Protein markers for diagnosing postmenopausal osteoporosis and use thereof

Abstract

The present application relates to a kind of protein markers for diagnosing postmenopausal osteoporosis and its application, belong to biological medicine technical field.The present application is screened, and it is found that NADH dehydrogenase 1 beta subcomplex subunit 4, NADH dehydrogenase 1 beta subcomplex subunit 5, NADH dehydrogenase 1 beta subcomplex subunit 6, NADH dehydrogenase 1, NADH dehydrogenase 1 alpha subcomplex subunit 8, cytochrome c oxidase subunit 7C, cytochrome c oxidase subunit 5A and cytochrome c oxidase subunit 7A2 can be used alone or in combination in the diagnosis test of postmenopausal osteoporosis, to accurately evaluate the PMOP state of subject.Further, the combination of NDUB5 and NDUA8 two protein markers shows excellent diagnostic sensitivity and specificity, which is helpful for early diagnosis of PMOP, screening of high-risk population of fracture and precise prevention and treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a protein biomarker for diagnosing postmenopausal osteoporosis and its application. Background Technology

[0002] The diagnosis of postmenopausal osteoporosis (PMOP) currently relies primarily on direct bone mineral density (DXA) testing, but it has significant limitations: DXA can only reflect the bone mineral density of local bones (such as the lumbar spine and hip), and cannot comprehensively show the overall morphology and subtle structural changes of the bones. For patients with severe spinal deformities, changes in spinal morphology can affect the accuracy of DXA measurements; and in obese patients, increased body thickness leads to complex attenuation of X-rays during penetration, also resulting in measurement errors.

[0003] In the early stages of osteoporosis, bone loss is silent, and patients often experience no symptoms. While DXA (decompression osmosis) is the gold standard, it is essentially a "densitometer," only diagnosing osteoporosis when bone mineral loss reaches a certain level. Furthermore, some individuals with seemingly "normal" bone density or only "reduced bone mass" may face fracture risk due to already deteriorated bone microstructure. Therefore, the ideal time to diagnose PMOP is to identify high-risk individuals early in the disease's development and before fractures occur. Screening for novel biomarkers for PMOP, establishing early diagnostic models, and identifying high-risk individuals are crucial for preventing and treating PMOP and reducing fracture risk. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of an early diagnostic biomarker for postmenopausal osteoporosis in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a protein biomarker for diagnosing postmenopausal osteoporosis and its application. Through screening, this invention has discovered that NADH dehydrogenase 1β subcomplex subunit 4 (NDUB4), NADH dehydrogenase 1β subcomplex subunit 5 (NDUB5), NADH dehydrogenase 1β subcomplex subunit 6 (NDUB6), NADH dehydrogenase 1 (NU1M), NADH dehydrogenase 1α subcomplex subunit 8 (NDUA8), cytochrome c oxidase subunit 7C (COX7C), cytochrome c oxidase subunit 5A (COX5A), and cytochrome c oxidase subunit 7A2 (COX7A2) can be used alone or in combination in diagnostic tests for postmenopausal osteoporosis, thereby accurately assessing the PMOP status of subjects.

[0006] The first objective of this invention is to provide an application of a reagent for detecting the content of a protein biomarker in the preparation of diagnostic products for postmenopausal osteoporosis, characterized in that the protein biomarker is selected from one or more of the following: NADH dehydrogenase 1β subcomplex subunit 4, NADH dehydrogenase 1β subcomplex subunit 5, NADH dehydrogenase 1β subcomplex subunit 6, NADH dehydrogenase 1, NADH dehydrogenase 1α subcomplex subunit 8, cytochrome c oxidase subunit 7C, cytochrome c oxidase subunit 5A, and cytochrome c oxidase subunit 7A2.

