Application of adjusting osteogenesis zinc finger protein Zfp260 phosphorylation site

Abstract

The invention relates to an application of a zinc finger protein Zfp260 phosphorylation site for regulating osteogenesis, and particularly discloses an application of a biomarker in preparation of a medicine for regulating bone regeneration and repair, and the biomarker comprises one or a combination of more of Y173 phosphorylation, S182 phosphorylation and S197 phosphorylation of the zinc finger protein Zfp260. The invention finds that any phosphorylation defect mutation of the three sites Y173, S182 and S197 can obviously change the binding force and nuclear translocation signal with kinase PKC alpha, and each mutant cannot save the cell mineralization defect caused by Zfp260 knockout, and prompts that the newly found Zfp260 phosphorylation sites Y173, S182 and S197 play important roles in Zfp260 related diseases and the regulation process of promoting bone regeneration and repair.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to an application of a zinc finger protein Zfp260 phosphorylation site for regulating osteogenesis. Background Art

[0002] The regeneration process after bone injury is finely regulated and is primarily driven by cells of the skeletal stem cell (SSC) lineage, which follow a unidirectional cascade of differentiation that begins with SSCs transforming into bone, cartilage, and stromal progenitor cells (BCSPs), followed by irreversible differentiation into cartilage lineage (PCP), osteogenic lineage (THY, BLSP), and stromal lineage (6C3, HEC) cells.

[0003] The temporal activation of transcription factors plays a key role in SSC lineage differentiation. For example, a large number of previous studies have confirmed that Runx2 and Sox9 are key transcription factors that regulate the fate of BCSP bone and cartilage differentiation. However, the functional transcription factors in the unidirectional differentiation process of SSC to BCSP have not yet been fully elucidated.

[0004] Recently, researchers have established a mouse model of maxillary sinus floor lift and fracture, and analyzed the single-cell sequencing and transcriptome sequencing data of the above models, and found that a transcription factor, zinc finger protein 260 (Zfp260), is significantly upregulated during the differentiation of SSC to BCSP. Furthermore, by isolating SSC, BCSP and osteoblast lineage cells from regenerated tissues after injury, using chromatin immunoprecipitation sequencing (ChIP-seq), cleavage under targets and tagmentation (CUT&Taq) and chromatin accessibility sequencing (Assay for Transposase-Accessible Chromatin using Sequencing, ATAC-seq) technology, the researchers found that Zfp260 formed a super enhancer complex, which regulates the fate of SSC to BCSP by regulating the expression of key transcription factors such as Runx2 and Zfhx4, and changes their ATAC signal accessibility at the BCSP stage.

[0005] Post-translational modifications (PTMs) of proteins are one of the key mechanisms for regulating protein function, activity and cell signaling. Among the various post-translational modifications, phosphorylation is one of the most common and important modifications. Phosphorylation usually occurs on serine, threonine and tyrosine residues, catalyzed by specific kinases, and can rapidly respond to changes in signals inside and outside the cell. Phosphorylation of transcription factors is a classic example of post-translational modification of proteins. Phosphorylation directly affects the expression pattern of genes by regulating the activity, nuclear entry and binding ability of transcription factors to DNA. Studying the phosphorylation mechanism of transcription factors is of great significance for understanding cell signaling networks, disease occurrence and the development of new therapeutic targets. However, as a newly discovered functional transcription factor that regulates osteogenic differentiation, it is not clear whether Zfp260 changes its expression pattern through phosphorylation modification and the site where it undergoes phosphorylation modification.

[0006] Therefore, there is an urgent need in the art to develop an application of zinc finger protein Zfp260 phosphorylation sites that regulate osteogenesis. Summary of the invention

[0007] In view of the above problems, the purpose of the present invention is to provide an application of the phosphorylation site of zinc finger protein Zfp260 for regulating osteogenesis.

[0008] In a first aspect of the present invention, a biomarker is provided for use in preparing a drug for regulating bone regeneration and repair, wherein the biomarker comprises one or more combinations of Y173 phosphorylation, S182 phosphorylation or S197 phosphorylation of zinc finger protein Zfp260.

[0009] In another preferred example, the amino acid sequence of the zinc finger protein Zfp260 is shown in SEQ ID NO:2.

[0010] In another preferred embodiment, the regulation is achieved by any of the following methods:

[0011] T1: Promote phosphorylation of at least one of the Y173, S182 or S197 sites of zinc finger protein Zfp260 to enhance bone regeneration and repair;

[0012] T2: Inhibit phosphorylation of at least one of the Y173, S182 or S197 sites of the zinc finger protein Zfp260 to reduce bone regeneration and repair.

[0013] In another preferred embodiment, the method T1 is achieved by overexpressing Zfp260 in cells; the method T2 is achieved by any of the following methods:

[0014] (i) inhibiting phosphorylation of at least one of Y173, S182 or S197 of zinc finger protein Zfp260 by an agent or drug;

[0015] (ii) subjecting the zinc finger protein Zfp260 to one or more mutations selected from the group consisting of Y173F, S182G and S197G;

[0016] (iii) overexpressing a zinc finger protein Zfp260 mutant in cells, wherein:

[0017] The amino acid sequence of the zinc finger protein Zfp260 mutant is a mutant in which any one of the following amino acid substitutions is performed on the basis of SEQ ID NO: 2: Y173F, S182G, S197G, Y173F and S182G, S182G and S197G, Y173F and S197G, Y173F and S182G and S197G.

