Marker LRPAP1 for diagnosing osteoporosis and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a marker LRPAP1 for diagnosing osteoporosis and application of the marker LRPAP1, and the marker LRPAP1 is low in expression quantity in osteoporosis patients. By quantitatively detecting the level of the LRPAP1, 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. According to the application, understanding of molecular mechanisms of the osteoporosis is promoted, the LRPAP1 serves as a novel diagnostic marker, changes of the expression level of the LRPAP1 are closely related to pathogenesis of the osteoporosis, the role of the LRPAP1 in the 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 particularly relates to a biomarker LRPAP1 for diagnosing osteoporosis and its application. Background Art

[0002] Osteoporosis is a systemic metabolic bone disease in the elderly, characterized by low bone mass, deteriorated bone microstructure and increased fracture risk. Bone metabolism occurs throughout life and maintains bone homeostasis through bone resorption and bone formation. Osteoporosis usually leads to fractures in the hip or spine, which can be painful, inconvenient, destructive, and even fatal. The International Osteoporosis Foundation estimates that osteoporosis affects more than 200 million people and is the most common age-related bone disease in the world, especially prevalent in postmenopausal women. Due to the rapid growth of the elderly population, the incidence of osteoporosis is also increasing, which poses a significant burden not only on public health but also on the economy. However, traditional osteoporosis treatments have led to mixed results and have many side effects, and classical serum markers are usually not effective in describing bone quality. Therefore, research on the mechanism of osteoporosis urgently needs to generate new research directions and lay a 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", and functional genomics has become the main research tool. Functional genomics includes structural genomics and proteomics research. In "proteomics", proteins are the research objects, and the focus is on the protein composition and protein-related changes in cells, tissues and organisms. The process of proteomics research involves comprehensive observation and analysis of the occurrence of diseases and cell metabolism processes, and these analyses are carried out at the protein level. Proteomics can provide an important theoretical basis for elucidating the mechanisms of many diseases and provide new molecular markers for the early diagnosis of diseases. In recent years, proteomics analysis related to osteoporosis has mainly focused on osteoblasts, osteoclasts, osteocytes and bone-related cells under model animals and different in vitro experimental conditions. However, the protein profile of human bone tissue is still unclear, and the analysis based on "human bone tissue proteomics" related to osteoporosis has not been carried out. So far, few proteomics studies on serum and bone marrow proteomics have focused on osteoporosis, but the proteomic information on human bone tissue and osteoporosis is still limited. Therefore, it is urgent to conduct research on osteoporosis in 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 object of the present invention is to provide a marker LRPAP1 for diagnosing osteoporosis and its application, and specifically provide a diagnostic marker that can be applied to the clinical diagnosis of osteoporosis. Using this diagnostic marker can be used for the early detection of the course of osteoporosis and has high sensitivity and high specificity during detection.

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

[0006] The present invention provides a marker for diagnosing osteoporosis, and the marker is LRPAP1.

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

[0008] The present invention also provides a product for diagnosing osteoporosis, and the product contains a reagent for quantitatively detecting the marker described in 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 a probe capable of quantitatively detecting LRPAP1.

[0011] The present invention also provides an application of the marker in the preparation of a product for diagnosing osteoporosis.

[0012] Beneficial effects:

[0013] The present invention provides a new diagnostic marker LRPAP1, and the expression level of this marker is significantly reduced in osteoporosis patients. By quantitatively detecting the level of LRPAP1, osteoporosis can be accurately identified at the early stage of the disease course, significantly improving the sensitivity and specificity of the diagnosis. This helps doctors take timely intervention measures, 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, the change in the expression level of LRPAP1 is closely related to the pathogenesis of osteoporosis. Based on the disclosure of the present invention, it is expected to reveal the role of LRPAP1 in osteoporosis and provide new ideas for the treatment of the disease.

[0014] The present invention focuses on the proteomic analysis related to osteoporosis, especially the research on the proteomics of human bone tissue, serum and bone marrow. By discovering this new diagnostic marker LRPAP1, the present invention fills the gap in the research in this field and provides new directions and ideas for future research. Description of the Drawings

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0016] Figure 1 It is a mass spectrometry result diagram of LRPAP1 protein in human bone tissue, where *** represents significant difference;

[0017] Figure 2 It is a result diagram of Lrpap1 mRNA in mouse bone tissue after ovariectomy, where ** represents significant difference;

[0018] Figure 3 It is a comparison diagram of osteogenic indexes after knocking down LRPAP1 in human osteoblasts, where *** represents significant difference;

[0019] Figure 4 It is a result diagram of alizarin red staining after knocking down LRPAP1;

[0020] Figure 5 It is a diagram of detecting the protein expression of LRPAP1 in human bone tissue of osteoporosis patients;

[0021] Figure 6 It is a bar chart of detecting the protein expression of LRPAP1 in human bone tissue of osteoporosis patients, where *** represents significant difference;

[0022] Figure 7 It is the analysis result of the sensitivity and specificity of LRPAP1 as a diagnostic marker for osteoporosis. Detailed implementation manners

[0023] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although only preferred methods and materials are described in this invention, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0026] Without departing from the scope or spirit of the present invention, various modifications and variations to the specific embodiments of the description of the present invention will be apparent to those skilled in the art. Other embodiments obtained from the description of the present invention will be apparent to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0027] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0028] The test methods used in the following examples are all common experimental methods in the art unless otherwise specified;

[0029] The test materials used in the following examples are all common experimental materials in the art unless otherwise specified.

