Marker ACY1 for diagnosing osteoporosis and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a marker ACY1 for diagnosing osteoporosis and application of the marker ACY1, and the marker ACY1 is low in expression quantity in osteoporosis patients. By quantitatively detecting the level of ACY1, osteoporosis can be accurately recognized in the early stage of the disease course, and the sensitivity and specificity of diagnosis are remarkably improved. Therefore, doctors can take intervention measures in time, the disease progression is delayed, and serious complications such as fracture are reduced. The implementation of the invention can promote the understanding of molecular mechanisms of osteoporosis, ACY1 is used as a new diagnostic marker, the change of the expression level of ACY1 is closely related to the pathogenesis of osteoporosis, the role of ACY1 in osteoporosis is expected to be revealed on the basis of the content disclosed by the invention, and a new thought is provided for disease treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a marker ACY1 for diagnosing osteoporosis and an application thereof. Background Art

[0002] Osteoporosis is a systemic metabolic bone disease of the elderly characterized by low bone mass, deterioration of bone microarchitecture, and increased fracture risk. Bone metabolism occurs throughout life, maintaining bone homeostasis through bone resorption and formation. Osteoporosis often results in fractures of the hip or spine, which can be painful, inconvenient, devastating, and even fatal. The International Osteoporosis Foundation estimates that osteoporosis affects over 200 million people worldwide and is the most common age-related bone disease, particularly in postmenopausal women. Due to the rapid growth of the elderly population, the incidence of osteoporosis is also increasing, posing a significant burden not only on public health but also on the economy. However, conventional osteoporosis treatments have resulted in mixed results and numerous side effects, and classic serum markers are generally ineffective in describing bone mass. Therefore, research into the mechanisms of osteoporosis is urgently needed to generate new research directions and lay the foundation for more effective clinical prevention and treatment of osteoporosis in the future.

[0003] With the progress of the Human Genome Project, life science research has gradually entered the "post-genomic era," with functional genomics becoming a major research tool. Functional genomics encompasses structural genomics and proteomics. In proteomics, proteins are the subject of study, focusing on protein composition and protein-related changes in cells, tissues, and organisms. Proteomics research involves comprehensive observation and analysis of disease pathogenesis and cellular metabolic processes at the protein level. Proteomics can provide an important theoretical basis for elucidating the mechanisms of many diseases and offer new molecular markers for early diagnosis. In recent years, proteomic analyses related to osteoporosis have primarily focused on osteoblasts, osteoclasts, osteocytes, and bone-related cells in model animals and under various in vitro experimental conditions. However, the protein profile of human skeletal tissue remains unclear, and osteoporosis-related "human skeletal tissue proteomic" analyses have yet to be conducted. To date, few proteomic studies of serum and bone marrow proteomes have focused on osteoporosis, and proteomic information on human skeletal tissue and osteoporosis remains limited. Therefore, there is an urgent need to study osteoporosis in terms of serum and bone marrow proteomics in order to discover key molecules closely related to osteoporosis, provide new molecular markers for the early diagnosis of osteoporosis, and provide potential therapeutic targets for the development of more effective therapeutic drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide a marker ACY1 for diagnosing osteoporosis and its application, and specifically to provide a diagnostic marker that can be used for clinical diagnosis of osteoporosis. The diagnostic marker can be used for early detection of osteoporosis and has high sensitivity and high specificity during detection.

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a marker for diagnosing osteoporosis, wherein the marker is ACY1.

[0007] Furthermore, the expression level of the marker is low in osteoporosis patients.

[0008] The present invention also provides a product for diagnosing osteoporosis, wherein the product contains a reagent for quantitatively detecting the marker according to claim 1.

[0009] Furthermore, the product includes a detection kit, a polymerase chain reaction reagent, a chip detection reagent or a sequencing reagent.

[0010] Furthermore, the reagent for quantitatively detecting the marker includes a primer or probe capable of quantitatively detecting ACY1.

[0011] The present invention also provides a use of the marker in preparing a product for diagnosing osteoporosis.

[0012] Beneficial effects:

[0013] The present invention provides a new diagnostic marker, ACY1, whose expression level is significantly reduced in osteoporosis patients. By quantitatively detecting the level of ACY1, osteoporosis can be accurately identified early in the course of the disease, significantly improving the sensitivity and specificity of the diagnosis. This helps doctors take timely intervention measures to delay the progression of the disease and reduce the occurrence of serious complications such as fractures. At the same time, the implementation of the present invention will promote the understanding of the molecular mechanism of osteoporosis. As a new diagnostic marker, changes in the expression level of ACY1 are closely related to the pathogenesis of osteoporosis. Based on the content disclosed in the present invention, it is expected to reveal the role of ACY1 in osteoporosis and provide new ideas for the treatment of the disease.

[0014] This study focuses on proteomic analysis related to osteoporosis, specifically targeting the proteomics of human bone tissue, serum, and bone marrow. By discovering ACY1, a new diagnostic marker, this study fills a gap in this field and provides new directions and insights for future research. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 This is the ACY1 protein spectrum result of human bone tissue, where *** represents significant difference;

[0017] Figure 2 The results of bone tissue in castrated mice are shown in Figure 2, where ** indicates significant differences.

[0018] Figure 3 Comparison of osteogenic markers after ACY1 knockdown in human osteoblasts, where *** indicates significant differences;

[0019] Figure 4 This is a bar graph showing the expression of ACY1 protein in human bone tissue from osteoporosis patients. *** indicates significant differences.

[0020] Figure 5 The sensitivity and specificity of ACY1 as a diagnostic marker for osteoporosis were analyzed. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] The experimental methods used in the following implementations, unless otherwise specified, are commonly used experimental methods in this field;

[0027] Unless otherwise specified, the experimental materials used in the following examples are all commonly used experimental materials in this field.