[0018] In another preferred embodiment, the method T1 is to enhance bone regeneration and repair by overexpressing Zfp260 in cells, promoting the binding of Zfp260 to kinase PKCα, and / or promoting nuclear translocation, and / or promoting calcium nodule formation;

[0019] The method T2 is to overexpress the zinc finger protein Zfp260 mutant in cells to inhibit the binding of Zfp260 to kinase PKCα, and / or inhibit nuclear translocation, and / or inhibit calcium nodule formation, so as to reduce bone regeneration and repair.

[0020] In a second aspect of the present invention, a use of a biomarker in preparing a bone injury diagnosis product is provided, wherein the biomarker comprises one or more combinations of Y173 phosphorylation, S182 phosphorylation or S197 phosphorylation of zinc finger protein Zfp260.

[0021] In another preferred example, the amino acid sequence of the zinc finger protein Zfp260 is shown in SEQ ID NO:2.

[0022] In another preferred embodiment, the product includes a reagent for detecting the expression level of a biomarker in a sample.

[0023] In the third aspect of the present invention, a zinc finger protein Zfp260 mutant is provided, wherein the amino acid sequence of the mutant is a mutant in which any one of the following amino acid substitutions is performed on the basis of SEQ ID NO: 2: Y173F, S182G, S197G, Y173F and S182G, S182G and S197G, Y173F and S197G, Y173F and S182G and S197G.

[0024] In the fourth aspect of the present invention, a gene of a zinc finger protein Zfp260 mutant is provided, encoding the zinc finger protein Zfp260 mutant described in the third aspect of the present invention.

[0025] In the fifth aspect of the present invention, a recombinant expression vector or a recombinant host cell containing the gene of the zinc finger protein Zfp260 mutant described in the fourth aspect of the present invention is provided.

[0026] In the sixth aspect of the present invention, there is provided a use of at least one of the zinc finger protein Zfp260 mutant described in the third aspect of the present invention, the zinc finger protein Zfp260 mutant gene described in the fourth aspect of the present invention, and the recombinant expression vector or recombinant host cell described in the fifth aspect of the present invention in screening or preparing a disease related to a Zfp260 gene defect or mutation or a drug for promoting bone regeneration and repair.

[0027] In another preferred embodiment, the related diseases caused by the Zfp260 gene defect or mutation include diseases caused by phosphorylation site modification.

[0028] In another preferred embodiment, the promoting bone regeneration and repair refers to improving the bone regeneration efficiency in the face of infection, trauma, genetic diseases or other factors that slow down the bone regeneration rate and subsequently affect the physiological state.

[0029] In the seventh aspect of the present invention, a method for screening or preparing drugs that promote bone regeneration and repair is provided, the method comprising the following steps: (a) constructing an animal model that overexpresses the zinc finger protein Zfp260 mutant described in the third aspect of the present invention; (b) administering a test substance to the animal model; and (c) detecting the formation of the zinc finger protein Zfp260 mutant in the animal model; if the formation of the zinc finger protein Zfp260 mutant is reduced compared to a control animal model to which the test substance is not administered, the test substance is a drug that promotes bone regeneration and repair.

[0030] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0031] Compared with the prior art, the present invention has the following technical effects:

[0032] The present invention uses tandem mass spectrometry (LC-MS / MS) to detect phosphorylation of Zfp260 protein, and discovers multiple Zfp260 phosphorylation sites for the first time. By performing point mutations on these tyrosine / serine sites one by one, two by two, or all of them, it is found that any phosphorylation defect mutations at the three sites of Y173, S182, and S197 can significantly change their binding force with the kinase PKCα and nuclear translocation signals, and each mutant cannot rescue the cell mineralization defect caused by Zfp260 knockout, suggesting that the newly discovered Zfp260 phosphorylation sites Y173, S182, and S197 play an important role in the regulation of Zfp260-related diseases and promoting bone regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Figure 1 shows the results of an embodiment of the present invention confirming that Prkca is a kinase of Zfp260, wherein Figure A shows the results of a Co-IP interaction experiment in which Zfp260 and Prkca expression plasmids were transiently transfected in 293T cells; Figure B shows the results of a Co-IP interaction experiment in target cells PSC (periosteal stem cells); Figure C shows the results of co-localization detection in an immunofluorescence experiment; Figure D shows the results of an in vitro phosphorylation experiment; and Figure E shows the results of osteogenic induction of PSCs to detect the effect of Prkca on the phosphorylation modification level of Zfp260.

[0034] Figure 2 The phosphorylation sites of Zfp260 discovered by tandem mass spectrometry (LC-MS / MS) in one embodiment of the present invention are shown, wherein the tyrosine at position 173 (Y173), the serine at position 182 (S182), and the serine at position 197 (S197) in the partial peptide of Zfp260 are found to be phosphorylation sites.

[0035] Figure 3 The graphs show the results of detecting the binding ability of wild-type and mutant Zfp260 with kinase PKCα in one embodiment of the present invention, wherein Figure A is a diagram of the construction process of Ctsk-creER; H11RSR-tdTomato-LSL-zsGreen; Zfp260fl / fl mice; Figures B and C are immunoprecipitation result graphs and relative quantitative result graphs, respectively, wherein the gray value of wild-type Zfp260-V5 (WT) is set to 1 for comparison.