[0007] Furthermore, the protein biomarker is selected from any combination of the following:

[0008] (1) NADH dehydrogenase 1β subcomplex subunit 4;

[0009] (2) NADH dehydrogenase 1β subcomplex subunit 5;

[0010] (3) NADH dehydrogenase 1β subcomplex subunit 6;

[0011] (4) NADH dehydrogenase 1;

[0012] (5) NADH dehydrogenase 1α subcomplex subunit 8;

[0013] (6) Cytochrome c oxidase subunit 7C;

[0014] (7) Cytochrome c oxidase subunit 5A;

[0015] (8) Cytochrome c oxidase subunit 7A2;

[0016] (9) NADH dehydrogenase 1β subcomplex subunit 5, NADH dehydrogenase 1β subcomplex subunit 6, NADH dehydrogenase 1α subcomplex subunit 8 and cytochrome c oxidase subunit 5A;

[0017] (10) NADH dehydrogenase 1β subcomplex subunit 5 and NADH dehydrogenase 1α subcomplex subunit 8.

[0018] Furthermore, the test sample for the diagnostic product for postmenopausal osteoporosis is exosomes.

[0019] Further, the Unirot ID of NADH dehydrogenase 1β subcomplex subunit 4 is O95168, the Unirot ID of NADH dehydrogenase 1β subcomplex subunit 5 is O43674, the Unirot ID of NADH dehydrogenase 1β subcomplex subunit 6 is O95139, the Unirot ID of NADH dehydrogenase 1 is P03886, the Unirot ID of NADH dehydrogenase 1α subcomplex subunit 8 is P51970, the Unirot ID of cytochrome c oxidase subunit 7C is P15954, the Unirot ID of cytochrome c oxidase subunit 5A is P20674, and the Unirot ID of cytochrome c oxidase subunit 7A2 is P14406.

[0020] A second objective of this invention is to provide a diagnostic kit for postmenopausal osteoporosis, the kit containing reagents for detecting the levels of protein biomarkers, wherein the protein biomarkers are selected from one or more of the following: NADH dehydrogenase 1β subcomplex subunit 4, NADH dehydrogenase 1β subcomplex subunit 5, NADH dehydrogenase 1β subcomplex subunit 6, NADH dehydrogenase 1, NADH dehydrogenase 1α subcomplex subunit 8, cytochrome c oxidase subunit 7C, cytochrome c oxidase subunit 5A, and cytochrome c oxidase subunit 7A2.

[0021] Furthermore, the postmenopausal osteoporosis diagnostic kit also includes reagents for extracting exosomes from plasma.

[0022] Furthermore, the reagents include those for detecting the concentration or content of the protein marker in the sample to be tested by nuclear magnetic resonance, chromatography, spectroscopy, mass spectrometry, or a combination thereof.

[0023] A third objective of this invention is to provide a reagent for detecting the concentration or content of a protein biomarker in a test sample, wherein the protein biomarker is selected from one or more of NADH dehydrogenase 1β subcomplex subunit 4, NADH dehydrogenase 1β subcomplex subunit 5, NADH dehydrogenase 1β subcomplex subunit 6, NADH dehydrogenase 1, NADH dehydrogenase 1α subcomplex subunit 8, cytochrome c oxidase subunit 7C, cytochrome c oxidase subunit 5A, and cytochrome c oxidase subunit 7A2, and the test sample is an exosome.

[0024] The fourth objective of this invention is to provide an application of a protein biomarker in the preparation of a diagnostic reagent product for postmenopausal osteoporosis, wherein the protein biomarker is selected from one or more of the following: NADH dehydrogenase 1β subcomplex subunit 4, NADH dehydrogenase 1β subcomplex subunit 5, NADH dehydrogenase 1β subcomplex subunit 6, NADH dehydrogenase 1, NADH dehydrogenase 1α subcomplex subunit 8, cytochrome c oxidase subunit 7C, cytochrome c oxidase subunit 5A, and cytochrome c oxidase subunit 7A2.

[0025] Furthermore, the diagnostic product includes equipment for analyzing the content of the protein markers.

