCGGATTGCCAAGCTTACTACGATCCCCTAGCAGCA; and amplified from mouse genomic DNA by PCR. The resulting fragment was subcloned into the EcoRV / HindIII restriction sites of the pGL4.14 [luc2 / Hygro] vector (Promega, USA) using a seamless cloning kit (TransGen Biotech, Beijing, China) to obtain the wild-type Snai1 promoter vector. To identify potential ZNF750 binding sites "CCTCAGG", bioinformatics analysis was performed using an online tool (https: / / jaspar.elixir.no / ). Subsequently, mutant primers were designed: Forward Primer: GGAAATCGCAGCGCACTGCGACTGCCCAACACCAACCTCA; Reverse Primer: GTTGGGCAGTCGCAGTGCGCTGCGATTTCCTGGCCTCGGG, and the ZNF750 binding site of the wild-type Snai1 promoter was deleted using a site-directed mutagenesis kit (Vazyme, Nanjing, China) to obtain the mutant Snai1 promoter vector.

[0058] (9) In vivo transplantation of Znf750-knockdown BMSCs

[0059] Eight-week-old female C57BL / 6 mice were purchased from Spf Biotechnology Co., Ltd. (Beijing, China) and housed in a SPF-class barrier environment with free access to food and water. The mice were randomly divided into two groups, namely the control lentivirus (Ctrl LV) group and the Znf750 knockdown lentivirus (Znf750 shRNA LV) group. The isolated primary BMSCs were infected with Znf750 shRNA LV and Ctrl LV respectively. After 24 hours, the cells of the two groups were digested with trypsin and resuspended in Hank's balanced salt solution, and 2.5x10 5 BMSCs were injected into the bone marrow cavity of the mice. One month after transplantation, the tibias of the mice were taken for bone phenotype analysis. The animal experiment was approved by the Animal Ethics Committee of Zhu Xianyi Memorial Hospital, Tianjin Medical University.

[0060] (10) μCT analysis

[0061] The tibial specimens of mice were scanned using a Scanco viva CT80 (Scanco Medical AG, Switzerland) with scanning parameters of 55 kVp and 145 μA. Three-dimensional reconstruction was performed using Scanco software. The region of interest was selected as a 1-mm area starting 0.1 mm below the growth plate.

[0062] (11) Immunohistochemistry

[0063] The dewaxed and hydrated tibial sections were subjected to the following steps in sequence: add trypsin to cover the tibial tissue, place it at 37 °C for 30 min for antigen retrieval, wash with PBS (5 min / time × 3 times); add 3% H2O2-methanol to cover the tibial tissue to eliminate endogenous peroxidase activity, let it stand at room temperature for 30 min, wash with PBS (5 min / time × 3 times); add 1% BSA-PBS solution and block at room temperature for 60 min; discard the blocking solution, add the ALP antibody (Hua'an Biotech, Hangzhou, China) diluted with 1% BSA-PBS solution to cover the bone tissue, incubate overnight at 4 °C; discard the primary antibody, wash with PBS (5 min / time × 3 times), then, incubate the sections with the enzyme-labeled secondary antibody at 37 °C for 1 hour, develop color with DAB solution, stain with hematoxylin for 3 s, dehydrate and mount the slides, observe under a microscope, take pictures and count the number of ALP + cells on the trabecular bone surface.

[0064] (12) HE staining of bone tissue

[0065] The dewaxed and hydrated tibial sections were subjected to the following steps in sequence: stain with hematoxylin for 3 s, wash with water until the waste liquid is colorless, differentiate with 1% hydrochloric acid ethanol for 3 s, stain with eosin for 3 min, wash with water until the waste liquid is colorless, dehydrate and mount the slides, observe under a microscope, take pictures and count the number and area of adipocytes.

[0066] (13) Statistical analysis

[0067] Statistical analysis was performed using GraphPad Prism 8.0 software. All data were expressed as mean ± standard deviation. Two-tailed unpaired independent Student t-test was used to compare the differences between two groups, while one-way ANOVA or two-way ANOVA was used to compare multiple groups, and Dunnett test or Least Significant Difference test was used for post hoc comparison respectively. A significance level of p < 0.05 was considered statistically significant.

