Peripheral blood mononuclear cell miR-150-5p as a molecular marker for metabolic-related fatty liver disease and application thereof

Abstract

A molecular marker for metabolic-associated fatty liver disease (MASLD) miR-150-5p in peripheral blood mononuclear cells and its application are disclosed. The expression of the miR-150-5p molecular marker is negatively correlated with the pathological severity of MASLD. It is applied in the preparation of diagnostic kits or expression chips for MASLD. By quantitatively detecting the expression level of miR-150-5p in mononuclear cells of peripheral blood samples, subjects can be diagnosed as healthy, with simple MASLD, with MASH, and with or without fibrosis. This enables non-invasive classification of the subject's liver disease status (healthy, simple MASLD, MASH, fibrosis) in clinical applications, with the advantages of being non-invasive, convenient, and highly reproducible.

Description

Technical Field

[0001] This invention belongs to the fields of molecular diagnostics and hepatology, specifically relating to miR-150-5p in peripheral blood mononuclear cells as a molecular marker for metabolic-related fatty liver disease and its application. Background Technology

[0002] Metabolic dysfunction associated steatohepatitis (MASH) is a leading cause of liver disease-related morbidity and mortality, characterized pathologically by hepatocellular steatosis, ballooning degeneration, intralobular inflammation, and progressive fibrosis. Persistent inflammation and fibrosis are the core factors driving the disease towards cirrhosis, liver failure, and even hepatocellular carcinoma. Currently, liver biopsy remains the "gold standard" for diagnosing MASH and assessing the degree of fibrosis. However, this invasive procedure suffers from sampling errors, inter-observer variability, bleeding risks, and poor patient compliance, making it completely inadequate for large-scale screening, long-term dynamic monitoring, and efficacy evaluation of this highly prevalent disease (approximately 25%-30% globally). Currently, the diagnosis of MASH primarily relies on imaging examinations and serological models. However, due to limitations such as the susceptibility of imaging results to interference from obesity and intercostal spaces, and the significant decrease in diagnostic accuracy of serological models within the critical value range, reliable diagnostic markers for MASH, especially reliable non-invasive diagnostic methods, remain lacking.

[0003] Therefore, developing a non-invasive molecular diagnostic tool that can directly, sensitively, and specifically reflect the degree of liver inflammation and thus accurately distinguish different stages of MASLD is a clinical bottleneck that urgently needs to be overcome in the field of liver disease. In recent years, circulating microRNAs, as stable biopsy biopsy markers, have provided a new approach to this problem. Among them, the research group, through previous high-throughput sequencing and screening, determined that the expression of miR-150-5p is significantly altered in MASH.

[0004] Existing literature reports that miR-150 plays an important role in immune regulation, inflammatory response, and metabolic homeostasis. At the metabolic and systemic inflammation level, studies have shown that circulating miR-150 expression is downregulated in overweight or obese individuals, negatively correlated with body mass index, and identified as a key variable molecule in the disease spectrum from obesity to type 2 diabetes. However, these studies mainly establish the correlation between miR-150 and metabolic state, failing to further elucidate its specific diagnostic value in MASH-related local liver inflammation, nor providing quantitative thresholds for clinical grading. At the liver pathology level, the role of miR-150 presents a complex and even seemingly contradictory picture. Some studies have found its upregulated expression in the livers of NAFLD patients and animal models, and its deficiency can improve steatosis; while other cell model-based studies suggest its dysregulated expression under lipotoxic stress. This inconsistency likely stems from differences in research models, test samples, and disease stages, but it also exposes a key limitation of existing research: the lack of a unified, standardized framework to define how dynamic changes in miR-150 correspond to specific points in the transition from metabolic dysfunction-associated steatotic liver (MASL) to steatohepatitis (MASH). At the level of liver fibrosis, serum miR-150-5p has been shown to effectively assess the severity of schistosomiasis-related liver fibrosis and is characteristically downregulated in viral hepatitis-related cirrhosis, as detailed in the report "Shaheen NMH, Zayed N, Riad NM, et al. Role of circulating miR-182 and miR-150 as biomarkers for cirrhosis and hepatocellular carcinoma post HCV infection in Egyptian patients. Virus Res. 2018;255:77-84." However, these studies focus on liver diseases with other causes, and it remains unknown whether their findings can be directly extrapolated to MASLD / MASH, which is dominated by metabolic causes, especially for the identification of early inflammatory activity (MASH).