[0028] Example 1 Osteoporosis Protein Osteological Analysis

[0029] 1. Sample Preparation

[0030] This study complies with ethical regulations for the use of human tissue samples and animal research. The use of tissue samples was approved by the Second Affiliated Hospital of Soochow University, ID-LK2020-027-01. All participants were recruited from the Department of Orthopedics, The Second Affiliated Hospital of Soochow University, China. The inventors recruited patients diagnosed with unilateral femoral neck fracture and requiring hip replacement surgery. Bone mineral density (BMD) was examined in each patient using dual-energy X-ray absorptiometry (Hologic Delphi A; Hologic, Bedford, MA, USA) 2–3 days before hip replacement surgery. Exclusion criteria included infection, tumor, hip dysplasia, femoral head necrosis, rickets, coagulopathy, renal insufficiency, history of lumbar spine surgery, history of anti-osteoporosis treatment, and diseases that affect bone metabolism, such as thyroid disease, parathyroid disease, adrenal disease, and diabetes. Patients were divided into three groups based on their hip T scores: normal (A) (T ≥ -1.0), osteopenia (B) (-2.5 < T < -1.0), and osteoporosis (C) (T ≤ -2.5). Appropriate amounts of femoral head tissue were obtained 0.5 cm below the ligamentous insertion of the femoral head, and all tissue samples were stored at -80°C. Frozen specimens were stored on dry ice, and approximately 50 mg of tissue was cut from each sample for proteomic analysis.

[0031] The sample was first ground in liquid nitrogen, and the powder was then transferred to a 5-ml centrifuge tube and sonicated three times on ice in lysis buffer (1% Triton X-100, 10 mM dithiothreitol, 1% protease inhibitor cocktail, 50 μM PR-619, 3 μM TSA, 50 mM NAM, and 2 mM EDTA) using a high-intensity ultrasonic processor (Scientz). An equal volume of Tris-saturated phenol (pH 8.0) was added, followed by further vortex mixing for 5 minutes. After centrifugation (4°C, 10 minutes, 5000 g), the upper phenol phase was transferred to a new centrifuge tube. Proteins were precipitated by adding at least four volumes of ammonium sulfate-saturated methanol and incubating at -20°C for at least 6 hours. After centrifugation at 4°C for 10 minutes, the supernatant was discarded. The remaining pellet was washed once with ice-cold methanol and then three times with ice-cold acetone. The protein was then resolubilized in 8 M urea, and the protein concentration was determined using a BCA kit according to the manufacturer's instructions.

[0032] 2. Sample Processing

[0033] 2.1 Trypsin digestion

[0034] The above protein solution was reduced with 5 mM dithiothreitol at 56 °C for 30 min, and then alkylated with 11 mM iodoacetic acid for 15 min at room temperature in the dark. The protein sample was then diluted to a urea concentration below 2 M by adding 100 mM TEAB. Finally, the first overnight digestion was performed at a trypsin to protein mass ratio of 1:50, and the second 4-h digestion was performed at a trypsin to protein mass ratio of 1:100.

[0035] 2.2 TMT Marking

[0036] After trypsin digestion, peptides were desalted using Strata X C18 SPE columns (Phenomenex) and dried under vacuum. Peptides were reconstituted in 0.5 M TEAB and treated with a TMT kit according to the manufacturer's protocol. Specifically, one unit of TMT reagent was thawed and reconstituted in acetonitrile. The peptide mixture was then incubated at room temperature for 2 hours, pooled, desalted, and dried by vacuum centrifugation.

[0037] 2.3HPLC separation

[0038] Tryptic peptides were separated by high pH reversed-phase HPLC using an Agilent 300Extend C18 column (5 μm particles, 4.6 mm inner diameter, 250 mm length). Specifically, the peptides were first separated into 60 fractions using a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 minutes. Then, the peptides were combined into 18 fractions and dried by vacuum centrifugation.

[0039] 2.4 LC-MS / MS analysis

[0040] Tryptic peptides were dissolved in 0.1% formic acid (solvent A) and loaded directly onto a homemade reversed-phase analytical column (15 cm length, 75 μm inner diameter). The gradient consisted of increasing from 6% to 23% solvent B (98% acetonitrile in formic acid) over 26 min, from 23% to 35% over 8 min, and then rising to 80% and holding at 800% over the final 3 min, all of which were performed at a constant flow rate of 400 nL / min on an EASY-nLC 1000 UPLC system.

[0041] Peptides were ionized using a nanospray ionization (NSI) source and then analyzed by tandem mass spectrometry (MS / MS) on a Q Exactive™ Plus mass spectrometer (Thermo) coupled to an online ultra-performance liquid chromatography (UPLC) instrument with an applied electrospray voltage of 2.0 kV. The m / z scan range was 350 to 1800 for full scans, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS / MS using a normalized collision energy (NCE) setting of 28. Fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure was used, alternating one MS scan followed by 20 MS / MS scans with a dynamic exclusion time of 15.0 seconds. Automatic gain control (AGC) was set to 5E4, and the fixed first mass was set to 100 m / z.

[0042] 2.5 Database Search

[0043] The MS / MS data were processed using the MaxQuant search engine (v.1.5.2.8). Tandem mass spectra were searched against a database and a reverse decoy database, specifying trypsin / P as the cleavage enzyme and allowing for up to two missed cleavages. The mass tolerance for precursor ions was set to 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set to 0.02 Da. Carbamidomethyl on Cys was specified as a fixed modification, and oxidation of Met was specified as a variable modification. The false discovery rate (FDR) was adjusted to <1%, and the minimum score for peptides was set to >40.

[0044] 3. Results

[0045] Bone tissue proteomic analysis of postmenopausal women A total of 83 bone tissue samples from postmenopausal women were evaluated. These samples and their data were divided into three groups according to hip T scores. In addition to the five participants assigned to the normal group, 46 had osteopenia and 32 had osteoporosis. Among the 83 women included in the analysis, their average age was 76.4±8.7 years and the average body mass index (BMI) was 21.94±3.24 kg / m 2 Unexpectedly, the levels of serum PINP and βCTX, known biomarkers of bone metabolism, did not differ significantly among the groups.