[0036] Figure 4The nuclear translocation and statistical results of wild-type Zfp260 and single-, double-, and triple-phosphorylation-deficient mutants of Zfp260 knocked out skeletal stem cells at 0 and 24 hours after osteogenic induction in one embodiment of the present invention are shown, wherein Mock represents Zfp260 knocked out in periosteal stem cells PSC (i.e., PSC Zfp260 - / - ), which was transfected with empty vector only, as a negative control.

[0037] Figure 5 In one embodiment of the present invention, after transfecting wild-type Zfp260 and Zfp260 single-point, double-point and triple-point phosphorylation-deficient mutants into Zfp260-knockout skeletal stem cells, ChIP-qPCR was used to detect the chromatin binding of wild-type and mutant Zfp260 at the P1 and P2 sites in the Runx2 super enhancer region.

[0038] Figure 6 In one embodiment of the present invention, after transfecting wild-type Zfp260 and Zfp260 single-point, double-point and triple-point phosphorylation-deficient mutants into Zfp260-knocked-out skeletal stem cells, ChIP-qPCR was used to detect the chromatin modification of each group of H3K27ac at the P1 and P2 sites in the Runx2 super enhancer region.

[0039] Figure 7 In one embodiment of the present invention, after transfecting wild-type Zfp260 and Zfp260 single-point, double-point and triple-point phosphorylation-deficient mutants into Zfp260-knockout skeletal stem cells, ChIP-qPCR was used to detect the chromatin binding of each group of Brd4 at the P1 and P2 sites in the Runx2 super enhancer region.

[0040] Figure 8 The figure shows that in one embodiment of the present invention, after transfection of wild-type Zfp260 and single-point, double-point and triple-point phosphorylation-deficient mutants of Zfp260 into Zfp260-knocked-out skeletal stem cells, osteogenic induction was observed to observe the rescue effect of each group on the mineralization defect caused by Zfp260 knockout; wherein, PSC OM 14d represents the detection result of wild-type PSC 14 days after osteogenic induction; OM represents osteogenic induction culture; ARS represents Alizarin red staining, which is the most direct standard for detecting the osteogenic effect of cells in vitro; Mock represents periosteal stem cell PSC with Zfp260 knocked out, which is only transfected with an empty vector as a negative control. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution, beneficial effects and significant progress of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, all the embodiments described are only partial embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] As used herein, the term "Prkca" is the protein kinase C alpha (PKC alpha) gene.

[0043] Example 1

[0044] The pGEX6p-Zfp60-GST plasmid was constructed, and the GST-tagged Zfp260 recombinant protein was purified using a prokaryotic expression system. A GST-Pulldown experiment was performed, and proteins that could bind to Zfp260 were observed in vitro. The captured proteins were subjected to mass spectrometry analysis, and a top-ranked protein kinase, Prkca, was enriched.

[0045] 293T cells were transiently transfected with Zfp260 and Prkca expression plasmids at the same time, and cell proteins were collected 72 hours later for Co-IP experiments. The results of the Co-IP interaction experiment are shown in Figure 1 As shown in Figure A: In the first lane, an empty vector was transferred, in the second lane, only Zfp260-V5 was transferred, in the third lane, only Prkca-Flag was transferred, and in the fourth lane, Prkca-Flag and Zfp260-V5 were co-transfected). By pulling down Flag to detect V5 and pulling down V5 to detect Flag, it was found that the two interacted.

[0046] In addition, in the target cell PSC (periosteal stem cell), by pulling down V5, the Prkca band was detected, indicating that Prkca and Zfp260 also interact in the target cell (PSC) ( Figure 1 B).

[0047] The co-localization of the two was also demonstrated in the immunofluorescence experiment. Figure 1 As shown in Figure C, the immunofluorescence signal distribution of Zfp260 and Prkca revealed that the signal peaks of the two had extensive overlap, indicating that Zfp260 can bind to Prkca.

[0048] In addition, the inventors verified whether Prkca can phosphorylate Zfp260. The specific experimental process is as follows:

[0049] (1) The in vitro phosphorylation experiment was divided into three systems. In system 1, only Zfp260-V5 recombinant protein (substrate) was added; in system 2, Zfp260-V5 recombinant protein and active PKCalpha kinase were added; in system 3, Zfp260-V5 recombinant protein, active PKCalpha kinase and kinase inhibitor GO 6976 were added.

[0050] The system buffer is: 40mM HEPES pH 7.6, 10mM MgCl2, 1mM EGTA, 1mM DTT, 2.5mM sodium β-glycerophosphate, 200μM ATP. The reaction conditions are 37℃ for 30 minutes. The amount of Zfp260-V5 recombinant protein input into each system is 5ug, the amount of active PKCalpha kinase protein input is 10ng, and the working concentration of kinase inhibitor GO 6976 is 1μM.

[0051] After the reaction was completed, WB detection was performed using Phos-PAGE WB and conventional WB detection. 8% Phos-PAGE gel was prepared by adding 5 mM Phos and 10 mM ZnCl2 to final concentrations of 25 μM and 50 μM, respectively. Electrophoresis was performed at a constant current of 30 mA. Before transfer, the gel was pretreated with 10 mM EDTA and washed before membrane transfer.

[0052] In vitro phosphorylation assay results Figure 1 As shown in Figure D, the first lane did not contain kinase, and no p-Zfp260-V5 band was observed; the second lane contained kinase, and p-Zfp260-V5 band was observed; the third lane contained kinase inhibitor, and no p-Zfp260-V5 band was observed, which proved that Prkca can phosphorylate Zfp260.

