Age-related plasma miRNA molecular marker and application thereof in evaluation of age-related state

Abstract

The application belongs to the technical field of biological health, and particularly relates to an age-related plasma miRNA molecular marker and application thereof in age-related state evaluation. The application fills the blank of age-related miRNA marker resources in the prior art, provides a group of newly discovered, high-specificity and high-stability age-related plasma miRNA molecular markers (hsa-miR-1294 and hsa-miR-6884-5p), simultaneously establishes a standardized detection and evaluation method based on the novel markers, solves the problems of lack of age-related markers and insufficient pertinence in the prior art, realizes non-invasive, accurate and convenient evaluation of the age-related state, and meets the application requirements in clinical, forensic and health management scenes.

Description

Technical Field

[0001] This invention belongs to the field of biohealth technology, specifically relating to an age-related plasma miRNA molecular marker and its application in age-related status assessment. Background Technology

[0002] In the molecular regulatory network of the aging process, miRNAs have been identified as core regulators and potential biomarkers, with related research covering multiple dimensions such as molecular pathways, multi-system aging, and signal transduction. Regarding core regulatory mechanisms, miR-19a-3p and miR-19b-3p regulate apoptosis and oxidative stress through the FoxO signaling pathway, and their expression characteristics are associated with the health status of centenarians; the p53-PVT1-miR-1204-MYLK pathway can induce the SASP phenotype in vascular smooth muscle cells, participating in cardiovascular aging-related pathologies; miR-144-3p and miR-149-5p carried by MSC-derived EVs can regulate the PI3K-Akt pathway, reversing organ epigenetic age and prolonging lifespan. In terms of biomarker applications, the age prediction model based on circulating sncRNA has extremely high accuracy (R²>0.96), clarifying the correlation between miRNA and age; the miR-21 / SOD3 axis regulates aging-related metabolic abnormalities through oxidative stress, providing a potential target for metabolic aging.

[0003] In the regulation of inflammation and multi-system aging, miR-146a and miR-223, carried by senescent EVs, mediate inflammatory aging through the NF-κB pathway; miR-155-5p, as a core molecule, regulates the senescence of different cell types; and miR-1273g-3p targets mitochondrial genes and participates in neurodegenerative aging. In specific disease and physiological state associations, miR-29b-5p is associated with cartilage aging in osteoarthritis, miR-107 and miR-132-3p are associated with neurodegenerative aging, and hsa-miR-126-5p and hsa-miR-146a-5p are associated with musculoskeletal aging. However, these discovered miRNA markers still have limitations in terms of specificity, stability, or applicable scenarios. Summary of the Invention

[0004] The first aspect of the present invention aims to provide the application of reagents for detecting biomarkers in the preparation of products for assessing age-related states.

[0005] A second aspect of the present invention is to provide a kit for assessing age-related conditions.

[0006] A third aspect of the present invention aims to provide a method for predicting and assessing age-related states for non-disease diagnosis and treatment purposes.

[0007] The fourth aspect of this invention aims to provide a method for constructing a model for assessing age-related states.

[0008] The fifth aspect of the present invention aims to provide a system for predicting and assessing age-related states.

[0009] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: A first aspect of the invention provides the use of reagents for detecting biomarkers in the preparation of products for assessing age-related states.

[0010] In some embodiments of the present invention, the marker includes at least one of hsa-miR-1294 or hsa-miR-6884-5p; Wherein: the sequence of hsa-miR-1294 is: UGUGAGGUUGGCAUUGUUGUCU (SEQ ID NO: 1); The sequence of hsa-miR-6884-5p is: AGAGGCUGAGAAGGUGAUGUUG (SEQ ID NO: 2).

[0011] In some embodiments of the present invention, the reagents for detecting the biomarkers are selected from reagents of one or more detection methods from the group consisting of: high-throughput sequencing, digital PCR, and quantitative real-time PCR.

[0012] In some embodiments of the present invention, the product includes a test kit, a test chip, or a test strip.

[0013] In some embodiments of the present invention, the test sample for the product includes blood.

[0014] In some embodiments of the present invention, the blood includes at least one of serum, plasma, and whole blood.

[0015] A second aspect of the invention provides a kit for assessing age-related status, comprising primers for detecting at least one of hsa-miR-1294 or hsa-miR-6884-5p.