[0030] Example 1 Osteoporosis Protein Osteological Analysis

[0031] 1. Sample Preparation

[0032] This study complied with the 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 under ID-LK2020-027-01. All participants were recruited from the Department of Orthopedics of the Second Affiliated Hospital of Soochow University in Suzhou, China. The inventors recruited patients diagnosed with unilateral femoral neck fractures who required hip replacement surgery. Two to three days before the hip replacement surgery, bone mineral density (BMI) was examined in each patient using dual-energy X-ray absorptiometry (Hologic Delphi A; Hologic, Bedford, MA, USA). Exclusion criteria included infection, tumors, hip dysplasia, osteonecrosis of the femoral head, osteomalacia, coagulopathy, renal insufficiency, history of lumbar spine surgery, history of anti-osteoporosis treatment, and diseases affecting bone metabolism, such as thyroid diseases, parathyroid diseases, adrenal diseases, and diabetes. According to their hip T-scores, the patients were divided into three groups: normal (A) group with T ≥ -1.0, osteopenia (B) group with -2.5 < T < -1.0, and osteoporosis (C) group with T ≤ -2.5. An appropriate amount of femoral head tissue was obtained 0.5 cm below the attachment point of the ligamentum teres femoris, and all tissue samples were stored at -80°C. The frozen specimens were stored on dry ice, and approximately 50 mg of tissue was cut from each sample for proteomic analysis.

[0033] The samples were first ground in liquid nitrogen, then the powder was transferred to 5-ml centrifuge tubes and sonicated three times on ice in lysis buffer (including 1% Triton X-100, 10 mM dithiothreitol, 1% protease inhibitor mixture, 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, and then vortexed for an additional 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 least at -20°C for 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 proteins were then redissolved in 8 M urea, and the protein concentration was determined using a BCA kit according to the manufacturer's instructions.

[0034] 2. Sample processing

[0035] 2.1 Trypsin digestion

[0036] The above protein solution was reduced with 5 mM dithiothreitol at 56 °C for 30 minutes, then alkylated with 11 mM iodoacetic acid in the dark at room temperature for 15 minutes, and then the protein sample was diluted to a urea concentration of less than 2 M by adding 100 mM TEAB. Finally, the first overnight digestion was carried out at a trypsin-to-protein mass ratio of 1:50, and the second 4-hour digestion was carried out at a trypsin-to-protein mass ratio of 1:100.

[0037] 2.2 TMT labeling

[0038] After trypsin digestion, the peptides were desalted using a Strata X C18 SPE column (Phenomenex) and vacuum dried. The peptides were redissolved in 0.5 M TEAB and processed using a TMT kit according to the manufacturer's protocol. Specifically, one unit of TMT reagent was thawed and redissolved in acetonitrile, and then the peptide mixture was incubated at room temperature for 2 hours, combined, desalted, and dried by vacuum centrifugation.

[0039] 2.3 HPLC separation

[0040] The 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 with a gradient of 8% to 32% acetonitrile (pH 9.0) in 60 minutes, and then the peptides were combined into 18 fractions and dried by vacuum centrifugation.

[0041] 2.4 LC-MS / MS analysis

[0042] The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm long, 75 μm inner diameter). The gradient included an increase from 6% to 23% of solvent B (98% acetonitrile in formic acid) in 26 minutes, an increase from 23% to 35% in 8 minutes, and then an increase to 80% and held at 80% in the last 3 minutes, all carried out at a constant flow rate of 400 nL / min on an EASY-nLC 1000 UPLC system.

[0043] The peptide segments were analyzed by tandem mass spectrometry (MS / MS) through a nano spray ionization (NSI) source and then by a Q ExactiveTM Plus mass spectrometer (Thermo), which was coupled with an online ultra-high performance liquid chromatography (UPLC) instrument. The applied electrospray voltage was 2.0 kV. The m / z scan range was from 350 to 1800 for full scan. Intact peptide segments were detected in the Orbitrap at a resolution of 70,000. The peptide segments were then selected for MS / MS using a normalized collision energy (NCE) set at 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure was adopted, alternating one MS scan and then 20 MS / MS scans, with a dynamic exclusion time of 15.0 seconds. The automatic gain control (AGC) was set to 5E4, and the fixed first mass was set to 100 m / z.

[0044] 2.5 Database search

[0045] The MS / MS data were processed using the MaxQuant search engine (v.1.5.2.8). The tandem mass spectra were searched against the database and the reverse decoy database. Trypsin / P was specified as the cleavage enzyme, allowing a maximum of 2 missed cleavages. The mass tolerance of precursor ions was set to 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance of 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 of peptide segments was set to >40.

[0046] 3. Results

[0047] For the postmenopausal women with bone tissue proteomics analysis, a total of 83 bone tissue samples from postmenopausal women were evaluated. These samples and their data were divided into three groups according to the hip T-score. Except for 5 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 / m2. Unexpectedly, the levels of serum PINP and βCTX, as known bone metabolism biomarkers, showed no significant differences among the groups.

[0048] Example 2 Screening of osteoporosis diagnostic markers

[0049] To identify proteins expressed in human bone tissue, the inventors extracted bone tissue from the femoral heads of postmenopausal women who had undergone hip replacement surgery and analyzed these samples by high-resolution mass spectrometry. For all 83 samples, the peptide length distribution identified by mass spectrometry and the mass accuracy distribution of the mass spectrometer met the quality control requirements. The inventors' assessment of the proteomic profiles of 83 bone tissue samples found a total of 3,743 proteins, of which 3,280 were specifically quantified. Pairwise proteomic comparisons of the three groups of bone tissue samples revealed multiple differentially expressed proteins (DEPs), including 353 upregulated and 387 downregulated proteins between the osteopenia and normal groups, 343 upregulated and 288 downregulated proteins between the osteoporosis and osteopenia groups, and 224 upregulated and 310 downregulated proteins between the osteoporosis and normal groups. Compared with previous bone marrow and serum proteomic studies, the inventors found a total of 1,222 DEPs (fold change > 1.2, p-value < 0.05), which is significantly more than the number identified in previous studies.