[0053] (2) Based on the process described in the above process (1), PSCs are induced to osteogenic state, and Prkca increases the phosphorylation modification level of Zfp260.

[0054] Zfp260-V5 of each treatment group was enriched by immunoprecipitation, and then the phosphorylation modification status of Zfp260 was detected by Phos-PAGE WB; the results are shown in Figure 1 As shown in Figure E, the first and second lanes are PSCs in a resting state, with GO-6976 added to the second lane; the third and fourth lanes are PSCs after 24 hours of osteogenic induction, with GO-6976 added to the fourth lane. By comparing the first and third lanes, it can be concluded that osteogenic induction increases the phosphorylation modification level of Zfp260; by comparing the third and fourth lanes, it can be concluded that PKCα kinase is the modification kinase of Zfp260.

[0055] The above results prove that Prkca is a kinase of Zfp260 and is involved in the regulation of Zfp260 on osteogenic differentiation.

[0056] Protein profiling analysis revealed three phosphorylation sites Y173, S182, and S197 in wild-type Zfp260 (nucleotide sequence as shown in SER ID NO: 1; amino acid sequence as shown in SER ID NO: 2). Figure 2 ). Further, through Alphafold2 protein structure simulation, it was found that Y173, S182 and S197 residues are spatially close to the catalytic domain of Prkca, which contradicts the mass spectrometry results.

[0057] Based on the three sites Y173, S182 and S197, a total of 7 Zfp260 protein structures were constructed, including 3 single-point mutations (Y173F, S182G and S197G), 3 double-point mutations (Y173F-S182G, Y173F-S197G and S182G-S197G) and 1 triple-point mutation (Y173F-S182G-S197G, referred to as Triple-Mut). Molecular docking analysis found that the Zfp260 residues with mutations at the phosphorylation sites were spatially separated from the catalytic domain of Prkca.

[0058] In 293T cells, Prkca plasmid and the above-mentioned wild-type, single-point, double-point and triple-point phosphorylation-deficient mutant Zfp260 plasmids were transiently transfected. Cell proteins were collected after 72 hours for Co-IP experiments. It was found that compared with wild-type Zfp260, the single-point, double-point and triple-point phosphorylation-deficient mutant Zfp260 had a decreased ability to bind to Prkca.

[0059] Example 2

[0060] (1) Preparation of Ctsk+ SSC skeletal stem cells:

[0061] The present invention commissioned Shanghai Model Organisms Technology Co., Ltd. to construct Ctsk-creER; H11RSR-tdTomato-LSL-zsGreen; Zfp260 fl / fl Mice; among them,

[0062] Ctsk-creER means that after mice were injected with tamoxifen, the cre enzyme was expressed and activated in the cells expressing the Ctsk gene, a molecule related to osteoclast function. The function of the cre enzyme is to cut off the gene between the two loxp sites.

[0063] H11-LSL-zsGreen-RSR-tdTomato is a dual-tracing tool mouse. In cells expressing the cre enzyme, the stop sequence is cut out, Zsgreen green fluorescent protein is expressed, and the cells are green. Figure 3 A)

[0064] Zfp260 fl / fl It means that a loxp site is inserted at both ends of the Zfp260 gene. In cells with activated Cre, the Zfp260 gene is cut out, achieving Zfp260 gene knockout.

[0065] Therefore, in this model mouse, after tamoxifen was injected into the mouse to activate cre, the Ctsk+ cells glowed green and the Zfp260 gene was knocked out. However, this effect did not occur without the injection of tamoxifen, so the knockout at different times could be achieved.

[0066] From 8-week-old Ctsk-creER;H11-LSL-zsGreen-RSR-tdTomato;Zfp260 fl / fl Zfp260 was isolated from intact femur, tibia, and maxillofacial bones of mice - / - PSCs cells.

[0067] The above mice were injected with tamoxifen (50 mg / kg) for 3 consecutive days at 7 weeks of age to achieve the purpose of labeling and conditional knockout of Zfp260 in PSCs.

[0068] Mice were anesthetized and the intact femur, tibia, and maxillofacial bones of the mice were dissected. After removing the attached soft tissue, the periosteum was scraped and digested with DPBS containing 0.3% type I collagenase, 0.3% type II neutral protease, and 0.01% DNase I in a shaking water bath at 37°C for 30 minutes. The cells were filtered using a 70um cell strainer. Centrifuged at 300g for 10 minutes at 4°C and the supernatant was discarded. The cells were lysed with red blood cell lysis buffer at 4°C for 5 minutes. After neutralization, centrifuged again. The cell pellet was resuspended in staining buffer containing CD16 / 32 blocking antibody and incubated for 30 minutes before subsequent antibody staining. The antibodies used were as follows: CD31-PE / DazzleTM 594, CD45-PE / DazzleTM 594, TER119-PE / DazzleTM 594, CD105-APC / Cy7, CD200-APC, CD51 (Itgav)-biotin, Ly-51-PE / Cy7. The cells were incubated on ice for 1 hour. After centrifugation, CD51 (Itgav)-biotin was labeled with BV421-streptavidin and incubated on ice for 30 minutes. ZombieAquaTM was then used to distinguish live and dead cells.

[0069] Cell sorting using BD FACSAriaTM III to isolate single live Lin - ZsGreen + Itgav +THY1 - 6C3 - CD105 - CD200 + Cells. Among them, the Lin- cell group is negative for the red blood cell marker ter119, negative for the endothelial cell marker CD31, and negative for the myeloid cell marker CD45. THY1 and 6C3 are two cell surface markers. The combination of these markers represents the SSC skeletal stem cells that have obtained Ctsk+, that is, Zfp260 - / - PSCs cells.