[0016] In some embodiments of the present invention, the primers include: The forward primer for hsa-miR-1294 has the sequence: TGTGAGGTTGGCATTGTTGTCT (SEQ ID NO: 3); The forward primer hsa-miR-6884-5p has the sequence: CAGGGCTGAGAAGGTGATGTTG (SEQ ID NO: 4). cel-miR-39 (external reference) forward primer: TCACCGGGTGUAAATCAGCTTG (SEQ ID NO:5); Universal reverse primer: AGTGCAGGGTCCGAGGTATT (SEQ ID NO:6).

[0017] A third aspect of the present invention provides a method for predicting and assessing age-related states for non-disease diagnosis and treatment purposes, comprising the following steps: Detect the expression level of at least one of hsa-miR-1294 or hsa-miR-6884-5p; If the expression level of at least one of hsa-miR-1294 or hsa-miR-6884-5p is higher than that of the reference sample, then the age is considered to be greater than that of the reference sample. Conversely, if the expression level of at least one of hsa-miR-1294 or hsa-miR-6884-5p is lower than that of the reference substance, then the age is considered to be younger than that of the reference substance.

[0018] In some embodiments of the present invention, the non-disease diagnosis and treatment purpose can be used for: 1) Clinical age assessment: Applicable to patients without a clear age record. By detecting any of the novel biomarkers of this invention, doctors can be assisted in assessing the patient's age-related physiological status and provide a reference for the formulation of treatment plans.

[0019] 2) Early warning of age-related diseases: If any new biomarker is detected in middle-aged people (40-60 years old) and they are determined to be in an "age-related state", it indicates that their physiological aging process is relatively fast and can be used as an early warning signal of age-related diseases.

[0020] In some embodiments of the invention, the reference material includes age-defined plasma samples (e.g., plasma from healthy men and women aged 20, 30, 60, and 70).

[0021] A fourth aspect of the present invention provides a method for constructing a model for assessing age-related states, comprising the following steps: Model building is based on the expression levels of biomarkers; The marker includes at least one of hsa-miR-1294 or hsa-miR-6884-5p; The sequence of hsa-miR-1294 is: UGUGAGGUUGGCAUUGUUGUCU (SEQ ID NO: 1); The sequence of hsa-miR-6884-5p is: AGAGGCUGAGAAGGUGAUGUUG (SEQ ID NO: 2).

[0022] In some embodiments of the present invention, the model construction algorithm includes at least one of logistic regression, linear discriminant analysis, support vector machine, random forest, and recursive partitioning tree.

[0023] A fifth aspect of the present invention provides a system for predicting and assessing age-related states, the system comprising a computing device for predicting and assessing age-related states based on the detection results of the expression levels of biomarkers; the biomarkers include at least one of hsa-miR-1294 or hsa-miR-6884-5p.

[0024] In some embodiments of the present invention, the detection results include RNA level results.

[0025] In some embodiments of the present invention, the system further includes any one or more of the following: 1) Detection result collection device, also known as detection result input device, can specifically be one or more of the following: mouse, keyboard, touch screen display, one or more buttons, one or more switches, one or more triggers, etc. 2) Diagnostic result output device, also known as diagnostic result display device, can specifically be one or more of the following: liquid crystal display (LCD), light-emitting diode (LED) display, plasma display, projection display, touch screen display, etc. 3) Diagnostic result sending device, which can send the subject's resolution results to an information communication terminal device that can be viewed by the patient or medical staff.

[0026] The beneficial effects of this invention are: 1. Discovery of novel biomarkers, filling a technological gap: This invention is the first to discover that hsa-miR-1294 and hsa-miR-6884-5p are related to age, enriching the resources of age-related miRNA biomarkers, solving the core problems of biomarker scarcity and insufficient targeting in existing technologies, and providing new molecular targets for age-related status assessment.

[0027] 2. High specificity and stability: Based on novel biomarkers, dedicated specific primers are designed and combined with exogenous reference RNA calibration to ensure the specificity and accuracy of the detection; the biomarkers are highly stable in plasma and the detection has good repeatability.

[0028] 3. Standardized technical solutions that can be directly applied: The novel biomarker sequences, specific primers, detection reaction conditions, and judgment logic have been clearly defined, forming a complete standardized technical system. It can be promoted and applied in various laboratories and testing institutions without additional optimization, thus driving the innovation of age assessment technology.

[0029] 4. Wide range of applications and strong practicality: The application process is designed for specific scenarios such as clinical testing, forensic identification, and risk warning of geriatric diseases, which can directly meet actual needs and has important social and economic value.