[0050] 1. Protein analysis of human bone tissue

[0051] Technical replicates of the 10 pooled samples in this example showed excellent reproducibility of proteomic measurements. The inventors performed principal component analysis on all samples. Subcellular localization analysis of the 3,743 identified proteins showed that 1,231 (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. The identified proteins were annotated with Gene Ontology (GO) terms for cellular components, biological processes, and molecular functions. These proteins were mainly enriched in the extracellular matrix GO cellular component category and in the biological process category for GDP, NAD, GTPase binding functions, and immune responses. In the enrichment analysis of KEGG pathways, bone tissue proteins were found to be significantly enriched in carbon metabolism, ECM-receptor interaction, and nervous system disease pathways, such as those related to Parkinson's disease, Alzheimer's disease, and Huntington's disease. Unsupervised k-means clustering analysis of the unique proteins in all human bone tissue revealed several pathways most abundant in bone mass, which were involved not only in classical oxidative activity, estrogen receptor binding, and calmodulin binding, but also in GTPase binding, serine-type endopeptidase activity, and threonine-type endopeptidase activity.

[0052] 2. Osteoporosis is closely related to aging

[0053] The inventors performed weighted gene co-expression network analysis (WGCNA), obtained 10 highly correlated protein modules, and found that the blue module containing 203 proteins had the highest correlation with age. These age-related proteins had similar subcellular localizations for all identified proteins. Gene Ontology (GO) term enrichment analysis indicated that these proteins might be related to extracellular exosomes, cell adhesion molecule binding, and NF-kappaB signaling. Proteins in this module were particularly enriched in the proteasome pathway, and previous studies have shown a strong relationship between this pathway and aging. In summary, this large-scale human bone tissue proteomics analysis revealed a preliminary profile of human bone proteins. Notably, bone proteins are related to neurological diseases, and age-related proteins are mainly enriched in the proteasome pathway.

[0054] 3. Identification of Differentially Expressed Proteins in Osteoporosis

[0055] To clarify the proteomic changes underlying the progression of bone loss, the inventors separately analyzed DEPs (differentially expressed proteins) in three groups of human bone tissues. 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 osteoporosis patients showed changes in structural molecule activity and ECM structural components. The inventors' analysis also revealed DEPs in bone samples from osteoporosis and osteopenia patients, which were mainly enriched in functional processes such as fibrillar collagen trimer, 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.

[0056] To more comprehensively understand the functional differences in the bones of osteoporosis patients and clarify the potentially unique protein characteristics, the inventors performed gene set enrichment analysis (GSEA) on the Molecular Signatures Database using signature gene sets and an additional set of genes for comparison between non-osteoporosis and osteoporosis. The GSEA enrichment map depicted specific differences in the pathways with the highest gene enrichment; these pathways included classical adipogenesis and hypoxia pathways, as well as newly discovered mTOR, coagulation, cholesterol homeostasis, and KRAS signaling pathways.

[0057] To further characterize the DEPs in osteoporosis, the inventors selected proteins that showed continuous changes in three groups because the bone mass of the patients in these groups decreased continuously. Finally, the inventors identified a total of 13 upregulated proteins and 18 downregulated proteins related to bone mass. Analysis of GO annotation, subcellular localization, and eukaryotic orthologous groups (KOG) categories showed no particularity. By performing functional enrichment analysis, the inventors found that these 31 proteins were related to ossification, endothelial cell proliferation, cartilage development, and ECM formation. Then the inventors performed protein interaction network analysis. These analyses together identified specific pathways in which these 31 DEPs might play a role in osteoporosis, and the inventors plan to conduct further functional studies on these DEPs.

[0058] Comparing the DEPs using database click-throughs and validating them in mouse models and osteoblasts, the inventors identified 31 DEPs as key molecules in osteoporosis. First, the inventors compared these 31 DEPs according to an authoritative phenotype database, with particular reference to the gene expression profiles of mouse osteoblasts and osteoclasts, positive controls for bone metabolism, data from the International Mouse Phenotyping 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 a part 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 a certain degree of innovation.

[0059] Bilateral ovariectomy is a classic method for constructing a mouse model of osteoporosis, and the femur bone mass of ovariectomized (OVX) mice is low. After extracting RNA from the bone tissue of the mice, the inventors measured the mRNA levels of all 31 DEPs. The inventors found that the levels of Afrf4, Matn3, Col1a1, Htra1, Col11a1, Lrpap1, Dag1, Anp32a, and Dmp1 changed significantly, and the expression of most DEPs was downregulated. Next, the inventors knocked out all 31 DEPs in human osteoblasts using small interfering RNA (siRNA). The inventors first observed the proliferation activity of osteoblasts after knockout and found that the cell proliferation activity decreased significantly after the expression of five molecules was knocked out; these five DEPs were C2CD2, COL1A1, KHSRP, LRPAP1, and DMP1. In addition, the inventors also observed the changes in the osteogenic indices of osteoblasts after knockout, including the expression levels of SP7, RUNX2, ALP, and OCN, as well as the mineralization changes of osteoblasts.

[0060] Identification and verification of key molecules for osteoporosis in human bone tissue and serum analysis Based on database comparison, OVX mouse bone tissue verification, and human osteoblast knockout results, the inventors comprehensively analyzed and identified 9 key molecules that could be verified in at least two studies. Based on this, the inventors selected LRPAP1 for further verification.

[0061] Example 3 Verification of the correlation between Lrpap1 expression and osteoporosis

[0062] 1. Mass spectrometry analysis of LRPAP1

[0063] The inventors collected serum samples from 80 physical examination subjects, explored the protein levels of LRPAP1 in these serum samples according to the method in Example 1, and found that the protein level of LRPAP1 was significantly decreased in osteoporosis patients. Specifically, as shown by Figure 1 the human bone tissue mass spectrometry results, LRPAP1 decreased successively in the normal group, bone mass decline group, and osteoporosis group.