[0046] Example 2 Screening of Osteoporosis Diagnostic Markers

[0047] To identify proteins expressed in human bone tissue, the inventors extracted bone tissue from the femoral heads of postmenopausal women undergoing hip replacement surgery and analyzed these samples by high-resolution mass spectrometry. For all 83 samples, the peptide length distributions identified by mass spectrometry and the mass accuracy distribution of the mass spectrometer met quality control requirements. The inventors' proteomic profile assessment of the 83 bone tissue samples identified a total of 3,743 proteins, of which 3,280 were specifically quantified. Pairwise proteome comparisons of the three groups of bone tissue samples revealed multiple differentially expressed proteins (DEPs), including 353 upregulated and 387 downregulated proteins between the osteopenic and normal groups, 343 upregulated and 288 downregulated proteins between the osteoporotic and osteopenic groups, and 224 upregulated and 310 downregulated proteins between the osteoporotic and normal groups. Compared with previous bone marrow and serum proteomic studies, the inventors found a total of 1222 DEPs (fold change > 1.2, P value < 0.05), which is significantly more than the number identified in previous studies.

[0048] 1. Human Bone Tissue Protein Analysis

[0049] The technical repetitions of 10 mixed samples in this example showed excellent repeatability of proteomic measurements. The inventors performed principal component analysis on all samples, and subcellular localization analysis of 3743 discovered proteins showed that 1231 (32.93%) proteins in human bone tissue were located in the cytoplasm, 783 (20.95%) in the extracellular space, 715 (19.13%) in the nucleus, and 418 (7.14%) in the mitochondria. These discovered proteins obtained gene ontology (GO) annotations for cellular components, biological processes, and molecular functions. These proteins were mainly enriched in the extracellular matrix GO cellular component category, and enriched in GDP, NAD, GTPase binding function, and immune response in the biological process category. In the KEGG pathway enrichment analysis, bone tissue proteins were found to be significantly enriched in carbon metabolism, ECM receptor interactions, and neurological disease pathways, such as pathways associated with Parkinson's disease, Alzheimer's disease, and Huntington's disease. Unsupervised k-means clustering analysis of unique proteins in all human bone tissues revealed several pathways most enriched for bone mass, which involved not only classical oxidative activity, estrogen receptor binding, and calmodulin binding, but also GTPase binding, serine-type endopeptidase activity, and threonine-type endopeptidase activity.

[0050] 2. Osteoporosis is closely related to aging

[0051] The inventors performed weighted gene co-expression network analysis (WGCNA) and obtained 10 highly correlated protein modules. They found that the blue module containing 203 proteins was most highly correlated with age. These age-related proteins had similar subcellular localization for all identified proteins. Gene ontology (GO) term enrichment analysis showed that these proteins may be related to extracellular exosomes, cell adhesion molecule binding, and NF-kappaB signaling. Proteins in this module were particularly enriched in the proteasome pathway, which previous studies have shown to be strongly associated with aging. In summary, this large-scale proteomic analysis of human bone tissue revealed a preliminary overview of human bone proteins. Notably, bone proteins are associated with neurological diseases, and age-related proteins are mainly enriched in the proteasome pathway.

[0052] 3. Identification of differentially expressed proteins in osteoporosis

[0053] To elucidate the proteomic changes underlying the progression of bone loss, the inventors analyzed DEPs (differentially expressed proteins) in three groups of human bone tissue. Compared with normal human bone tissue, DEPs in the bones of patients with osteopenia were mainly enriched in ECM (extracellular matrix) and estrogen receptor binding, while the bones of patients with osteoporosis showed changes in structural molecular activity and ECM structural components. The inventors' analysis also revealed DEPs in bone samples from patients with osteoporosis and osteopenia, which were mainly enriched in the functional processes of fibrillar collagen trimers, collagen fiber organization, and osteoblast proliferation. These results indicate that the DEPs obtained through proteomic analysis of human bone tissue are indeed related to bone metabolism, and many new molecules and pathways related to osteoporosis have been discovered.

[0054] To gain a more comprehensive understanding of the functional differences in bone in osteoporosis patients and to elucidate possible unique protein signatures, the inventors performed gene set enrichment analysis (GSEA) on the molecular signature database using a signature gene set and an additional set of genes for comparison between non-osteoporotic and osteoporotic conditions. The GSEA enrichment map depicted specific differences in pathways with the highest gene enrichment; these pathways included classic adipogenesis and hypoxia pathways, as well as newly discovered mTOR, coagulation, cholesterol homeostasis, and KRAS signaling pathways.

[0055] To further characterize DEPs in osteoporosis, the inventors selected proteins that showed continuous changes in the three groups because patients in these groups had a continuous decline in bone mass. Ultimately, the inventors identified a total of 13 upregulated proteins and 18 downregulated proteins associated with bone mass. Analysis of GO annotation, subcellular localization, and eukaryotic homologous group (KOG) categories did not reveal specificity. By performing functional enrichment analysis, the inventors found that these 31 proteins were related to ossification, endothelial cell proliferation, cartilage development, and ECM formation. The inventors then performed protein interaction network analysis. Together, these analyses identified specific pathways in which these 31 DEPs may play a role in osteoporosis, and the inventors plan to conduct further functional studies on these DEPs.

[0056] By comparing DEPs using database hits and validating them in mouse models and osteoblasts, the inventors identified 31 DEPs as key molecules for osteoporosis. First, the inventors compared these 31 DEPs based on authoritative phenotypic databases, with particular reference to the gene expression profiles of mouse osteoblasts and osteoclasts, positive controls for bone metabolism, data from the International Mouse Phenotype Consortium, and data from international genome-wide association studies (GWAS). As expected, all 31 proteins were included in the expression profile of mouse osteoblasts, but not in the expression profile of osteoclasts. These 31 DEPs were identified in at least some of these databases, and the inventors performed GO biological process enrichment analysis on the proteins found in these authoritative databases. The results showed that the 31 DEPs screened by the inventors were indeed related to bone metabolism and had certain innovations.