[0030] 5. Simple operation and controllable cost: It adopts conventional real-time PCR technology, and universal reverse primers and exogenous reference RNA are readily available and low in cost. No complicated equipment is required, making it suitable for use in primary healthcare institutions and small and medium-sized testing institutions. Attached Figure Description

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The expression levels of hsa-miR-1294 and hsa-miR-6884-5p in plasma of elderly and young people respectively; (Old represents plasma samples from elderly people, Young represents plasma samples from young people, P<0.01).

[0032] Figure 2 This is a comparison of the expression levels of hsa-miR-1294 (a newly discovered biomarker) in the plasma of elderly and young people (Old represents plasma samples from elderly people, and Young represents plasma samples from young people).

[0033] Figure 3 This is a comparison of the expression levels of hsa-miR-6884-5p (a newly discovered biomarker) in the plasma of elderly and young people (Old represents plasma samples from elderly people, and Young represents plasma samples from young people).

[0034] Figure 4 ROC curves for assessing aging status based on hsa-miR-1294 and hsa-miR-6884-5p. Detailed Implementation

[0035] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0036] Example 1: Clinical Sample Collection and Processing 1. Sample source and grouping To verify the effectiveness of age-related plasma miRNA molecular markers, this embodiment designed an age-stratified sample system for healthy individuals, including plasma samples from 106 healthy individuals. These samples were divided into two age groups of 53 individuals each, with the sample size meeting the requirements for statistical difference analysis. The specific grouping criteria are as follows: 1) Young group: age ≤30 years, average age 25.1 years, covering the period from childhood to adolescence, aiming to establish the expression baseline of age-related miRNAs in a non-aging state; all subjects were confirmed to have no abnormalities through health check-ups, no bad lifestyle habits such as smoking and excessive drinking, no history of chronic diseases such as hypertension, diabetes, and cardiovascular diseases, have not taken any medications in the past 6 months, have regular living habits (7-8 hours of sleep per day, no long-term stay-at-home orders), and have normal liver and kidney function tests.

[0037] 2) Elderly group: age ≥ 60 years, average age 69.2 years; inclusion criteria are the same as the younger group, except for pathological diseases such as Alzheimer's disease and severe organ dysfunction, and only age-related physiological decline manifestations (without pathological lesions) are allowed.

[0038] 2. Raw materials and reagents Materials and reagents used in the experiment: EDTA anticoagulant tubes, high-speed refrigerated centrifuge with a rotation speed range of 0-16000×g, -80℃ ultra-low temperature freezer, exogenous reference RNA (cel-miR-39, concentration 10nM). All reagents met the quality standards for clinical trials and were free from RNase contamination.

[0039] 3. Sample collection and processing procedures All sample collection and processing procedures strictly adhere to clinical sample collection ethical guidelines. The specific operational steps are as follows: 1) Subject preparation: All subjects fasted for 12 hours before blood collection to avoid interference with plasma miRNA expression caused by factors such as diet and exercise.

[0040] 2) Blood collection and plasma separation: 5 mL of peripheral venous blood was collected from the subject using an EDTA anticoagulant tube. After collection, the blood was gently inverted and mixed to avoid blood clotting. The blood was immediately transported at 4°C and centrifuged at 3000 rpm for 10 min (centrifugation temperature 4°C) to separate the upper plasma layer.

[0041] 3) Impurity removal and aliquoting for cryopreservation: Take the plasma supernatant after centrifugation and centrifuge it again at 4℃ and 3000rpm for 10min to completely remove residual blood cells and impurities; aliquot the purified plasma into 0.2mL / tube and quickly place it in an ultra-low temperature freezer at -80℃ for later use. Avoid repeated freeze-thaw cycles throughout the process (freeze-thaw cycles ≤ 1 time) to prevent plasma miRNA degradation.

[0042] 4) Quality control: During subsequent RNA extraction, exogenous reference RNA (cel-miR-39, 10 nM) was added to each tube of plasma sample to monitor the stability of the entire RNA extraction and subsequent detection process. After extraction, the purity and concentration of RNA were detected by ultraviolet spectrophotometry to ensure that the purity A260 / A280 value was between 1.8 and 2.0 and the concentration was ≥50 ng / μL, which met the experimental requirements for the screening and validation of miRNA molecular markers.

[0043] 4. Experimental Results 106 qualified plasma samples (53 from the elderly group and 53 from the young group) were successfully collected and standardized. All samples were free from hemolysis, contamination, and degradation. RNA quality testing showed that the purity and concentration of RNA extracted from all samples met the experimental standards. The recovery rate of exogenous reference RNA was stable, providing a stable and reliable sample source for the subsequent screening, identification, and validation of novel age-related plasma miRNA molecular markers.