[0064] 2. Explore the content of Lrpap1 mRNA in the bone tissue of osteoporosis mice

[0065] 2.1 Construction of osteoporosis mouse model

[0066] Sixteen 8-week-old female C57B1 / 6 mice purchased were randomly divided into two groups, namely: osteoporosis group (OVX group, n = 8). At 8 weeks of age, the mice in this group were anesthetized by intraperitoneal injection of tribromoethanol, the hair on the ventral and dorsal sides of the mice was removed, the skin, muscle, and peritoneum were incised to enter the pelvic cavity of the mice, the bilateral ovaries of the mice were explored, ligated and excised, and the muscle layer and skin were sutured; the other group was the control group (Ctrl group, n = 8). After anesthesia, the ventral and dorsal skin was incised and sutured. Eight weeks after the operation, all mice were intraperitoneally injected with tribromoethanol again. After the mice were completely anesthetized and lost pain sensation, they were sacrificed by cervical dislocation. After the mice were sacrificed, the bilateral femurs and tibias of the two groups of mice were quickly collected and dissected. The soft tissues such as muscles around the lower limb bones of the mice were completely removed with ophthalmic scissors and sterile gauze. The bone tissue specimens were wrapped with gauze moistened with physiological saline and stored in a -80°C refrigerator for subsequent Micro-CT detection analysis and QPCR detection.

[0067] 2.2 RNA extraction from mouse bone tissue

[0068] ①. Take out the mouse femur samples from the -80°C refrigerator and quickly cool them in liquid nitrogen. Grind the mouse femurs thoroughly in a mortar filled with liquid nitrogen until they are in powder form. Pay attention to adding liquid nitrogen in time during the grinding process to prevent RNA degradation due to excessive temperature.

[0069] ②. Transfer the femoral powder into a pre-chilled EP tube. Add 1000 μL of Trizol lysis buffer to each EP tube to fully immerse the bone tissue. Use a 1000 μL pipette to blow and mix well, and let it stand at room temperature for 3 - 5 minutes for sufficient lysis and digestion.

[0070] ③. Transfer the mouse bone tissue lysate into a 1.5 mL RNA-free EP tube. Centrifuge in a pre-chilled centrifuge at 4°C at a speed of 12000 rpm for 5 minutes.

[0071] ④. Gently and slowly pipette the supernatant after centrifugation into a 1.5 mL RNA-free EP tube. Discard the precipitate and add 100 μL of chloroform. Invert the tube vigorously up and down and shake well for 15 seconds.

[0072] ⑤. After shaking, a milky white liquid can be seen. Let it stand at room temperature for 5 minutes. Then centrifuge in a 4°C centrifuge at 12000 rpm for 15 minutes.

[0073] ⑥. Carefully take out the EP tube from the centrifuge. It can be seen that the liquid in the EP tube has separated into three layers. The top layer is a clear and colorless aqueous phase. Transfer it to a new RNA-free EP tube with a volume of about 200 μL. Immediately add 200 μL of isopropanol. Flocculates will precipitate. Invert the tube vigorously up and down and shake well to mix. Let it stand at room temperature for 10 minutes. Then place the EP tube back into a 4°C centrifuge and centrifuge under the conditions of 12000 rpm for 10 minutes.

[0074] ⑦. Gently take out the EP tube from the centrifuge. A small amount of white precipitate can be seen at the bottom of the tube. Aspirate the supernatant clean, add 500 μL of 75% ethanol, being careful not to impact the white precipitate. Then place the EP tube back into a 4°C centrifuge and centrifuge under the conditions of 7500 rpm for 5 minutes.

[0075] ⑧. After centrifugation, carefully aspirate the supernatant clean with a 1000 μL RNA-free pipette tip. Invert the EP tube on absorbent paper and air-dry the precipitate at room temperature for 10 minutes. Add 30 - 40 μL of DEPC water and dissolve the precipitate at 4°C for at least 6 hours.

[0076] ⑨. Use a nucleic acid detector to measure the RNA concentration of each bone tissue sample, measure and analyze the OD value, and take an appropriate amount of RNA for gel electrophoresis to observe the RNA band situation.

[0077] 2.3 QPCR detection of mouse bone tissue

[0078] All operations during this experimental process should be carried out on ice to prevent RNA degradation.

[0079] ①. Residual genomic DNA will seriously affect the accuracy of QPCR test results. Before performing the RNA reverse transcription reaction, it is necessary to remove the genomic DNA in the RNA extraction solution first. The specific reaction system is shown in Table 1.

[0080] Table 1 Reaction System

[0081] Reagent Dosage 5X gDNA Eraser Buffer 2.0 μL gDNA Eraser 1.0 μL Total RNA Appropriate amount <![CDATA[RNase Free dH2O]]> Up to 10 μL

[0082] React at 42°C for 2 min, 4°C +∞

[0083] 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 the reverse transcription reaction according to the standard reaction conditions.

[0084] Table 2 Reaction System

[0085] Reagent Dosage PrimeScript RT Enzyme Mix 1.0 μL RT Primer Mix 1.0 uL 5X PrimeScript Buffer 2 4.0 uL <![CDATA[RNase Free dH2O]]> 4.0 uL Reaction solution of Step ① 10 μL

[0086] React at 37°C for 15 min, 85°C for 5 s, 4°C +∞

[0087] ③. After the reverse transcription reaction is completed, 20 μL of cDNA can be obtained for each bone tissue sample. The cDNA of each sample needs to be prepared according to the system shown in Table 3 on ice and in the dark, and the PCR reaction is carried out according to the standard reaction conditions.