[0068] Example 1

[0069] This example analyzed the effect of ZNF750 on the osteogenic differentiation of stromal cell line ST2 cells. The ZNF750 overexpression plasmid (Znf750 Construct) was transfected into ST2 cells, and the changes in osteogenic differentiation after overexpressing ZNF750 were analyzed. It was detected by qRT-PCR that the Znf750 mRNA was significantly increased in the Znf750 Construct group ( Figure 1 A). After osteogenic induction, it was found that the ALP staining in the Znf750 Construct group was deepened ( Figure 1 B), and the mRNA levels of key osteogenic differentiation factors Runx2, Osterix, Alp, and Opn ( Figure 1 C) and the protein levels of Runx2, Osterix, and ALP ( Figure 1 D) were all increased, indicating that overexpressing ZNF750 promoted the osteogenic differentiation of ST2 cells.

[0070] Example 2

[0071] This example analyzed the effect of ZNF750 on the adipogenic differentiation of mesenchymal cell line C3H10T1 / 2 cells. The ZNF750 overexpression plasmid (Znf750 Construct) was transfected into C3H10T1 / 2 cells, and the changes in adipogenic differentiation after overexpressing ZNF750 were analyzed. It was detected by qRT-PCR that the Znf750 mRNA was significantly increased in the Znf750 Construct group ( Figure 2 A). After adipogenic induction, it was found that the Oil Red O staining in the Znf750 Construct group was weakened ( Figure 2 B,C), and the mRNA levels of key adipogenic differentiation factors Pparγ, C / ebpα, Fabp4, and adipsin ( Figure 2 D) and the protein levels of PPARγ, C / EBPα, and FABP4 ( Figure 2 E) were significantly decreased, indicating that ZNF750 inhibited the adipogenic differentiation of C3H10T1 / 2 cells.

[0072] Example 3

[0073] This example analyzed the effect of Znf750 gene silencing on the osteogenic / adipogenic differentiation of primary BMSCs. The constructed Znf750 knockdown lentivirus (Znf750 shRNA LV) and its control virus (Ctrl LV) were respectively used to infect primary BMSCs. It was detected by qRT-PCR that the Znf750 mRNA level in the BMSCs infected with Znf750 shRNA LV was significantly decreased ( Figure 3 A). After osteogenic induction, compared with the control, the ALP staining of the cells infected with Znf750 shRNA LV became lighter ( Figure 3B), mRNA of key factors for osteogenic differentiation, Runx2, Osterix, Alp, and Opn( Figure 3 C) and Runx2, Osterix, and ALP proteins( Figure 3 D) showed a significant decrease in expression, indicating that knockdown of Znf750 inhibited osteogenic differentiation of primary BMSCs. After adipogenic induction, compared with the control, the intensity of Oil Red O staining in cells infected with Znf750 shRNA LV increased( Figure 3 E,F), mRNA of key factors for adipogenic differentiation, Pparγ, C / ebpα, Fabp4, and adipsin( Figure 3 G) and PPARγ, C / EBPα, and FABP4 proteins( Figure 3 H) showed a significant increase in expression, indicating that knockdown of Znf750 promoted adipogenic differentiation of primary BMSCs.

[0074] Example 4

[0075] In this example, the role of ZNF750 in transcriptional regulation of Snai1 gene expression and its effect on the activity of the Wnt / β-catenin signaling pathway were analyzed. The mRNA level of Snai1 in ST2 cells overexpressing Znf750 was detected to be decreased by qRT-PCR( Figure 4 A). Through bioinformatics analysis, we found a potential binding site of ZNF750 at the -658nt position of the Snai1 promoter( Figure 4 B). The dual-luciferase reporter gene assay demonstrated that ZNF750 inhibited the transcriptional activity of Snai1, and this transcriptional inhibitory effect disappeared after mutating the expected binding site( Figure 4 C). Overexpression of ZNF750 in ST2 cells led to a significant increase in the expression levels of Wnt / β-catenin signaling pathway-related proteins p-GSK3β(S9) and non-p-β-catenin( Figure 4 D), indicating that overexpression of ZNF750 could enhance the activity of the Wnt / β-catenin signaling pathway. Overexpression of Snai1 in ST2 cells led to a significant decrease in the expression levels of Wnt / β-catenin signaling pathway-related proteins p-GSK3β(S9) and non-p-β-catenin( Figure 4 E), indicating that overexpression of Snai1 could inhibit the activity of the Wnt / β-catenin signaling pathway.