[0026] The fifth objective of this invention is to provide an application of a protein biomarker in constructing a predictive model for postmenopausal osteoporosis, wherein the protein biomarker is selected from one or more of the following: NADH dehydrogenase 1β subcomplex subunit 4, NADH dehydrogenase 1β subcomplex subunit 5, NADH dehydrogenase 1β subcomplex subunit 6, NADH dehydrogenase 1, NADH dehydrogenase 1α subcomplex subunit 8, cytochrome c oxidase subunit 7C, cytochrome c oxidase subunit 5A, and cytochrome c oxidase subunit 7A2.

[0027] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0028] This invention has screened and obtained a highly efficient protein biomarker for diagnosing postmenopausal osteoporosis. Applying this biomarker to the preparation of diagnostic products for postmenopausal osteoporosis can yield diagnostic products with good diagnostic efficacy. Specifically, this invention discovers that NADH dehydrogenase 1β subcomplex subunit 4 (NDUB4), NADH dehydrogenase 1β subcomplex subunit 5 (NDUB5), NADH dehydrogenase 1β subcomplex subunit 6 (NDUB6), NADH dehydrogenase 1 (NU1M), NADH dehydrogenase 1α subcomplex subunit 8 (NDUA8), cytochrome c oxidase subunit 7C (COX7C), cytochrome c oxidase subunit 5A (COX5A), and cytochrome c oxidase subunit 7A2 (COX7A2) can be used alone or in combination in diagnostic tests for postmenopausal osteoporosis, thereby accurately assessing the PMOP status of subjects. Furthermore, this invention found that when two protein biomarkers, NDUB5 and NDUA8, are used in combination to construct a diagnostic model, the AUC is 0.818 (95% CI: 0.725-0.912), the cutoff value is 0.561, the sensitivity is 0.750, and the specificity is 0.750, demonstrating excellent diagnostic sensitivity and specificity. This provides a core basis for the construction of PMOP diagnostic models and the preparation of diagnostic products. Its translational application is helpful for the early diagnosis of PMOP, the screening of high-risk groups for fractures, and precise prevention and treatment. Attached Figure Description

[0029] Figure 1 These are images for identifying exosome characteristics; where A is an electron micrograph of the exosome; and B is a particle size distribution of the exosome.

[0030] Figure 2 This is a volcano diagram of 127 differentially expressed proteins identified in plasma exosomes in Example 1;

[0031] Figure 3 This is a graph showing the KEGG enrichment analysis results of 127 differentially expressed proteins. A represents the KEGG enrichment analysis; B is a schematic diagram of mitochondrial electron transport chain proteins, where NADH represents reduced coenzyme I, and NAD... + FADH2 represents coenzyme I, FAD represents flavin adenine dinucleotide, FADH2 represents flavin adenine dinucleotide hydrogen carrier, CoQ represents coenzyme Q, and Cyt C represents cytochrome C.

[0032] Figure 4 The bar chart shows the expression levels of eight differentially expressed proteins in the case and control groups of Example 1. The expression levels were significantly downregulated in the PMOP case group. * indicates P < 0.05, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0033] Figure 5 These are ROC analysis curves for eight proteins, where the integrated model refers to the model constructed using NDUB5, NDUB6, NDUA8, and COX5A in combination.

[0034] Figure 6 This is a bar chart showing the expression levels of NDUB5 and NDUA8 proteins in the validation case group and validation control group of Example 2;

[0035] Figure 7 These are ROC analysis curves for NDUB5 and NDUA8, where the integrated model refers to the model constructed using NDUB5 and NDUA8 together. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0037] Example 1: Screening of PMOP plasma exosomal protein biomarkers

[0038] 1. Research subjects and research design

[0039] This example is from the prospective cohort study "Prevention and Intervention of Osteoporosis" initiated by our research group in 2015 (Chinese Clinical Trial Registry - Registration No.: ChiCTR2000040832). This cohort has completed the collection of baseline data and biological samples from 22,000 participants. This example includes 50 PMOP patients as the case group and 50 healthy controls as the control group. The inclusion and exclusion criteria for the subjects are as follows:

[0040] (1) Includes postmenopausal Han Chinese women aged ≥65 years;

[0041] (2) The inclusion criteria for osteoporosis are based on bone mineral density measured by dual-energy X-ray absorptiometry (T-value of femoral neck bone mineral density is less than -2.5).