[0088] Table 3 Reaction System

[0089] Reagent Dosage 2X SYBR 10 μL PCR Forward Primer 0.8 μL PCR Reverse Primer 0.8 uL 50X ROX 0.4 uL cDNA 2.0 uL <![CDATA[dH2O]]> 6.0 μL

[0090] ④. The PCR reaction conditions are as follows:

[0091] First step: Pre-denaturation, 95°C for 30 s, 1× cycle;

[0092] Second step: PCR reaction, 95°C for 5 s, 60°C for 31 s, 40× cycle.

[0093] 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.

[0094] The specific primers used are shown in Table 4.

[0095] Table 4 Primers Used

[0096]

[0097] The final results are as Figure 2 shown. From the results of the bone tissue of castrated mice in Figure 2 , it can be seen that Lrpap1 mRNA is significantly reduced in the bone tissue of OVX mice.

[0098] 3. Investigation of osteogenic indices after knocking down LRPAP1 in osteoblasts

[0099] 3.1 Osteogenic induction culture of Saos-2 cells

[0100] ①. When the cells reached 70%-80% confluence, they were digested and passaged using trypsin, and the cells were seeded in a 12-well culture plate at a density of 2×10 5 and cultured normally in an incubator.

[0101] ②. After overnight incubation until the osteoblasts were completely adherent, the osteogenic induction medium containing 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate was replaced.

[0102] ③. The cells were cultured in complete medium containing osteogenic inducer for 21 days. The medium was changed every other day for the first 7 days and every day for the next 14 days.

[0103] 3.2 si-RNA transfection of Saos-2 osteoblasts

[0104] ①. According to the ratio that 0.25 μL of Lipofectamine RNAi MAX reagent needs to be added with 25 μL of serum-free transfection medium Opti-MEM, it was mixed well in an EP tube and left at room temperature for 5 min.

[0105] ②. 15 pmol of siRNA was added to 25 μL of serum-free transfection medium Opti-MEM and mixed well to dissolve.

[0106] ③. The diluted solutions obtained in ① and ② were gently mixed well with a 200 μL pipette and left at room temperature for 15 min.

[0107] ④. The medium of Saos-2 cells with a cell density of 70%-80% was aspirated, washed with PBS, and then complete medium was added. The reagent prepared in ③ was added to the culture dish and mixed well.

[0108] ⑤. After culturing the osteoblasts in the culture plate for 24-48 h, the transfection effect and transfection efficiency were verified by extracting cell RNA and performing QPCR detection.

[0109] 3.3 RNA extraction of Saos-2 osteoblasts

[0110] ①. After completing the culture and intervention of Saos-2 osteoblasts in each group, the cells in each group were taken out of the incubator, the residual medium was aspirated with a pipette, and the cells were washed with PBS at room temperature 3 times, 3 min each time.

[0111] ②. After washing, osteoblasts were lysed with Trizol lysis solution. 500 μL of lysis solution was added to each well of osteoblasts, and the culture dish was gently shaken up, down, left, and right to ensure that all cells were completely covered with Trizol lysis solution. After a moment, a 1000 μL pipette was used to pipette the cells, and the mixture was left standing at room temperature for 5 min.

[0112] ③. After the osteoblasts in the culture dish were completely digested, they were transferred to a 1.5 mL RNA-free EP tube and centrifuged at a rate of 12000 rpm for 5 min in a pre-cooled 4 °C centrifuge.

[0113] ④. The EP tube was carefully taken out of the centrifuge, and the upper supernatant was carefully aspirated with a pipette. Then 100 μL of chloroform was added, and the tube was vigorously inverted up and down for 15 s.

[0114] ⑤. After shaking, the mixture was left standing at room temperature for 5 min and then centrifuged again at a rate of 12000 rpm for 15 min in a pre-cooled 4 °C centrifuge.

[0115] ⑥. The EP tube was carefully taken out of the centrifuge. It could be seen that the liquid in the EP tube was separated into three layers. The top layer was a clear and colorless aqueous phase, which was transferred to a new RNA-free EP tube with a volume of about 200 μL. Immediately, 200 μL of isopropanol was added, and flocculates were visible. The tube was vigorously inverted up and down, shaken well, and then left standing at room temperature for 10 min. The EP tube was then placed in a 4 °C centrifuge and centrifuged under the conditions of 12000 rpm and 10 min.

[0116] ⑦. The EP tube was gently taken out of the centrifuge, and a small amount of white precipitate could be seen at the bottom of the tube. The supernatant was aspirated clean, 500 μL of 75% ethanol was added, taking care not to impact the white precipitate. The EP tube was then placed in a 4 °C centrifuge and centrifuged under the conditions of 7500 rpm and 5 min.

[0117] ⑧. After centrifugation, the supernatant was carefully aspirated clean with a 1000 μL RNA-free pipette tip, and the EP tube was placed upside down on absorbent paper. The precipitate was air-dried at room temperature for 10 min, and 30 - 40 μL of DEPC water was added. The precipitate was dissolved at 4 °C for no less than 6 h.

[0118] ⑨. The RNA concentration of each bone tissue sample was detected using a nucleic acid detector, the OD value was measured and analyzed, and an appropriate amount of RNA was taken for gel electrophoresis to observe the RNA banding pattern.

[0119] 3.4 RT-PCR detection of osteoblast-related gene expression

[0120] All operations in this experimental procedure should be carried out on ice to prevent RNA degradation, and all operations should be strictly carried out in accordance with the requirements of the kit instructions.

[0121] ①. Residual genomic DNA will seriously affect the accuracy of QPCR test results. Before performing the RNA reverse transcription reaction, it is necessary to remove the genomic DNA in the RNA extraction solution first. The specific reaction system is shown in Table 5 below.