[0076] Example 5

[0077] This example analyzed the corrective effect of Snai1 overexpression on the dysregulated differentiation of mesenchymal progenitor cells induced by ZNF750. ST2 was co-transfected with the Znf750 and Snai1 overexpression vectors. The increased levels of p-GSK3β(S9) and non-p-β-catenin caused by Znf750 overexpression were attenuated by SNAI1 ( Figure 5 A). After osteogenic induction, the osteoblast differentiation caused by Znf750 overexpression was attenuated by SNAI1 ( Figure 5 B-D). Conversely, C3H10T1 / 2 was co-transfected with the Znf750 and Snai1 overexpression vectors. After adipogenic induction, the inhibitory effect of Znf750 overexpression on adipogenic differentiation of cells was attenuated by SNAI1 ( Figure 5 E-H). In summary, it is suggested that ZNF750 promotes osteogenic differentiation and inhibits adipogenic differentiation of mesenchymal cells by downregulating the expression of the SNAI1 gene and enhancing the activity of the Wnt / β-catenin signaling pathway.

[0078] Example 6

[0079] This example analyzed the changes in bone mass in mice after knocking down Znf750 in BMSCs in vivo. To study the in vivo role of ZNF750, we transplanted BMSCs infected with Znf750 shRNA lentivirus (ZNF750 shRNA LV) or control lentivirus (Ctrl LV) into the tibial bone marrow of mice. One month after transplantation, compared with the control, the trabecular bone volume fraction (Tb.BV / TV), trabecular thickness (Tb.Th), and trabecular bone mineral density (Tb.BMD) in the ZNF750 shRNA LV group of mice were significantly decreased ( Figure 6 A-D), and the structure model index increased ( Figure 6 E). We found by immunohistochemistry (IHC) that the number of ALP-positive osteoblasts in the ZNF750 shRNA LV group was significantly decreased compared with the control group ( Figure 7 A,B); while the HE staining results showed that the number and area percentage of adipocytes in the ZNF750 shRNA LV group increased ( Figure 7 C-E).

[0080] In summary, ZNF750 can promote osteoblast differentiation and inhibit adipocyte differentiation, affect bone homeostasis, and contribute to an increase in bone mass.

[0081] Therefore, drugs prepared using the ZNF750 gene or its encoded protein as a target have the potential to prevent and treat osteoporosis.

[0082] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the present invention product, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. Application of zinc finger protein 750 (ZNF750) in the preparation of a drug for preventing and treating osteoporosis. ZNF750 is used to regulate the osteogenic / adipogenic differentiation of mesenchymal stem / progenitor cells by downregulating the expression of the snail family transcriptional inhibitor 1 (SNAI1) gene, enhancing the activity of the Wnt / β-catenin signaling pathway, increasing the expression of osteogenic differentiation marker genes Runx2, Osx, Alp, and Opn, and inhibiting the expression of adipogenic differentiation marker genes Pparγ, C / ebpα, Fabp4, and adipsin, thereby increasing the number of osteoblasts, reducing the number of adipocytes, and promoting bone mass increase.

2. The application according to claim 1, wherein The drug is a ZNF750 regulator, which is used to increase the number and activity of osteoblasts, thereby preventing and treating osteoporosis.

3. The application according to claim 1, characterized in that The drug is a ZNF750 regulator, which is used to reduce the number of fat cells and thus prevent and treat osteoporosis.

4. The application according to claim 1, characterized in that The drug is a ZNF750 regulator, which inhibits SNAI1 gene expression and enhances the activity of the Wnt / β-catenin signaling pathway, thereby preventing and treating osteoporosis.

5. The application according to any one of claims 1-4, characterized in that, The drug includes at least one of an overexpression drug targeting the ZNF750 encoding gene or a promoter or activator drug targeting the ZNF750 encoding protein.

6. The use according to claim 5, characterized in that The overexpression drug for the ZNF750 encoding gene includes an overexpression vector targeting the ZNF750 encoding gene, a cell overexpressing ZNF750, or a recombinant protein.

7. The application according to claim 5, characterized in that, The promoter or activator drug for ZNF750 protein includes any one of small molecule compounds, nucleic acids, amino acids, and polypeptides.

8. The application according to claim 5, wherein The dosage forms of the drug include oral solution, injection, tablet, pill, dispersion, capsule, drop, granule, suspension or emulsion.

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