[0070] After a two-week culture period, all clones were selected and passaged to P1.

[0071] (2) Cell expansion: To obtain a large number of cells required for co-immunoprecipitation and chromatin immunoprecipitation, P1 PSCs were plated into 24-well plates. When the cell density reached 50%, they were infected with lentivirus expressing human telomerase reverse transcriptase (hTERT) at an MOI of 100 for amplification and then further cultured to meet the cell number requirements for subsequent experiments and ChIP analysis.

[0072] (3) Zfp260-overexpressing PSCs and point mutant Zfp260-overexpressing stable Zfp260 - / - Construction of PSC:

[0073] The coding sequence (CDS) of mouse wild-type Zfp260 was cloned and fused into the MCS sites (EcoRI and BamHI) of the pCDH-V5-Puro plasmid by homologous recombination to construct the pCDH-Zfp260-V5 lentiviral expression vector.

[0074] The lentivirus packaging used a three-plasmid system (pol / gag, VSV-G, and pCDH-V5 / pCDH-Zfp260-V5) and transfected it into the 293T cell line. The lentivirus was concentrated and purified by ultracentrifugation. These viruses were used to infect wild-type PSCs with a multiplicity of infection (MOI) of 100. After 72 hours, cells with high fluorescence intensity were flow-sorted, single clones were plated, and the number of cells was expanded by continued subculture to obtain a stable strain overexpressing wild-type Zfp260.

[0075] In order to perform point mutations on the basis of pCDH-Zfp260-V5, the QuickMutation Plus Point Mutation Kit was used and primers were designed according to the instructions as shown in Table 1.

[0076] Table 1

[0077]

[0078] Using pCDH-Zfp260-V5 as a template, the following plasmids were constructed:

[0079] 1) pCDH-Y173F-V5 (relative to the wild-type Zfp260 nucleotide sequence shown in SEQ ID NO.1, adenine A at position 518 is replaced by adenine T, i.e., 518A-T),

[0080] 2) pCDH-S182G-V5 (relative to the wild-type Zfp260 nucleotide sequence shown in SEQ ID NO.1, the adenine A at position 544 is replaced with guanine G, i.e., 544A-G),

[0081] 3) pCDH-S197G-V5 (relative to the wild-type Zfp260 nucleotide sequence shown in SEQ ID NO.1, adenine A at position 589 is replaced by guanine G, i.e., 589A-G),

[0082] 4) pCDH-Y173F-S182G-V5 (relative to the wild-type Zfp260 nucleotide sequence shown in SEQ ID NO.1, the adenine A at position 518 and the adenine A at position 544 were replaced with adenine T and guanine G, i.e., 518A-T; 544A-G),

[0083] 5) pCDH-Y173F-S197G-V5 (relative to the wild-type Zfp260 nucleotide sequence shown in SEQ ID NO.1, the adenine A at position 518 and the adenine A at position 589 were replaced with adenine T and guanine G, i.e., 518A-T; 589A-G),

[0084] 6) pCDH-S182G-S197G-V5 (relative to the wild-type Zfp260 nucleotide sequence shown in SEQ ID NO.1, adenine A at position 544 and adenine A at position 589 were replaced with guanine G, i.e., 544A-G; 589A-G) and

[0085] 7) pCDH-triple-mut-V5 (relative to the wild-type Zfp260 nucleotide sequence shown in SEQ ID NO.1, adenine A at positions 518, 544 and 589 are replaced by adenine T, guanine G and guanine G, respectively, i.e., 518A-T; 544A-G; 589A-G).

[0086] The subsequent lentiviral packaging was performed according to the above method. These viruses were used to infect Zfp260 at an MOI of 100 - / -PSCs were passaged after 72 hours, cells with high fluorescence intensity were selected by flow cytometry, single clones were plated, and subcultured to expand the cell number, thereby obtaining stable transgenic lines overexpressing the above-mentioned mutant Zfp260.

[0087] Example 3

[0088] This example detects the binding of wild-type Zfp260 and Zfp260 single-site, double-site and triple-site phosphorylation defective mutants to kinase PKCα. The specific process is as follows:

[0089] 3.1 The wild-type Zfp260 and Zfp260 single-site, double-site and triple-site phosphorylation-deficient mutants obtained in Example 2 were stably transfected - / - PSCs were plated at 70% density in 6 cm cell culture dishes.

[0090] 3.2 After the cells adhered for 24 hours, the cells were subjected to osteogenic induction culture (OM) for 24 hours. The ratio of osteogenic induction medium was: α-MEM containing 10% FBS, 10 nM dexamethasone, 50 μg / mL ascorbic acid, 1% penicillin / streptomycin and 10 mM β-glycerophosphate.

[0091] 3.3 Immunoprecipitation: Wash cells twice with ice-cold PBS, and then lyse cells with IP lysis buffer containing 1 mM PMSF and a mixture of protease inhibitors. Incubate cell lysates on ice for 30 minutes, sonicate at 25% power for 1 minute, start for 1 second, stop for 2 seconds, and then centrifuge at 13,000×g for 10 minutes at 4°C. Collect the supernatant as whole-cell protein lysate. Take 1 / 10 volume of lysate into a new EP tube, add SDS loading buffer, and after 95°C water bath for 10 minutes, mark it as input sample, cool it on ice, and store the sample in a -80°C refrigerator.