[0057] Bilateral ovariectomy is a classic method for establishing an osteoporosis mouse model, and ovariectomized (OVX) mice have low femoral bone mass. After extracting RNA from the mouse bone tissue, the inventors measured the mRNA levels of all 31 DEPs. The inventors found that the levels of Arf4, Matn3, Colla1, Htra1, Coll1a1, Lrpap1, Dag1, Chid1, Acy1, and Dmp1 changed significantly, among which the expression of most DEPs was downregulated. Next, the inventors used small interfering RNA (siRNA) to knock down all 31 DEPs in human osteoblasts. The inventors first observed the proliferation activity of osteoblasts after knockout and found that cell proliferation activity was significantly reduced after the expression of five molecules was knocked down; these five DEPs are C2CD2, COL1A1, KHSRP, LRPAP1, and DMP1. In addition, the inventors also observed the changes in osteoblastic indicators after knockout, including the expression levels of SP7, RUNX2, ALP and OCN, as well as the mineralization changes of osteoblasts.

[0058] Identification and validation of key molecules for osteoporosis in human bone tissue and serum analysis Based on database comparison, OVX mouse bone tissue validation and human osteoblast knockout results, the inventors comprehensively analyzed and identified 9 key molecules that could be validated in at least two studies, based on which the inventors selected ACY1 for further validation.

[0059] Example 3 Verification of the Correlation between ACY1 Expression and Osteoporosis

[0060] 1. Mass Spectrometry Analysis of ACY1

[0061] The inventors collected serum samples from 80 physical examination subjects and investigated the protein levels of ACY1 in these serum samples according to the method in Example 1. They found that the protein level of ACY1 was significantly reduced in osteoporosis patients. Figure 1 The results of the human bone tissue protein spectrum shown in the figure show that ACY1 increases in the normal group, the bone loss group, and the osteoporosis group in this order.

[0062] 2. Exploring the ACY1 mRNA content in bone tissue of osteoporotic mice

[0063] 2.1 Construction of osteoporosis mouse model

[0064] Sixteen 8-week-old female C57Bl / 6 mice were randomly divided into two groups: an osteoporosis group (OVX group, n=8). At 8 weeks of age, mice in this group were anesthetized with intraperitoneal injection of tribromoethanol. The pelvic cavity was then accessed through the skin, muscle, and peritoneum. Both ovaries were then ligated and removed, and the muscular layer and skin were sutured. The other group served as a control group (Ctrl group, n=8). After anesthesia, the ovaries were incised and the skin was sutured. Eight weeks after surgery, all mice were again intraperitoneally injected with tribromoethanol. After complete anesthesia and loss of pain, they were sacrificed by cervical dislocation. Following sacrifice, bilateral femurs and tibias were quickly collected from both groups. Muscles and soft tissue surrounding the lower limb bones were thoroughly removed using ophthalmic scissors and sterile gauze. Bone tissue specimens were wrapped in saline-soaked gauze and stored at -80°C for subsequent micro-CT analysis and qPCR analysis.

[0065] 2.2 RNA extraction from mouse bone tissue

[0066] ① Remove the mouse femur sample from the -80°C freezer and rapidly cool it in liquid nitrogen. Grind the mouse femur thoroughly in a mortar filled with liquid nitrogen until it becomes a powder. During the grinding process, be sure to add liquid nitrogen regularly to prevent RNA degradation due to excessive temperature.

[0067] ②. Transfer the femoral powder to a pre-cooled EP tube. Add 1000 μL of Trizol lysis buffer to each EP tube, allowing the Trizol lysis buffer to fully submerge the bone tissue. Use a 1000 μL pipette to thoroughly mix the mixture. Let it stand at room temperature for 3-5 minutes to fully lyse and digest the tissue.

[0068] ③. Transfer the mouse bone tissue lysate to an RNA-free 1.5 mL EP tube and centrifuge at 12,000 rpm for 5 minutes in a 4°C pre-cooled centrifuge.

[0069] ④. Gently and slowly transfer the supernatant after centrifugation to a 1.5 mL RNA-free EP tube, discard the precipitate, add 100 μL of chloroform, and vigorously invert the tube for 15 seconds.

[0070] ⑤. After shaking, a milky white liquid is visible. Let it stand at room temperature for 5 minutes. After standing, centrifuge it at 4℃, 12000rpm, and centrifuge for 15 minutes.

[0071] ⑥ Carefully remove the EP tube from the centrifuge. You will see that the liquid in the EP tube has separated into three layers. The top layer is a clear, colorless aqueous phase. Transfer this layer to a new RNA-free EP tube with a volume of approximately 200 μL. Immediately add 200 μL of isopropanol. Flocculent material will precipitate. Invert the tube vigorously, shake thoroughly to mix, and let it stand at room temperature for 10 minutes. Place the EP tube back into a 4°C centrifuge and centrifuge at 12,000 rpm for 10 minutes.

[0072] ⑦. Gently remove the EP tube from the centrifuge. A small amount of white precipitate may be seen at the bottom of the tube. Aspirate the supernatant and add 500 μL of 75% ethanol, being careful not to impact the white precipitate. Place the EP tube back into the centrifuge at 4°C and centrifuge at 7500 rpm for 5 minutes.

[0073] ⑧. After centrifugation, carefully aspirate the supernatant with a 1000μL RNA-free pipette tip, place the EP tube on absorbent paper, air-dry the precipitate at room temperature for 10 minutes, add 30-40μL DEPC water, and dissolve the precipitate at 4℃ for at least 6 hours.