[0044] Example 2 Screening and Validation of Novel Molecular Markers 1. Small RNA sequencing and screening of novel biomarkers: 1) Total RNA extraction: Total RNA was randomly extracted from 6 plasma samples (3 from the Old group and 3 from the Young group). RNA integrity was detected by agarose gel electrophoresis (28S:18S>=1.5); RNA purity was detected by Nanodrop (OD260 / 280 ratio of 1.8~2.2); and RNA concentration was accurately quantified by Qubit (>=500ng / ul) to ensure that the sample quality was up to standard.

[0045] 2) Sequencing Library Construction: First, adapters are added. Utilizing the unique structure of the 3' and 5' ends of small RNA (the 5' end has a complete phosphate group, and the 3' end has a hydroxyl group), adapters are directly added to both ends of the small RNA starting sample. Then, cDNA is synthesized through reverse transcription. Next, the target DNA fragment is isolated using QIAseq miRNA NGS Beads and purified to obtain the cDNA library. Finally, library detection and quantification are performed to obtain the raw sequencing data.

[0046] 3) Sequencing: First, bridge PCR amplification is performed on the cBot instrument to generate clusters. Then, sequencing by synthesis is performed using the Illumina sequencing platform in 2*150 sequencing mode. Four fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flowcell for amplification. Each added fluorescently labeled dNTP releases corresponding fluorescence as the complementary strand of each sequencing cluster is extended. The sequencer captures the fluorescence signal and converts it into sequencing peaks using computer software, thus obtaining the sequence information of the fragment. The image data obtained by the high-throughput sequencer is converted into sequence data (reads) using CASAVA base recognition in FASTQ format, which mainly includes the sequence information of the sequencing fragment and its corresponding sequencing quality information.

[0047] 4) Data preprocessing: The raw sequencing data is filtered to remove adapter sequences, low-quality reads, and contaminating sequences such as ribosomal RNA (rRNA) and transfer RNA (tRNA) to obtain clean reads.

[0048] 5) Differential expression analysis: Cleanreads were aligned to the human miRNA database (miRBase22.0) for miRNA identification and quantification; DESeq2 software was used for inter-group differential expression analysis, with the screening thresholds set as |log2FC|≥0.58 and P<0.05, to screen for differentially expressed miRNAs between the elderly and young groups.

[0049] 6) Differential Expression Heatmap Construction: Select the screened differentially expressed miRNAs and use the R package pheatmap (version 1.0.13) to construct a target differential expression heatmap (e.g., Figure 1As shown in the figure, the R version is 4.5.1. The heatmap visually displays the expression levels of each miRNA in different samples using color gradients. Among them, hsa-miR-1294 and hsa-miR-6884-5p both show significant inter-group clustering characteristics in the heatmap (clustering in the Old group and clustering in the Young group), and have not been reported to be related to age in existing technologies, thus being identified as candidate novel biomarkers.

[0050] The nucleotide sequences of hsa-miR-1294 and hsa-miR-6884-5p are as follows: hsa-miR-1294:UGUGAGGUUGGCAUUGUUGUCU (SEQ ID NO: 1); hsa-miR-6884-5p: AGAGGCUGAGAAGGUGAUGUUG (SEQ ID NO: 2).

[0051] 2. RT-qPCR validation 1) Total RNA was randomly extracted from 60 plasma samples (30 from the Old group and 30 from the Young group). During extraction, 1 μL of 10 nM exogenous reference RNA was added to each sample. The purity and concentration of RNA were detected using Nanodrop to ensure sample quality. hsa-miR-1294 and hsa-miR-6884-5p, selected through sequencing, were individually validated using corresponding specific primers and universal reverse primers, with cel-miR-39 as the exogenous reference. Three replicate wells were set for each sample.

[0052] It should be noted that cel-miR-39 serves as a good exogenous reference for miRNAs, and its expression level is used for relative quantification of the expression level of the target miRNA. The CT value is obtained using a real-time PCR instrument, and then the expression level of the miRNA molecular marker is obtained using the following formula: ΔCT = CT(target miRNA) – CT(external reference cel-miR-39) Here, ΔCT represents the expression level of the miRNA molecular marker.