[0092] Add 15ul of Pierce Protein A / G Magnetic Beads to the remaining whole-cell protein lysate, rotate the sample on a rotating mixer, and pre-clear for 2 hours at 4°C. After 2 hours, place the sample on a magnetic rack, discard the magnetic beads, and then incubate the supernatant of each group after pre-clearing with V5 tag antibody overnight, then add magnetic beads and rotate and incubate on a rotating mixer at 4°C for 3 hours to capture the antibody. Place the sample on a magnetic rack, discard the supernatant, wash the magnetic beads 3 times with 4°C pre-cooled TBST, add a certain volume of SDS loading buffer according to the number of cells, boil at 95°C water bath for 5 minutes, place the sample on a magnetic rack, aspirate the supernatant and put it into a new EP tube, and mark it as an IP sample.

[0093] 3.4 Immunoblotting: First, select a constant voltage of 80V for concentrated gel electrophoresis for about 20 minutes until the sample forms a horizontal straight line. Then adjust the voltage to 130V and continue for about 40 minutes until the bromoaniline blue front runs to the bottom of the gel plate, and then turn off the power. Soak the PVDF membrane in methanol for 2 minutes to activate it, and then put it in the wet transfer buffer for use. Prepare a sandwich clamp, first place a black transfer sponge at the bottom, then a layer of thick filter paper, then the SDS-PAGE gel, then the PVDF membrane, then two layers of thin filter paper, and finally the white transfer sponge. Put these materials into the "sandwich clamp" in turn, and use a roller to drive away bubbles when putting each layer. Pay attention to the direction of the positive and negative electrodes. After installing the transfer instrument, put it in the transfer buffer to ensure that the gel and membrane clamp are completely covered. Adjust the current to 300mA, continue the transfer for 50 minutes, and keep it in an ice bath throughout the process. After the transfer is completed, use a quick blocking solution and block it on a shaker at room temperature for 15 minutes. The primary antibody was diluted at a ratio of 1:2000 and incubated overnight at 4°C on a shaker.

[0094] The primary antibodies used in this example include: rabbit anti-V5 tag, mouse anti-Prkca, rabbit anti-β-Actin. The membrane incubated with the primary antibody was washed with TBST, and each washing was done on a shaker for 5 minutes, for a total of 3 times. In order to avoid interference from light and heavy chains, HRP-conjugated VeriBlot for IP Detection secondary antibodies were used to detect immunoprecipitated samples at a dilution ratio of 1:200.

[0095] For input samples, conventional HRP-conjugated secondary antibodies were used for WB signal detection at a dilution ratio of 1:2000 for the corresponding species. The secondary antibodies were incubated on a shaker at room temperature for 1 hour, and the membrane was washed with TBST solution for 5 minutes each time, and the washing was performed 3 times on a shaker. The ultrasensitive ECL chemiluminescent colorimetric solution was exposed and the color was developed on the machine. The V5 protein expression levels between groups were adjusted to be relatively consistent. After analyzing the IPv5 of each group using image j software, the grayscale ratio of the Prkca / v5 band was semi-quantitatively analyzed.

[0096] The results showed that Zfp260 transfected with wild-type Zfp260 - / - The gray value of the Prkca / v5 band in PSCs was significantly higher than that in other groups, indicating that single-point, double-point, and triple-point phosphorylation defective mutants of Zfp260 could not rescue the Zfp260 - / - The binding ability of Zfp260 to protein kinase Prkca in PSCs ( Figure 3 B-3C).

[0097] Example 4

[0098] In this example, immunofluorescence detection was used to detect Zfp260 - / -PSCs were transfected with wild-type Zfp260 and single-site, double-site and triple-site phosphorylation-deficient mutants of Zfp260, and then cultured for osteogenic induction to observe the nuclear translocation of Zfp260.

[0099] 4.1 Place a cell slide in a 24-well plate and transfect the wild-type Zfp260 and the Zfp260 single-site, double-site and triple-site phosphorylation-deficient mutants obtained in Example 2 with - / - PSCs were plated at 50% density;

[0100] 4.2 After 24 hours, the cells were cultured for another 24 hours using osteogenic induction medium. The ratio of the osteogenic induction medium was as follows: α-MEM containing 10% FBS, 10 nM dexamethasone, 50 μg / mL ascorbic acid, 1% penicillin / streptomycin, and 10 mM β-glycerophosphate.

[0101] 4.3 Fix with 4% PFA at room temperature for 10 minutes, wash 3 times with PBS, remove PFA, then punch with PBS containing 0.5% Triton X100 at room temperature for 10 minutes, wash 3 times with PBS, block with immunostaining blocking solution at room temperature for 15 minutes, dilute rabbit anti-V5 antibody at 1:150, and incubate at 4°C overnight. The next day, return the slide to room temperature, wash 3 times with PBS, incubate with fluorescent secondary antibody at room temperature for 60 minutes, wash 3 times with PBS, counterstain with DAPI, incubate at 37°C for 4 minutes, wash 3 times with PBS, seal with anti-fading sealing agent, and take pictures with a fluorescence microscope.

[0102] 4.4 Using Image J software, select the nuclear or cytoplasmic region and establish the corresponding region of interest (ROI). After measuring the mean fluorescence intensity (MFI) values ​​of the above regions, substitute these values ​​into the formula MFInucleus / MFIcytosol to calculate the translocation efficiency. 30 cells were randomly selected from each group for calculation.