[0042] (3) Exclude patients with a history of fractures, liver and kidney dysfunction, thyroid dysfunction, chronic inflammation and infection, malignant tumors, diabetes, etc.

[0043] All participants obtained written informed consent before participating in the study. Venous blood was collected from participants in the morning on an empty stomach, plasma was separated, and stored at -80°C.

[0044] 2. Isolation and identification of plasma exosomes

[0045] Exosomes were separated from plasma samples using a plasma exosome affinity extraction kit (catalog number: EV02-05-01). Particle size was calculated using nanoparticle tracking analysis, and exosome morphology was detected by transmission electron microscopy. The exosomes exhibited a typical "coffee tray" structure, with sizes mostly ranging from 60 nm to 80 nm. Figure 1 ).

[0046] 3. Extraction of total protein from exosomes

[0047] Total protein was extracted from exosome samples using SDT lysis buffer (containing sodium dodecyl sulfate (SDS), DL-dithiothreitol (DTT), and Tris-HCl buffer) for lysing mammalian cells and tissues upon heating. Total protein concentration was determined using a BCA (diquinoline carboxylic acid) protein assay kit (catalog number: E112-01) and a microplate reader.

[0048] 4. Liquid chromatography-mass spectrometry analysis of exosomal proteomic expression profiles

[0049] Exosomal proteomics were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Extracted exosomal proteins were denatured, reduced, and alkylated, then digested overnight with trypsin at 37°C. After desalting, the digested peptides were redissolved in mobile phase A containing 0.1% formic acid for LC-MS / MS analysis. Following chromatographic separation, detection was performed using a timsTOF-HT mass spectrometer (equipped with a Captive Spray ion source) in both data-independent acquisition (DIA) and data-dependent acquisition (DDA) modes. The DIA mode scan range was 300–1500 m / z with a capillary voltage of 1.5 kV; the DDA mode scan range was 100–1700 m / z with a capillary voltage of 1.6 kV.

[0050] 5. Mass spectrometry data analysis

[0051] The raw data were analyzed using Spectronaut software, based on the Homo sapiens SP subset of the UniProt database. A spectral library was constructed by integrating DDA and DIA data through the built-in DIA search engine (Pulsar) module. A total of 45,199 peptides and 5,056 proteins were identified in the library. Further searching of the DIA data based on this library resulted in the quantification of 40,783 peptides and 4,130 proteins. Finally, the number of quantified proteins for each sample in the "*.intst.xls" results file was statistically analyzed to evaluate the protein identification performance among samples.

[0052] 6. Screening of disease biomarkers

[0053] (1) The T-test was used for differential analysis. Significantly different proteins between the PMOP case group and the control group were screened (P ≤ 0.05, FC ≥ 1.5-fold, where FC (Fold-Change) is the fold change value). A total of 127 differentially different proteins were finally obtained. Figure 2 ).

[0054] (2) To identify the core pathway most relevant to the occurrence of postmenopausal osteoporosis, KEGG enrichment analysis was performed on 127 differentially expressed proteins. Statistical significance was tested based on hypergeometric distribution, and the p-value reflecting the enrichment of differentially expressed proteins in a specific KEGG pathway was calculated. A p-value ≤ 0.05 was considered statistically significant (results are shown in Figure 1). Figure 3 (As shown in A in the diagram).

[0055] (3) Oxidative phosphorylation, as a core process of cellular energy metabolism, is a key mechanism leading to osteoporosis when its function is disordered; therefore, the oxidative phosphorylation pathway should be a focus. For example... Figure 3 The eight differentially expressed proteins (NDUB4, NDUB5, NDUB6, NU1M, NDUA8, COX7C, COX5A, and COX7A2) in the oxidative phosphorylation pathway shown in B are all electron transport chain proteins and are significantly downregulated in postmenopausal osteoporosis cases. Figure 4 Therefore, these 8 proteins were selected as protein biomarkers.