[0074] ⑨. Use a nucleic acid detector to detect the RNA concentration of each bone tissue sample, measure and analyze the OD value, take an appropriate amount of RNA for gel electrophoresis, and observe the RNA bands.

[0075] 2.3 QPCR detection of mouse bone tissue

[0076] All operations during this experiment should be performed on ice to prevent RNA degradation.

[0077] ① Genomic DNA residue will seriously affect the accuracy of QPCR test results. Before performing RNA reverse transcription reaction, genomic DNA in RNA extract must be removed first. The specific reaction system is shown in Table 1.

[0078] Table 1 Reaction system

[0079] Reagents Usage 5X gDNA Eraser Buffer 2.0 μL gDNA Eraser 1.0μL Total RNA appropriate amount <![CDATA[RNase Free dH2O]]> Up to 10uL

[0080] 42℃ for 2 min, 4℃+∞

[0081] ②. Pipette 10 μL of the RNA solution obtained from the genomic DNA removal reaction in the previous step, prepare it according to the system shown in Table 2, and perform reverse transcription reaction according to standard reaction conditions.

[0082] Table 2 Reaction system

[0083] Reagents Usage Prim eScript RT Enzyme Mix 1.0 μL RT Primer Mix 1.0μL 5X PrimeScript Buffer 2 4.0μL <![CDATA[RNase Free dH2O]]> 4.0μL Step ① reaction solution 10uL

[0084] 37℃ for 15min, 85℃ for 5s, 4℃+∞

[0085] ③ After the reverse transcription reaction is completed, 20 μL of cDNA can be obtained from each bone tissue sample. The cDNA of each sample must be prepared on ice in the dark according to the system shown in Table 3, and the PCR reaction must be performed according to standard reaction conditions.

[0086] Table 3 Reaction system

[0087] Reagents Usage 2X SYBR 10 μL PCR Forward Primer 0.8μL PCR Reverse Primer 0.8μL 50X ROX 0.4μL cDNA 2.0 μL <![CDATA[dH2O]]> 6.0μL

[0088] ④ PCR reaction conditions are:

[0089] Step 1: pre-denaturation, 95°C for 30 s, 1× cycle;

[0090] Step 2: PCR reaction, 95℃5s, 60℃31s, 40× cycles.

[0091] ⑤. After the reaction is completed, promptly confirm the amplification curve and melting curve of Real Time PCR, and compare and analyze the experimental results.

[0092] The specific primers used are shown in Table 4.

[0093] Table 4 Primers used

[0094]

[0095] The final result is as follows Figure 2 As shown by Figure 2 The results showed that ACY1 mRNA was significantly reduced in the bone tissue of OVX mice after castration.

[0096] 3. Exploring osteoblastic markers after knockdown of ACY1 in osteoblasts

[0097] 3.1 Osteogenic Induction Culture of Saos-2 Cells

[0098] ① When the cells were 70%-80% confluent, they were digested and passaged using trypsin. The cells were plated at 2×10 5 The cells were seeded at a density of 100 μg / ml in a 12-well culture plate and placed in an incubator for normal culture.

[0099] ② After the osteoblasts have completely adhered overnight, replace the osteogenic induction medium containing 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate.

[0100] ③. Culture the cells with complete culture medium containing osteogenic inducers for 21 days. Change the culture medium every other day for the first 7 days and then every day for the next 14 days.

[0101] 3.2 si-RNA transfection of Saos-2 osteoblasts

[0102] ①. Add 25 μL of serum-free transfection medium Opti-MEM to every 0.25 μL of Lipofectamine RNAi MAX reagent in an EP tube, mix well, and let it stand at room temperature for 5 minutes.

[0103] ②. Add 15 pmol of siRNA to 25 μL of serum-free transfection medium Opti-MEM and mix thoroughly to dissolve.

[0104] ③. Gently mix the diluted solutions obtained in ① and ② thoroughly with a 200μL pipette and let stand at room temperature for 15 minutes.

[0105] ④. Remove the culture medium from Saos-2 cells with a cell density of 70% to 80%, wash with PBS, add complete culture medium, and add the reagent prepared in ③ to the culture dish and mix thoroughly.

[0106] ⑤. After culturing osteoblasts in the culture plate for 24-48 hours, the transfection effect and efficiency are verified by extracting cell RNA and performing QPCR detection.

[0107] 3.3 RNA extraction from Saos-2 osteoblasts

[0108] ① After completing the culture and intervention of each group of Saos-2 osteoblasts, remove each group of cells from the incubator, aspirate the remaining culture medium with a pipette, and wash the cells with room temperature PBS three times, each time for 3 minutes.

[0109] ② After washing, use Trizol lysis buffer to lyse the osteoblasts. Add 500 μL of lysis buffer to each well of osteoblasts. Gently shake the culture dish up and down to allow the Trizol lysis buffer to completely cover all cells. After a while, use a 1000 μL pipette to blow the cells and let them stand at room temperature for 5 minutes.

[0110] ③ After the osteoblasts in the culture dish are completely digested, transfer them to an RNA-free 1.5 mL EP tube and centrifuge in a pre-cooled 4°C centrifuge at 12,000 rpm for 5 minutes.

[0111] ④. Carefully remove the EP tube from the centrifuge, carefully aspirate the supernatant with a pipette, add 100 μL of chloroform, and shake vigorously upside down for 15 seconds.

[0112] ⑤. After shaking, let it stand at room temperature for 5 minutes, and then centrifuge again in a pre-cooled 4℃ centrifuge at 12000 rpm for 15 minutes.

[0113] ⑥ Carefully remove the EP tube from the centrifuge. You will see that the liquid in the EP tube has separated into three layers. The top layer is a clear, colorless aqueous phase. Transfer this layer to a new RNA-free EP tube with a volume of approximately 200 μL. Immediately add 200 μL of isopropanol. Flocculent material will precipitate. Invert the tube vigorously, shake thoroughly to mix, and let it stand at room temperature for 10 minutes. Place the EP tube back into a 4°C centrifuge and centrifuge at 12,000 rpm for 10 minutes.