[0103] The results showed that after 24 hours of osteogenic induction, Zfp260 cells transfected with wild-type Zfp260 - / - PSCs showed an increased MFI ratio of Zfp260 in the nucleus and cytoplasm; however, Zfp260 transfected with single-, double-, and triple-phosphorylation-deficient mutants of Zfp260 - / - PSCs did not show significant changes in MOI ratios ( Figure 4 This indicates that single-site, double-site, and triple-site phosphorylation-deficient mutants of Zfp260 cannot rescue the phosphorylation of Zfp260. - / - Nuclear translocation of Zfp260 in PSCs.

[0104] Example 5

[0105] Enhancers are DNA sequences on genes that enhance the transcriptional activity of corresponding genes. Super enhancers are specific regions of genes that are rich in a large number of enhancer sequences and significantly regulate the transcription and expression of genes. Super enhancer regions are usually modified by histones, which will be enriched in H3K27ac. When ChIP-qPCR detects a high relative enrichment value of H3K27ac, it indicates that the transcriptional activity of the target gene is enhanced. Brd4 is a common co-activator of super enhancers. ChIP-qPCR shows that the high enrichment of Brd4 also indicates that the transcriptional activity of the target gene is enhanced. Runx2 is a classic transcription factor that promotes osteogenic differentiation of stem cells. P1 and P2 are two sites of known Runx2 super enhancers. After osteogenic induction of PSCs, Chip-seq was performed and it was found that both sites were enriched in H3K27ac histone modification. Zfp260 can bind to these two sites to regulate the expression of Runx2.

[0106] In this example, ChIP-qPCR was used to detect the expression of Zfp260 in the wild-type Zfp260 and single-, double-, and triple-phosphorylation-deficient mutants. - / - Chromatin binding of wild-type and mutant Zfp260, chromatin modification of H3K27ac, and chromatin binding of Brd4 at the P1 and P2 sites in the Runx2 super enhancer region of PSCs.

[0107] 5.1 Preparation of Zfp260 carrying wild-type Zfp260 and Zfp260 single-site, double-site and triple-site phosphorylation-deficient mutants obtained in Example 2 - / - PSCs

[0108] 5.2 Perform chromatin immunoprecipitation (ChIP) experiments using the Thermo Scientific Pierce Magnetic ChIP Kit: The specific process includes:

[0109] The above PSCs cells were crosslinked with 1% formaldehyde for 10 minutes, and then the reaction was terminated with 125mM glycine for 5 minutes. Subsequently, the cells were washed three times with ice-cold PBS and the precipitate was collected. Cell lysis was performed on ice for 10 minutes. The nuclei were collected by centrifugation and digested with MNase to ensure that the DNA fragment length was between 150bp and approximately 1000bp. Soluble chromatin was incubated with magnetic antibodies using V5, H3K27ac and Brd4 antibodies and corresponding isotype controls on a rotating mixer at 4°C overnight.

[0110] 5.3 Collect the immunoprecipitated beads, followed by protein digestion and DNA cleaning. Then perform quantitative PCR (qPCR) analysis on the purified ChIP and total DNA at the target site and compare the enrichment with the corresponding isotype control IgG. The primer sequences of the target site are shown in Table 2:

[0111] Table 2

[0112]

[0113] The results showed that Zfp260 - / - After PSCs were transfected with wild-type Zfp260, Zfp260 showed strong chromatin binding ability at the P1 and P2 sites of the Runx2 super enhancer ( Figure 5 ), accompanied by higher levels of H3K27ac histone modification ( Figure 6 ), and the co-activator BRD4 also has a strong binding ability at these two sites ( Figure 7 When single-site, double-site, and triple-site phosphorylation-deficient mutants of Zfp260 were transfected, the chromatin binding abilities of Zfp260 and BRD4 at P1 and P2 sites were weakened, and the level of H3K27ac histone modification was significantly decreased.

[0114] These results indicate that single-site, double-site, and triple-site phosphorylation-deficient mutants of Zfp260 cannot rescue the - / - The chromatin binding of Zfp260 to the Runx2 super enhancer regions P1 and P2, histone modification of H3K27ac, and chromatin binding of BRD4 in PSCs result in the single-site, double-site, and triple-site phosphorylation-deficient mutants of Zfp260 having weaker regulatory effects on Runx2 gene expression than wild-type Zfp260.

[0115] Example 6

[0116] In this example, Alizarin red staining was used to observe the transfection of wild-type Zfp260 and Zfp260 single-site, double-site and triple-site phosphorylation-deficient mutants. - / - Calcium nodule formation in PSCs

[0117] 6.1 The wild-type Zfp260 and the single-, double- and triple-phosphorylation-deficient mutants of Zfp260 obtained in Example 2 were stably transfected - / - PSCs were plated at 70% density in 6-well plates;

[0118] 6.2 After the cells adhered for 24 hours, PSCs were induced to osteogenic state. The ratio of osteogenic induction solution was: α-MEM containing 10% FBS, 10 nM dexamethasone, 50 μg / mL ascorbic acid, 1% penicillin / streptomycin and 10 mM β-glycerophosphate. After 14 days of induction culture, the cells were fixed with 4% paraformaldehyde at room temperature for 10-15 minutes, washed 3 times with PBS, stained with 1% alizarin red stain at 37°C for 30-50 minutes so that all cells were immersed in the stain, washed 3 times with ultrapure water, and photographed with a camera.