[0056] 7. ROC Analysis

[0057] To further analyze the efficacy of the above eight protein biomarkers in PMOP diagnosis, a linear regression method with L1 regularization and 10-fold cross-validation (LASSO regression) was used. Specifically, the dataset was randomly divided into 10 similarly sized subsets. Nine of these subsets were combined as the training set, and the remaining subset was used as the test set. On the training set, the optimal regularization parameter λ (lambda) for LASSO regression was determined through 10-fold cross-validation. This parameter was determined by the criterion of minimizing the error (lambda.min) under the cross-validation curve. A comprehensive model was established based on the selected variables and their corresponding coefficients: Log(odds) = 2.0776 - 18.5267 * X NDUB5 -2.3476 *X NDUB6 - 2.8688 *X NDUA8 -12.6449 *X COX5A Where X represents the relative abundance value of the corresponding protein measured in mass spectrometry analysis. This model suggests that four proteins—NDUB5, NDUB6, NDUA8, and COX5A—make significant contributions to the diagnosis of PMOP, and the negative coefficients indicate that they may be risk factors in the current cohort.

[0058] The predicted probability of each sample was calculated based on the model, and the predicted probabilities of all test sets were compared with the true labels in 10 cross-validation iterations. A receiver operating characteristic (ROC) curve of the comprehensive model was plotted, and the area under the curve (AUC) was calculated to evaluate the overall discriminative ability of the model. The results are as follows: Figure 5 As shown, the comprehensive model composed of NDUB5, NDUB6, NDUA8, and COX5A exhibits significantly better diagnostic efficacy than any single biomarker, with an area under the curve (AUC) of 0.810. At a cutoff value of 0.556, the sensitivity is 0.720 and the specificity is 0.740. This suggests that combined detection has superior diagnostic value for PMOP.

[0059] Example 2: Validation of PMOP plasma exosomal protein markers

[0060] 1. Validation of the correlation between electron transport chain proteins in plasma exosomes and PMOP

[0061] The samples in this embodiment are independent of Example 1 and are also from a prospective cohort for osteoporosis prevention and intervention, including 40 PMOP cases (validation case group) and 40 healthy controls (validation control group). Peripheral blood was collected and plasma exosomes were isolated. Four electron transport chain proteins (NDUB5, NDUB6, NDUA8, and COX5A) from the above optimal integrated model were selected, and their contents in plasma exosomes were determined by enzyme-linked immunosorbent assay (ELISA), and inter-group differences were analyzed. The results showed that the protein levels of NDUB5 and NDUA8 in the validation case group were significantly lower than those in the validation control group. Figure 6 This result is consistent with the direction of difference observed in Example 1, supporting the correlation between electron transport chain proteins and PMOP. No significant inter-group differences were observed for the other two proteins.

[0062] 2. ROC analysis

[0063] To evaluate the specificity of NDUB5 and NDUA8 in the diagnosis of PMOP, a comprehensive model was constructed using LASSO regression with 10-fold cross-validation: Log(odds) = 3.8179 - 1.8367 * X NDUB5 - 0.2924 * X NDUA8 Where X represents the relative abundance value of the corresponding protein measured in mass spectrometry. This model suggests that NDUB5 and NDUA8 proteins make significant contributions to the diagnosis of PMOP, and their negative coefficients indicate that they may be risk-related factors in the current cohort. Further ROC curve analysis results ( Figure 7 The results showed that the area under the curve (AUC) of the combined NDUB5 and NDUA8 diagnostic model was 0.818 (95% confidence interval (CI): 0.725-0.912). At a cutoff value of 0.561, the sensitivity and specificity were 0.750, which were higher than the diagnostic performance of the single indicator (AUC of NDUB5 = 0.813; AUC of NDUA8 = 0.622).

[0064] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of reagents for detecting protein biomarker content in the preparation of diagnostic products for postmenopausal osteoporosis, characterized in that, The protein biomarker is selected from one or more of the following: NADH dehydrogenase 1β subcomplex subunit 4, NADH dehydrogenase 1β subcomplex subunit 5, NADH dehydrogenase 1β subcomplex subunit 6, NADH dehydrogenase 1, NADH dehydrogenase 1α subcomplex subunit 8, cytochrome c oxidase subunit 7C, cytochrome c oxidase subunit 5A, and cytochrome c oxidase subunit 7A2.