[0114] ⑦. Gently remove the EP tube from the centrifuge. A small amount of white precipitate may be seen at the bottom of the tube. Aspirate the supernatant and add 500 μL of 75% ethanol, being careful not to impact the white precipitate. Place the EP tube back into the centrifuge at 4°C and centrifuge at 7500 rpm for 5 minutes.

[0115] ⑨. After centrifugation, carefully aspirate the supernatant with a 1000μL RNA firee pipette tip, place the EP tube on absorbent paper, air-dry the precipitate at room temperature for 10 minutes, add 30-40μL DEPC water, and dissolve the precipitate at 4℃ for at least 6 hours.

[0116] ⑨. Use a nucleic acid detector to detect the RNA concentration of each bone tissue sample, measure and analyze the OD value, take an appropriate amount of RNA for gel electrophoresis, and observe the RNA bands.

[0117] 3.4 RT-PCR detection of osteoblast-related gene expression

[0118] All operations during the experiment should be performed on ice to prevent RNA degradation, and all operations should be strictly in accordance with the kit instructions.

[0119] ① Genomic DNA residue will seriously affect the accuracy of QPCR test results. Before performing RNA reverse transcription reaction, genomic DNA in RNA extract must be removed first. The specific reaction system is shown in Table 5.

[0120] Table 5 Reaction system

[0121] Reagents Usage 5X gDNA Eraser Buffer 2.0 μL gDNA Eraser 1.0 μL Total RNA appropriate amount <![CDATA[RNase Free dH2O]]> Up to 10μL

[0122] 42℃ for 2 min, 4℃+∞

[0123] ②. Pipette 10 μL of the RNA solution obtained from the genomic DNA removal reaction in the previous step, prepare it according to the system shown in Table 6, and perform reverse transcription reaction according to standard reaction conditions.

[0124] Table 6 Reaction system

[0125] Reagents Usage Prim eScript RT Enzyme Mix 1.0 μL RT Primer Mix 1.0 μL 5X PrimeScript Buffer 2 4.0μL <![CDATA[RNase Free dH2O]]> 4.0μL Step ① reaction solution 10 μL

[0126] 37℃ for 15min, 85℃ for 5s, 4℃+∞

[0127] ③. The 20 μL liquid obtained in the previous step is the cDNA of each group of osteoblasts. Each group of cDNA is subjected to PCR reaction according to the system shown in Table 7 (all reaction solutions must be prepared on ice).

[0128] Table 7 Reaction system

[0129]

[0130]

[0131] ④ PCR reaction conditions are:

[0132] Step 1: pre-denaturation, 95°C for 30 s, 1× cycle;

[0133] Part II: PCR reaction, 95°C for 5s, 60°C for 31s, 40× cycles.

[0134] ⑤. After the reaction is completed, promptly confirm the amplification curve and melting curve of Real Time PCR, and compare and analyze the experimental results.

[0135] The specific primers used are shown in Table 8.

[0136] Table 8 Primers used

[0137]

[0138] The final result is as follows Figure 3 As shown by Figure 3 It can be seen from the results of the study on the osteoblastic indicators after knocking down ACY1 in human osteoblasts that the osteoblastic indicators increased significantly after knocking down ACY1.

[0139] 4. Extraction of human bone protein and detection of ACY1 protein expression level by western blot

[0140] 4.1 Extraction of human bone tissue protein

[0141] ① Remove the human bone tissue sample from a -80°C freezer and rapidly cool it in liquid nitrogen. Grind it thoroughly in a mortar filled with liquid nitrogen until it becomes a powder. During the grinding process, be sure to add liquid nitrogen promptly to prevent protein degradation due to excessive temperature.

[0142] ②. Transfer the bone tissue powder to a pre-cooled EP tube, add 500 μL of RIPA reagent (protein lysis and extraction solution, containing 10×PI) to each tube of bone tissue powder, gently shake the EP tube to allow the protein lysis solution RIPA to fully immerse the bone tissue powder and mix the reaction thoroughly. Digest and lyse on ice for 30 minutes, inverting and mixing once every 5 minutes.

[0143] ③. Transfer the completely cleaved and digested human bone tissue protein solution to a new 1.5 mL EP tube and centrifuge at 12,000 rpm in a 4°C centrifuge for 5 minutes.

[0144] ④ After centrifugation, carefully remove the EP tube, aspirate the supernatant, freeze it in liquid nitrogen, and immediately store it in a -80℃ refrigerator for later use.

[0145] 4.2 Western Blot of Human Bone Tissue-Related Proteins

[0146] 4.2.1 Preparation of standard curve and detection of sample protein content

[0147] ①. Take out the BCA kit standard: 30 mg BSA from the -20℃ refrigerator, fully dissolve it in standard solvent and prepare a 25 mg / mL protein solution standard.

[0148] ② Take 10 μL of 25 mg / mL protein standard solution and dilute it at a ratio of 1:500 to obtain a BSA protein solution with a final concentration of 0.5 mg / mL.

[0149] ③. Take out 50X BCA solution and dilute it to 1X BCA working solution for subsequent testing.

[0150] ④. Prepare a transparent 96-well plate and add 0.5 mg / mL BSA protein solution into the standard wells at volumes of 0, 1, 2, 4, 8, 12, 16, and 20 μl. Fill up to 20 μl with protein diluent.

[0151] ⑤. After all sample protein solutions are diluted appropriately, add them into a 96-well plate at a volume of 20 μL per well.

[0152] ⑥. Add 200 μL of BCA working solution to each well and incubate at room temperature for 30 minutes (the specific incubation time should be determined according to the protein concentration and dilution factor, and multiple observations can be made during this period).