[0053] hsa-miR-1294 forward primer: TGTGAGGTTGGCATTGTTGTCT (SEQ ID NO:3); hsa-miR-6884-5p forward primer: CAGGGCTGAGAAGGTGATGTTG (SEQ ID NO:4); cel-miR-39 (external reference) forward primer: TCACCGGGTGUAAATCAGCTTG (SEQ ID NO:5) Universal reverse primer: AGTGCAGGGTCCGAGGTATT (SEQ ID NO:6).

[0054] 2) Quantitative Real-Time PCR Detection: A 20 μL reaction system was used, containing 10 μL SYBR Green quantitative PCR enzyme, 1 μL cDNA, 0.5 μL of 10 μM target miRNA forward primer, 0.5 μL of 10 μM universal reverse primer, and 8 μL RNase-free water. The reaction conditions were: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 40 cycles. These standardized reaction conditions ensured the specificity and repeatability of the detection.

[0055] 3) Results of quantitative real-time PCR detection of collected plasma samples from healthy individuals are as follows: Figures 2-3 As shown.

[0056] pass Figure 2 , Figure 3 and Figure 4 It was found that the expression levels of hsa-miR-1294 and hsa-miR-6884-5p in the plasma of the elderly group were significantly higher than those in the young group, and showed a significant upregulation trend with increasing age, which is consistent with the results of high-throughput sequencing, indicating that these three miRNAs have significant age-related expression characteristics.

[0057] Example 3 Performance Validation of Evaluation Method Based on Novel Biomarkers 1. ROC curve analysis 1) Operational steps: Forty plasma samples (20 from the Old group and 20 from the Young group) independent of the samples used in Example 2 were selected as the validation set. The expression levels of three target miRNA molecular markers were detected by qPCR technology. Age grouping was used as the gold standard. ROC curves were plotted using statistical software. The area under the curve (AUC), sensitivity, and specificity of each marker and the combined marker were analyzed to evaluate their efficacy in assessing age-related status.

[0058] 2) Judgment logic: When the relative expression level of the target miRNA in the sample to be tested is significantly higher than that of the young control group (i.e., ΔΔCT<0, relative expression level>1), it is judged as "age-related state"; otherwise, it is judged as "non-age-related state".

[0059] 3) Results: The areas under the ROC curves (AUCs) of the three target miRNA molecular markers were 0.788 for miR-210-3p, 0.748 for miR-1294, and 0.76 for miR-6884-5p. All three target miRNAs exhibited stable age-related status discrimination ability, effectively distinguishing age-related expression differences between young and elderly samples. This indicates that each miRNA molecular marker described in this invention has reliable assessment efficacy for age-related status.

[0060] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of reagents for detecting biomarkers in the preparation of products for assessing age-related status; The marker includes at least one of hsa-miR-1294 or hsa-miR-6884-5p; The sequence of hsa-miR-1294 is: UGUGAGGUUGGCAUUGUUGUCU; The sequence of hsa-miR-6884-5p is: AGAGGCUGAGAAGGUGAUGUUG.

2. The application according to claim 1, characterized in that: The reagents for detecting the biomarkers are selected from reagents of one or more detection methods from the group consisting of: high-throughput sequencing, digital PCR, and quantitative real-time PCR.

3. The application according to claim 1, characterized in that: The products include test kits, test chips, or test strips.

4. The application according to claim 3, characterized in that: The test samples for the product include blood.

5. A kit for assessing age-related status, characterized in that: Includes primers for detecting markers in any one of the applications described in claims 1 to 4.

6. The reagent kit according to claim 5, characterized in that: The primers include: hsa-miR-1294 forward primer, sequence: TGTGAGGTTGGCATTGTTGTCT; The forward primer for hsa-miR-6884-5p has the sequence: CAGGGCTGAGAAGGTGATGTTG.

7. A method for predicting and assessing age-related statuses for non-disease diagnosis and treatment purposes, comprising the following steps: Detect the expression level of at least one of hsa-miR-1294 or hsa-miR-6884-5p; If the expression level of at least one of hsa-miR-1294 or hsa-miR-6884-5p is higher than that of the reference sample, then the age is considered to be greater than that of the reference sample.

8. A method for constructing a model for assessing age-related states, comprising the following steps: Model building is based on the expression levels of biomarkers; The markers include at least one of hsa-miR-1294 or hsa-miR-6884-5p.

9. The construction method according to claim 8, characterized in that: The model construction algorithm includes at least one of logistic regression, linear discriminant analysis, support vector machine, random forest, and recursive partitioning tree.

10. A system for predicting and assessing age-related states, the system comprising a computational device for predicting and assessing age-related states based on the detection results of the expression levels of the biomarkers according to claim 8.

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