[0119] The results showed that Zfp260 transfected with wild-type Zfp260 - / - The calcium nodule formation of PSCs was similar to that of wild-type PSCs, while the Zfp260 transfected with single-, double-, and triple-phosphorylation-deficient mutants of Zfp260 - / - The formation of calcium nodules in PSCs was significantly reduced ( Figure 8 ), indicating that single-site, double-site, and triple-site phosphorylation defective mutants of Zfp260 cannot rescue Zfp260 - / - Mineralized nodules of PSCs are generated.

[0120] The above results show that, compared with the wild-type Zfp260, any phosphorylation-defective mutation (single-point, double-point or triple-point mutation) at the three sites of Y173, S182 and S197 can significantly change the binding ability of Zfp260 with kinase PKCα and affect the nuclear translocation signal. In addition, the above Zfp260 mutants cannot rescue the cell mineralization defect caused by Zfp260 knockout, suggesting that the newly discovered Zfp260 phosphorylation sites Y173, S182 and S197 play an important role in the regulation of Zfp260-related diseases and promoting bone regeneration and repair.

[0121] The relevant sequences involved in the present invention are as follows:

[0122] SEQ ID NO.1: (Zfp260 original nucleotide sequence)

[0123]

[0124] SEQ ID NO.2: (Zfp260 original amino acid sequence)

[0125] MLESLQPESHLLHDEPDPGESVYECNECKETFSLEQNFVEHKKTHSGEKSPECTGCGEESSQASSLTLHLRSRPRRESYKCGECGKAFSQRGNFLSHQKQHTEERPSESKKTPVPMTTTVRNQRNTGNKPYACKECGKAFNGKSYLKEHEKIHTGEKPFECSQCGRAFSQKQYLIKHQNIHSGKKPFKCNECGKAFSQKENLI IHQRIHTGEKPYECKGCGKAFIQKSSLIRHQRSHTGEKPYTCKECGKAFSGKSNLTEHEKIHIGEKPYKCNECGTIFRQKQYLIKHHNIHTGEKPYECNKCGKAFSRITSLIVHVRIHTGDKPYECKICGKAFCQSSSLTVHMRSHTGEKPYGCNECGKAFSQFSTLALHMRIHTGEKPYQCSECGKAFSQKSHHIRHQRIHIH

[0126] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. The use of biomarkers in the preparation of drugs for regulating bone regeneration and repair, characterized in that: The biomarkers include: one or more combinations of Y173 phosphorylation, S182 phosphorylation or S197 phosphorylation of zinc finger protein Zfp260.

2. The use according to claim 1, characterized in that: The amino acid sequence of the zinc finger protein Zfp260 is shown in SEQ ID NO:

2.

3. The use according to claim 1, characterized in that: The control is achieved by any of the following methods: T1: Promote phosphorylation of at least one of the Y173, S182 or S197 sites of zinc finger protein Zfp260 to enhance bone regeneration and repair; T2: Inhibit phosphorylation of at least one of the Y173, S182 or S197 sites of the zinc finger protein Zfp260 to reduce bone regeneration and repair.

4. The use according to claim 3, characterized in that: The method T1 is achieved by overexpressing Zfp260 in cells; the method T2 is achieved by any of the following methods: (i) inhibiting phosphorylation of at least one of Y173, S182 or S197 of zinc finger protein Zfp260 by an agent or drug; (ii) subjecting the zinc finger protein Zfp260 to one or more mutations selected from the group consisting of Y173F, S182G and S197G; (iii) overexpressing a zinc finger protein Zfp260 mutant in cells, wherein: The amino acid sequence of the zinc finger protein Zfp260 mutant is a mutant in which any one of the following amino acid substitutions is performed on the basis of SEQ ID NO: 2: Y173F, S182G, S197G, Y173F and S182G, S182G and S197G, Y173F and S197G, Y173F and S182G and S197G.

5. Application of biomarkers in the preparation of bone injury diagnosis products, characterized in that: The biomarkers include: one or more combinations of Y173 phosphorylation, S182 phosphorylation or S197 phosphorylation of zinc finger protein Zfp260.

6. A zinc finger protein Zfp260 mutant, characterized in that: The amino acid sequence of the mutant is a mutant in which any one of the following amino acid substitutions is made based on SEQ ID NO: 2: Y173F, S182G, S197G, Y173F and S182G, S182G and S197G, Y173F and S197G, Y173F and S182G and S197G.

7. A gene of a zinc finger protein Zfp260 mutant, characterized in that: Encoding the zinc finger protein Zfp260 mutant according to claim 6.

8. A recombinant expression vector or recombinant host cell containing the gene of the zinc finger protein Zfp260 mutant according to claim 7.

9. Use of the zinc finger protein Zfp260 mutant according to claim 6, the gene of the zinc finger protein Zfp260 mutant according to claim 7, and at least one of the recombinant expression vector or recombinant host cell according to claim 8 in screening or preparing drugs for diseases related to Zfp260 gene defects or mutations or for promoting bone regeneration and repair.

10. A method for screening or preparing a drug for promoting bone regeneration and repair, characterized in that: The method comprises the following steps: (a) constructing an animal model overexpressing the zinc finger protein Zfp260 mutant according to claim 6; (b) administering a test substance to the animal model; and (c) detecting the formation of the zinc finger protein Zfp260 mutant in the animal model; If the formation of the zinc finger protein Zfp260 mutant is reduced compared with a control animal model to which the test substance is not administered, the test substance is a drug for promoting bone regeneration and repair.

Images