[0153] ⑦ After incubation, use a microplate reader to measure the OD value at a wavelength of 540-595 nm. Based on the OD value and the protein concentration of the standard, fit a linear trend line to complete the construction of the standard curve.

[0154] ⑨. According to the linear fitting formula of the standard curve, substitute the OD value of the sample protein solution into the formula to calculate the protein content of the sample.

[0155] 4.2.2 Protein denaturation

[0156] ①. Determine the approximate loading amount of all samples based on the sample protein solution concentration detected by BCA, and add 4×SDS sample buffer solution (containing 2-mercaptoethanol) to the sample solution.

[0157] ② After fully mixing the sample protein solution and sample buffer solution, place it in a 95℃ water bath and cook for 3 minutes and 30 seconds to denature the protein. The denatured protein solution can be stored in a -20℃ refrigerator for subsequent experimental use.

[0158] 4.2.3 Glue making

[0159] ①. Select two complete glass plates, rinse them with clean water first, then use detergent to repeatedly clean them until the water neither gathers into drops nor flows down in streams on the glass surface. Finally, rinse both sides with distilled water, place them in a ventilated place at room temperature, and let them dry naturally.

[0160] ② After the glass plates have dried naturally, align the two glass plates first, then place them into the special card slot for making glue. Confirm that the glass plates are aligned again and fasten them, then install the entire card slot vertically on the glue rack.

[0161] ③. Prepare a separation gel of appropriate concentration according to the molecular weight of the target protein to be detected. After the preparation is completed according to the standard system, add TEMED in the last step and immediately mix it thoroughly by pipetting. Immediately use a pipette to slowly and vertically pour the system solution into the glass plate. After the liquid surface is slightly flat, gently and slowly add a layer of alcohol on the gel to flatten it and drive out bubbles.

[0162] The glue formulas for each concentration are shown in Tables 9-10.

[0163] Table 9 Glue formulas for various concentrations (I)

[0164] Separation gel / reagents 6% concentration 8% concentration 10% concentration 12.5% concentration Double distilled water (mL) 2.925 2.675 2.475 2.19 40% acrylamide (mL) 0.75 1 1.275 1.56 1.5M Tris-HCl (mL) 1.25 1.25 1.25 1.25 10% SDS (μL) 50 50 50 50 10% APS (μL) 30 30 30 30 TEMED (μL) 5 5 5 5

[0165] Table 10 Glue formulas for various concentrations (II)

[0166] Stacking gel / reagents 3% concentration 4% concentration Double distilled water (mL) 2.025 1.95 40% acrylamide (mL) 0.225 0.3 0.5M Tris-HCl (mL) 0.75 0.75 10% SDS (μL) 30 50 10% APS (μL) 25 30 TEMED (μL) 3 5

[0167] ④. After standing for 20 minutes, when the separation gel is fully solidified, if the alcohol on the separation gel has not completely evaporated, the alcohol can be discarded, and then rinsed with double distilled water and dried with absorbent paper.

[0168] ⑤. Continue preparing 4% stacking gel according to the standard recipe. After adding TEMED in the final step, mix thoroughly by pipetting. Immediately, use a pipette to slowly and vertically fill the glass plate with the solution. Simultaneously, insert a comb of appropriate size into the glass plate, inserting it diagonally downward to minimize bubble formation. Wait 20 minutes for the stacking gel to solidify before removing the comb and proceeding with electrophoresis.

[0169] 4.2.4SDS-PAGE electrophoresis

[0170] ① After pulling out the comb, rinse the sample well with double distilled water to remove the residual concentrated gel and impurities, then place it in the electrophoresis tank. Make sure the glass plate opening is facing inwards when placing it.

[0171] ② Before electrophoresis, make sure the electrodes of the electrophoresis tank are correct, then pour the electrophoresis fluid into the tank and ensure that the liquid level of the electrophoresis fluid exceeds the level of the electrodes.

[0172] ③. Take the denatured protein sample out of the -20℃ refrigerator. After it is fully dissolved, use a 10μL pipette to slowly add the protein sample vertically into the sample well. At the same time, add appropriate protein marker reagents to the spotting wells on both sides.

[0173] ④. Set the starting voltage of gel electrophoresis to 110V. When the bromophenol blue indicator buffer runs out of the stacking gel layer and the protein bands are relatively neat, lower the voltage to 90V to avoid excessive voltage and heat generation that may affect the experimental results.

[0174] ⑤. During the electrophoresis process, pay attention to real-time observation of the electrophoresis situation. When the bromophenol blue indicator at the bottom is about to run out of the entire separation gel, the electrophoresis can be terminated.

[0175] 4.2.5 Transfer

[0176] ①. Take out the separation gel from the electrophoresis tank after electrophoresis. Rinse the glass plate with double-distilled water. Peel off the entire separation gel from the glass plate. Gently rinse the separation gel in double-distilled water and then transfer it to the transfer buffer for buffering.

[0177] ②. Cut a 6cm×9cm PVDF membrane and 6 pieces of 7cm×10cm filter paper. Place the PVDF membrane in 100% methanol to fully immerse it for a few seconds for activation, and then immediately transfer it to double-distilled water for immersion. After activation, the PVDF membrane must not dry out and must be kept moist.

[0178] ③. Thoroughly clean the clips and sponges needed for transfer and place them in the transfer solution.

[0179] ④. Place the separation gel and PVDF membrane in the transfer clamp in the following order, from negative to positive, namely the first sponge, 3 filter papers, separation gel, PVDF membrane, 3 filter papers and the second sponge. During the placement process, be sure to ensure that the PVDF membrane and separation gel are completely aligned, and the order must not be wrong. The entire process must be completed while soaking in transfer solution.

[0180] ⑤. Place the transfer clip quickly in the transfer tank filled with transfer solution according to the principle of positive to positive and negative to negative. The transfer conditions are generally constant voltage 30V, 6h or constant current 200mA, 1h30min (the specific conditions should be adjusted appropriately according to the size of the target protein).