2. The application according to claim 1, characterized in that, The test sample for the diagnostic product for postmenopausal osteoporosis is exosomes.

3. The application according to claim 1, characterized in that, The Unirot ID of NADH dehydrogenase 1β subcomplex subunit 4 is O95168, the Unirot ID of NADH dehydrogenase 1β subcomplex subunit 5 is O43674, the Unirot ID of NADH dehydrogenase 1β subcomplex subunit 6 is O95139, the Unirot ID of NADH dehydrogenase 1 is P03886, the Unirot ID of NADH dehydrogenase 1α subcomplex subunit 8 is P51970, the Unirot ID of cytochrome c oxidase subunit 7C is P15954, the Unirot ID of cytochrome c oxidase subunit 5A is P20674, and the Unirot ID of cytochrome c oxidase subunit 7A2 is P14406.

4. A diagnostic kit for postmenopausal osteoporosis, characterized in that, The diagnostic kit for postmenopausal osteoporosis contains reagents for detecting the levels of protein biomarkers, wherein the protein biomarkers are selected from one or more of the following: NADH dehydrogenase 1β subcomplex subunit 4, NADH dehydrogenase 1β subcomplex subunit 5, NADH dehydrogenase 1β subcomplex subunit 6, NADH dehydrogenase 1, NADH dehydrogenase 1α subcomplex subunit 8, cytochrome c oxidase subunit 7C, cytochrome c oxidase subunit 5A, and cytochrome c oxidase subunit 7A2.

5. The diagnostic kit for postmenopausal osteoporosis according to claim 4, characterized in that, The diagnostic kit for postmenopausal osteoporosis also includes reagents for extracting exosomes from plasma.

6. The diagnostic kit for postmenopausal osteoporosis according to claim 4, characterized in that, The reagents include those for detecting the concentration or content of the protein markers in the sample to be tested by nuclear magnetic resonance, chromatography, spectroscopy, mass spectrometry, or a combination thereof.

7. A reagent for detecting the concentration or content of protein markers in a sample, characterized in that, The protein biomarker is selected from one or more of the following: NADH dehydrogenase 1β subcomplex subunit 4, NADH dehydrogenase 1β subcomplex subunit 5, NADH dehydrogenase 1β subcomplex subunit 6, NADH dehydrogenase 1, NADH dehydrogenase 1α subcomplex subunit 8, cytochrome c oxidase subunit 7C, cytochrome c oxidase subunit 5A, and cytochrome c oxidase subunit 7A2, and the sample to be tested is an exosome.

8. The application of protein biomarkers in the preparation of diagnostic reagents for postmenopausal osteoporosis, characterized in that, The protein biomarker is selected from one or more of the following: NADH dehydrogenase 1β subcomplex subunit 4, NADH dehydrogenase 1β subcomplex subunit 5, NADH dehydrogenase 1β subcomplex subunit 6, NADH dehydrogenase 1, NADH dehydrogenase 1α subcomplex subunit 8, cytochrome c oxidase subunit 7C, cytochrome c oxidase subunit 5A, and cytochrome c oxidase subunit 7A2.

9. The application according to claim 8, characterized in that, The diagnostic reagent product includes equipment for analyzing the content of the protein markers.

10. The application of protein biomarkers in constructing a predictive model for postmenopausal osteoporosis, characterized in that, The protein biomarker is selected from one or more of the following: NADH dehydrogenase 1β subcomplex subunit 4, NADH dehydrogenase 1β subcomplex subunit 5, NADH dehydrogenase 1β subcomplex subunit 6, NADH dehydrogenase 1, NADH dehydrogenase 1α subcomplex subunit 8, cytochrome c oxidase subunit 7C, cytochrome c oxidase subunit 5A, and cytochrome c oxidase subunit 7A2.

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