[0122] Table 5 Reaction System

[0123] Reagent Dosage 5X gDNA Eraser Buffer 2.0 μL gDNA Eraser 1.0 μL Total RNA Appropriate amount <![CDATA[RNase Free dH2O]]> Upto 10 μL

[0124] React at 42°C for 2 min, 4°C + ∞

[0125] 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 the reverse transcription reaction according to the standard reaction conditions.

[0126] Table 6 Reaction System

[0127] Reagent Dosage PrimeScript RT Enzyme Mix 1.0 μL RT Primer Mix 1.0 μL 5X PrimeScript Buffer 2 4.0 μL <![CDATA[RNase Free dH2O]]> 4.0 μL Reaction solution of Step ① 10 μL

[0128] React at 37°C for 15 min, 85°C for 5 s, 4°C + ∞

[0129] ③. The 20 μL of liquid obtained in the previous step is the cDNA of each group of osteoblasts. Perform the PCR reaction on the cDNA of each group according to the system shown in Table 7 (all reaction solutions must be prepared on ice).

[0130] Table 7 Reaction System

[0131]

[0132]

[0133] ④. The PCR reaction conditions are as follows:

[0134] First step: Pre-denaturation, 95°C for 30 s, 1 cycle;

[0135] Second step: PCR reaction, 95°C for 5 s, 60°C for 31 s, 40 cycles.

[0136] 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.

[0137] The specific primers used are shown in Table 8 below.

[0138] Table 8 Primers Used

[0139]

[0140] The final results obtained are as Figure 3 shown, byFigure 3 It can be seen from the osteogenic index after knocking down Lrpap1 in human osteoblasts that the osteogenic index decreased significantly after knocking down Lrpap1.

[0141] 4. Explore the mineralization function of Saos-2 osteoblasts

[0142] ① After culturing Saos-2 osteoblasts in each group with osteogenic induction medium for 21 days, since the cell number is very large, carefully aspirate the medium of each well of cells, and add 1000 μL of PBS buffer to each well for washing, 3 times in total, 3 minutes each time.

[0143] ② After washing, aspirate the PBS buffer, add 500 μL of cell fixative (PFA, 4% paraformaldehyde) to each well for fixation, at room temperature, and the fixation duration is 20 minutes.

[0144] ③ After PFA fixation, add 1000 μL of PBS buffer to each well for washing, 1 time for 3 minutes, and then continue to wash with 1000 μL of double-distilled water (without calcium salt ions), wash 3 times in total, 5 minutes each time.

[0145] ④ After washing with double-distilled water, fully aspirate the residual double-distilled water, add 300 μL of alizarin red S staining solution (PH = 4.0) to each well of cells, gently shake the culture plate up, down, left and right to ensure that the staining can fully cover all cell samples, incubate at room temperature in the dark, and the incubation duration is about 15 minutes.

[0146] ⑤ After incubation, fully aspirate the remaining alizarin red S staining solution with a pipette, add 500 μL of double-distilled water to each well for repeated washing, wash 5 times, 5 minutes each time, and be careful during the washing process not to make the cells fall off.

[0147] ⑥ Add 300 μL of McGee-Russell differentiation solution to each well, incubate at room temperature for 10 s, aspirate the McGee-Russell differentiation solution, and then add 200 μL of Mayer hematoxylin staining solution to each well to lightly stain the cell nuclei, incubate at room temperature, and the duration is 1 - 2 minutes.

[0148] ⑦ Fully aspirate the Mayer hematoxylin staining solution, add 500 μL of double-distilled water to each well for thorough washing 2 times, 3 minutes each time, to terminate the relevant color reaction.

[0149] ⑨ Observe the results of the staining experiment and collect images for relevant quantitative analysis.

[0150] The final alizarin red staining results are as Figure 4 shown, from Figure 4From the results of alizarin red staining, it can be seen that the mineralization ability of osteoblasts is significantly weakened after LRPAP1 knockdown.

[0151] 5. Extraction of human bone proteins and detection of LRPAP1 protein expression level by Western blotting

[0152] 5.1 Extraction of human bone tissue proteins

[0153] ①. Take out the human bone tissue sample from the -80°C refrigerator and quickly cool it in liquid nitrogen. Grind it thoroughly in a mortar filled with liquid nitrogen until it becomes powdery. During the grinding process, pay attention to adding liquid nitrogen in a timely manner to prevent protein degradation due to excessive temperature.

[0154] 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 ensure that the protein lysis solution RIPA can fully immerse the bone tissue powder, and mix well to react. Digest and lyse on ice for 30 min, and invert and mix once every 5 minutes during this period.

[0155] ③. Transfer the completely lysed and digested human bone tissue protein solution to a new 1.5 mL EP tube. Centrifuge at a speed of 12000 rpm for 5 min in a 4°C centrifuge.

[0156] ④. After centrifugation, carefully take out the EP tube, aspirate the centrifuged supernatant, quickly freeze it in liquid nitrogen, and immediately store it in the -80°C refrigerator for later use.

[0157] 5.2 Western Blot of human bone tissue-related proteins

[0158] 5.2.1 Making a standard curve and detecting the protein content of samples

[0159] ①. Take out the standard product of the BCA kit from the -20°C refrigerator: 30 mg BSA. After fully dissolving it with the standard solvent, prepare a protein solution standard product with a concentration of 25 mg / mL.

[0160] ②. Take 10 μL of the 25 mg / mL protein standard solution and dilute it according to a ratio of 1:500 to obtain a BSA protein solution with a final concentration of 0.5 mg / mL.

[0161] ③. Take out the 50X BCA solution and dilute it to 1X BCA working solution for subsequent detection.

[0162] ④. Prepare a transparent 96-well plate. Add the 0.5 mg / mL BSA protein solution to the standard product wells in volumes of 0, 1, 2, 4, 8, 12, 16, 20 μL, and make up to 20 μL with protein diluent.