[0181] 4.2.6 Immune response (primary and secondary antibody incubation)

[0182] ① After the transfer is completed, take out the membrane and rinse it with TBS 2 to 3 times, each time for 5 minutes.

[0183] ② After washing, use TBST to prepare 5% BSA or 5% skim milk for blocking. The blocking conditions are: room temperature, decolorization shaker, and time for 1 hour.

[0184] ③. Prepare the primary antibody using 5% BSA or 5% skim milk and dilute it accordingly according to the Western blot requirements in the primary antibody instructions. Cut the PVDF membrane to the appropriate size according to the size of the target protein and the marker on the PVDF membrane. Then immerse the PVDF membrane in the diluted primary antibody solution and incubate. The general incubation conditions are: room temperature for 1-2 hours or 4°C for 6 hours.

[0185] ④ After the primary antibody incubation is completed, transfer the PVDF membrane to TBST for washing, washing twice, each time for 10 minutes, and recover the primary antibody dilution solution at the same time.

[0186] ⑤. After washing, incubate with secondary antibody at room temperature. The choice of secondary antibody mainly depends on the host source of the primary antibody. The incubation conditions are generally: room temperature, duration of 1 hour; after incubation, wash 3 times with TBST, each time for 5 minutes.

[0187] 4.2.7 Chemiluminescence imaging

[0188] ①. Prepare the developer solution by mixing developer A and developer B in a ratio of 1:1 to obtain the developer working solution. Keep away from light.

[0189] ②. Add an appropriate amount of developing working solution to the PVDF membrane that has completed the immune reaction to carry out the color development reaction. The specific exposure time should be determined by various factors such as the titer of the antibody and the protein concentration of the sample solution.

[0190] ③. Expose and capture images of the PVDF membrane after color development using an ultra-sensitive multifunctional imager, and observe and analyze changes in protein expression.

[0191] The final protein expression was as follows Figure 4 As shown by Figure 4 The expression of ACY1 protein in human bone tissue of patients with osteoporosis was significantly reduced (ACY1 protein expression was detected by ACY1 antibody (Abcam, ab277463)).

[0192] It can be seen that the present invention has confirmed the potential of ACY1 protein as a diagnostic marker for osteoporosis through a series of experiments. First, in a Western blot analysis comparing normal bone mass and bone tissue of osteoporosis patients, it was found that the ACY1 protein level in osteoporosis patients was significantly reduced, suggesting that ACY1 may be related to the onset of osteoporosis. Further, by collecting serum samples from 80 physical examination personnel and evaluating the ACY1 protein level by bone density group, the significant reduction of ACY1 in osteoporosis patients was verified again. In addition, in the study of human bone tissue protein spectrum, ACY1 mRNA expression in bone tissue of castrated mice, and osteogenic index after knocking down ACY1 in human osteoblasts, a positive correlation between ACY1 and osteoporosis was shown. In summary, changes in ACY1 protein levels can be used as an effective marker for diagnosing osteoporosis, providing a new potential target for the early diagnosis and treatment of osteoporosis.

[0193] Example 4 Analysis of the sensitivity and specificity of ACY1 as a diagnostic marker for osteoporosis

[0194] The receiver operating characteristic curve (ROC curve) is a graphical method for evaluating the performance of a binary classification model. It is formed by connecting the points corresponding to sensitivity and specificity at different cutoff values with sensitivity (i.e., true positive rate) as the vertical axis and 1-specificity (i.e., false positive rate) as the horizontal axis. The curve shows the model's ability to distinguish between positive and negative individuals at different judgment thresholds. ROC curves are often used in diagnostic or prognostic studies to measure the model's ability to predict the probability of an event and to help determine the optimal critical point, that is, to make the curve as close to the upper left corner as possible. Its performance is often expressed as the area under the curve (AUC). AUC reflects a quantitative indicator of the overall discrimination accuracy of the model. The value ranges from 0.5 to 1.0. The closer the value is to 1, the more effectively the model can distinguish between individuals with and without a certain outcome. The ROC curve using ACY1 as a diagnostic marker for osteoporosis is shown below. Figure 5 shown.

[0195] The specific method for analysis using GraphPad Prism is as follows:

[0196] (1) Open GraphPad Prism. On the left side of the welcome screen that pops up, select New table & graph, select Column. In Data table, select Enter or import data into a new table. In Options, select Enter replicate values, stacked into columns. Click Create.

[0197] (2) Enter the required data in Group A and B columns, representing the normal population group and the osteoporosis patient group

[0198] (3) Select the Analyze command icon in the Analysis option tool group in the toolbar. In the newly popped-up Analyze Data command box, select Column analyses. Continue to select the ROC Curve option. Check the two data sets A: Normal group and B: Osteoporosis group in the right box, and click OK.

[0199] (4) In the pop-up ROC curve parameter setting window, keep the default options and click OK

[0200] (5) In the Results, check the Area and P value. The Area is 0.8592 and the P value is less than 0.05, indicating that the expression level of ACY1 protein can effectively distinguish between normal people and osteoporosis patients.

[0201] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A marker for diagnosing osteoporosis, characterized in that: The marker is ACY1.

2. The marker for diagnosing osteoporosis according to claim 1, wherein The expression level of the marker is low in osteoporosis patients.

3. A product for diagnosing osteoporosis, characterized in that: The product contains a reagent for quantitatively detecting the marker according to claim 1.

4. The product according to claim 3, characterized in that The products include detection kits, polymerase chain reaction reagents, chip detection reagents or sequencing reagents.

5. The product according to claim 3, characterized in that The reagent for quantitatively detecting the marker according to claim 1 comprises primers and probes capable of quantitatively detecting ACY1.

6. Use of the marker according to any one of claims 1 to 2 in the preparation of a product for diagnosing osteoporosis.

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