[0163] ⑤. After diluting all the sample protein solutions appropriately, add 20 μL of each solution into a 96-well plate according to the specification of 20 μL per well.

[0164] ⑥. Add 200 μL of BCA working solution to each well, and incubate at room temperature for 30 min (the specific incubation time should be determined according to the protein concentration and dilution factor, and multiple observations can be made during this period).

[0165] ⑦. After incubation, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value at a wavelength of 540 - 595 nm. According to the OD value and the protein concentration value of the standard product, fit a linear trend line to complete the drawing of the standard curve.

[0166] ⑨. According to the fitting linear 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.

[0167] 5.2.2 Protein denaturation

[0168] ①. Determine the approximate loading amount of all samples based on the protein solution concentration detected by BCA, and add 4×SDS sample buffer solution (containing 2-mercaptoethanol) to the sample solution.

[0169] ②. After thoroughly mixing the sample protein solution and the sample buffer solution, place it in a 95°C water bath for 3 min 30 s for protein denaturation. The denatured protein solution can be stored in a -20°C refrigerator for subsequent experiments.

[0170] 5.2.3 Gel preparation

[0171] ①. Select two intact glass plates. First, rinse the glass plates with clean water, and then repeatedly wash them with a cleaning agent until the running water neither forms drops nor flows in a stream on the glass surface. Finally, rinse them twice with distilled water and place them in a well-ventilated area at room temperature to dry naturally.

[0172] ②. After the glass plates are naturally dried, align the two glass plates first, then place them in a special slot for gel preparation. After reconfirming that the glass plates are aligned, fasten them, and then vertically install the entire slot on the gel preparation rack.

[0173] ③. Prepare a separation gel with an appropriate concentration according to the molecular weight of the target protein to be detected. After preparing the solution according to the standard system, add TEMED in the last step and immediately blow and mix it thoroughly. Immediately use a pipette to slowly and vertically pour the system solution into the glass plates. After the liquid surface is slightly flat, gently and slowly add a layer of alcohol on the gel to flatten it and expel air bubbles.

[0174] The formula for gels with different concentrations is shown in Table 9 - 10.

[0175] Table 9 Formula for gels with different concentrations (I)

[0176]

[0177]

[0178] Table 10 Formulation of Gels with Different Concentrations (II)

[0179] Concentrating gel / Reagent 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

[0180] ④. After standing for 20 min, when the separating gel has fully solidified, if the alcohol on the upper layer of the separating gel has not completely evaporated, the alcohol can be poured out, then slightly rinsed with double-distilled water and blotted dry with absorbent paper.

[0181] ⑤. Continue to prepare the stacking gel with a concentration of 4% according to the standard formula. Immediately after adding TEMED in the last step, fully pipette and mix well. Immediately use a pipette to slowly and vertically fill the glass plate with the system solution. At the same time, insert a comb of appropriate size into the glass plate. Note that inserting it obliquely downward can reduce the formation of bubbles. Wait for 20 min. After the stacking gel has solidified, the comb can be removed and electrophoresis can be carried out.

[0182] 5.2.4 SDS-PAGE Electrophoresis

[0183] ①. After removing the comb, rinse the sample wells with double-distilled water to wash away the remaining stacking gel and impurities, and then place it in the electrophoresis tank. Note that when placing it, the opening of the glass plate faces inward.

[0184] ②. Before performing electrophoresis, be sure to confirm whether the electrodes of the electrophoresis tank are correct, and then pour the electrophoresis buffer into the electrophoresis tank to ensure that the liquid level of the electrophoresis buffer exceeds the electrode level.

[0185] ③. Take out the denatured protein sample from the -20°C refrigerator. After it has fully dissolved, use a 10-μL pipette to slowly and vertically add the protein sample to the sample wells, and at the same time add an appropriate protein Marker reagent to the sample wells on both sides.

[0186] ④. Set the initial voltage of the gel electrophoresis to 110 V. When the bromophenol blue indicator buffer runs out of the stacking gel layer and the protein bands are relatively neat, the voltage can be adjusted to 90 V to avoid too high voltage and excessive heat generation affecting the experimental results.

[0187] ⑤. During electrophoresis, pay attention to observing the electrophoresis situation in real time. When the bromophenol blue indicator at the bottom is about to run out of the entire separating gel, electrophoresis can be terminated.

[0188] 5.2.5 Blotting

[0189] ①. Take out the separating gel that has completed electrophoresis from the electrophoresis tank. After slightly rinsing the glass plate with double-distilled water, peel the whole separating gel out of the glass plate completely. Gently immerse the separating gel in double-distilled water for washing, and then transfer it to the transfer buffer for buffering.

[0190] ②. Cut a PVDF membrane of 6 cm × 9 cm and six filter papers of 7 cm × 10 cm. Immerse the PVDF membrane completely in 100% methanol for several seconds for activation, and then immediately transfer it to double-distilled water for rinsing. After activation, the PVDF membrane cannot be dried and must be kept moist.

[0191] ③. Thoroughly clean the clips and sponges required for membrane transfer and place them in the membrane transfer solution.

[0192] ④. Sandwich the separated gel and the PVIDF membrane in the membrane transfer clip in the following order, from the negative pole to the positive pole: the first sponge, three filter papers, the separated gel, the PVDF membrane, three filter papers, and the second sponge. During the placement process, make sure that the PVDF membrane and the separated gel are completely aligned and the order must not be wrong. The whole process must be completed under the condition of being immersed in the membrane transfer solution.

[0193] ⑤. According to the principle of positive pole to positive pole and negative pole to negative pole, quickly place the membrane transfer clip into the membrane transfer tank filled with the membrane transfer solution. The membrane transfer conditions are generally constant voltage of 30 V for 6 h or constant current of 200 mA for 1 h 30 min (the specific conditions need to be appropriately adjusted according to the size of the target protein).

[0194] 5.2.6 Immune reaction (incubation with primary antibody and secondary antibody)

[0195] ①. After membrane transfer is completed, take out the membrane and rinse it 2 - 3 times with TBS for 5 min each time.

[0196] ②. After rinsing, block it with 5% BSA or 5% skim milk prepared with TBST. The blocking conditions are: at room temperature, on a shaker for decolorization, for 1 h.

[0197] ③. The primary antibody is prepared with 5% BSA or 5% skim milk and diluted according to the requirements of Western blot in the primary antibody instruction manual; according to the size of the target protein and the marker indication on the PVDF membrane, cut the PVDF membrane to an appropriate size, and then immerse the PVDF membrane in the diluted primary antibody solution for incubation. The general incubation conditions are: at room temperature,

[0198] 1 - 2 h or at 4°C for 6 h.

[0199] ④. After the incubation of the primary antibody is completed, transfer the PVDF membrane to TBST for washing, wash it 2 times for 10 min each time, and recycle the primary antibody dilution at the same time.

[0200] ⑤. After washing is completed, incubate the secondary antibody at room temperature. The selection of the secondary antibody mainly depends on the host source of the primary antibody. The incubation conditions are generally: at room temperature, for 1 h; after incubation is completed, wash it 3 times with TBST for 5 min each time.

[0201] 5.2.7 Chemiluminescence imaging

[0202] 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.

[0203] 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.

[0204] 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.

[0205] The final protein expression was as follows Figure 5 - 6 As shown by Figure 5 - 6 The expression of LRPAP1 protein in human bone tissue of osteoporosis patients was significantly reduced (LRPAP1 protein expression was detected by LRPAP1 antibody (Abcam, #ab76500, UK)).

[0206] Thus, the present invention demonstrates the potential of LRPAP1 protein as a diagnostic marker for osteoporosis through a series of experiments. First, Western blot analysis comparing bone tissue from normal bone mass and osteoporosis patients revealed significantly reduced LRPAP1 protein levels in osteoporosis patients, suggesting a possible association between LRPAP1 and the pathogenesis of osteoporosis. Furthermore, serum samples from 80 individuals undergoing physical examinations were collected and LRPAP1 protein levels were assessed by bone density grouping, further confirming the significant reduction of LRPAP1 in osteoporosis patients. Furthermore, multiple studies, including protein profiling in human bone tissue, LRPAP1 mRNA expression in bone tissue of castrated mice, osteogenic markers and mineralization capacity after LRPAP1 knockdown in human osteoblasts, and LRPAP1 protein expression in human bone tissue from osteoporosis patients, consistently demonstrated a negative correlation between LRPAP1 and osteoporosis. In summary, changes in LRPAP1 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.

[0207] Example 4: Analysis of the Sensitivity and Specificity of LRPAP1 as a Diagnostic Marker for Osteoporosis

[0208] The Receiver Operating Characteristic Curve (ROC curve) is a graphical method for evaluating the performance of a binary classification model. By taking sensitivity (i.e., true positive rate) as the vertical axis and 1 - specificity (i.e., false positive rate) as the horizontal axis, the points corresponding to sensitivity and specificity at different cut-off values are connected. This curve demonstrates the ability of the model to distinguish between positive and negative individuals at different judgment thresholds. The ROC curve is commonly used in diagnostic or prognostic studies to measure the discriminatory ability of a model in predicting the probability of an event occurring and to help determine the optimal cut-off point, that is, to make the curve as close as possible to the upper left corner. Its performance is often represented by the Area Under the Curve (AUC). The AUC is a quantitative indicator reflecting the overall discriminative accuracy of the model, with a value range of 0.5 to 1.0. The closer the value is to 1, the better the model can effectively distinguish individuals with and without a certain outcome. The ROC curve using L RPAP1 as an osteoporosis diagnostic marker is as Figure 7 shown.

[0209] The specific method of analysis using GraphPad Prism is as follows:

[0210] (1) Open GraphPad Prism. In the New table&graph (New table or picture) on the left side of the pop-up welcome interface, select Column (Vertical list). In the Data table (Data table), select Enter or import data into a new table (Write data in a new data table). In Options (Options), select Enter replicate values, stacked into columns (Enter replicate values, stacked into columns), and click create (Create).

[0211] (2) Enter the required data in columns Group A and B, representing the normal population group and the osteoporosis patient group

[0212] (3) In the Analysis (Analysis) option tool group in the toolbar, select the Analyze (Analyze) command icon below it. In the newly popped-up Analyze Data (Data Analysis) command box, select Column analyses (Vertical list analysis), and then continue to select the ROC Curve (ROC curve) option. Check the two data sets of A: Normal population group and B: Osteoporosis patient group in the right box, and click OK.

[0213] (4) Keep the default options in the popped-up ROC curve parameter setting window and click OK

[0214] (5) View Area and P value in Results. The Area is 0.9333 and the p value is less than 0.05, indicating that the expression level of the LRPAP1 protein can effectively distinguish normal people from osteoporosis patients.

[0215] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A biomarker for diagnosing osteoporosis, characterized in that, The biomarker is LRPAP1.

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

3. A product for diagnosing osteoporosis, characterized in that, The product contains a reagent for quantitatively detecting the biomarker recited in claim 1.

4. The product according to claim 3, characterized in that, The product includes a detection kit, polymerase chain reaction reagent, chip detection reagent, or sequencing reagent.

5. The product according to claim 3, wherein The reagent for quantitatively detecting the biomarker recited in claim 1 includes primers and probes capable of quantitatively detecting LRPAp1.

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

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