[0005] While numerous studies have revealed the association between miR-150 and liver disease and metabolic disorders from different perspectives, these studies all have limitations to varying degrees. First, existing work is largely exploratory and association-based, lacking a dedicated design for prospective diagnosis, and failing to systematically evaluate and validate the diagnostic efficacy (such as sensitivity and specificity) of miR-150 in differentiating between "healthy-MASLD-MASH". Second, variations in detection techniques, sample sources, and data processing methods lead to fragmented results, hindering the establishment of standardized testing procedures for clinical application. Finally, existing literature completely fails to provide a rigorously validated, clearly defined cut-off value or continuous scoring model, preventing clinicians from directly translating miR-150 test values ​​into specific grading diagnostic conclusions, significantly limiting its clinical applicability. Therefore, there is currently no reliable clinical tool for grading MASLD / MASH.

[0006] Therefore, there is an urgent need in this field for an innovative solution that can not only confirm the diagnostic value of miR-150-5p, but also break through existing bottlenecks and provide a solution specifically designed for accurate MASLD / MASH grading, from sample processing and detection standardization to result interpretation optimization. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies in lacking a non-invasive, accurate, and standardized diagnostic method for differentiating the disease activity of metabolic-associated fatty liver disease (MASLD) (i.e., distinguishing between healthy individuals, simple MASL, MASH, and liver fibrosis), this invention aims to provide a molecular marker for metabolic-associated fatty liver disease, miR-150-5p in peripheral blood mononuclear cells (PBMCs), and its application. Based on the quantitative detection of miR-150-5p in peripheral blood, a non-invasive classification of the liver disease status of subjects (healthy individuals, simple MASL, MASH, and fibrosis) can be achieved in clinical applications, which has the advantages of being non-invasive, convenient, and highly reproducible.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A molecular marker for metabolic-associated fatty liver disease using miR-150-5p in peripheral blood mononuclear cells, wherein the nucleotide sequence of the miR-150-5p gene fragment is SEQ ID NO.1.

[0009] Furthermore, the expression of the miR-150-5p molecular marker is negatively correlated with the pathological severity of metabolism-associated fatty liver disease (MASLD).

[0010] Furthermore, the nucleotide sequence of the reverse transcription primer for the miR-150-5p molecular marker is SEQ ID NO.2, the nucleotide sequence of the forward amplification primer is SEQ ID NO.3, and the reverse amplification primer is a universal primer with the sequence SEQ ID NO.4.

[0011] An application of miR-150-5p in peripheral blood mononuclear cells as a molecular marker for metabolic-associated fatty liver disease (MASLD) is proposed. This application is used in the preparation of diagnostic kits or expression chips for MASLD. By quantitatively detecting the expression level of miR-150-5p in mononuclear cells of peripheral blood samples, subjects can be diagnosed as healthy, in simple MASLD, in metabolic-associated steatohepatitis (MASH), or as to whether fibrosis is present.

[0012] The specific application is as follows: Step 1: RNA was extracted from peripheral blood mononuclear cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to obtain the Ct values ​​of miR-150-5p and the internal reference gene snRNA U6. The Ct value of snRNA U6 was subtracted from the Ct value of miR-150-5p to obtain ΔCt, and the relative quantitative value of miR-150-5p was obtained. (-ΔCt) ; The nucleotide sequence of the reverse transcription primer for the internal reference gene snRNA U6 is SEQ ID NO.5, the nucleotide sequence of the forward amplification primer is SEQ ID NO.6, and the nucleotide sequence of the reverse amplification primer is SEQ ID NO.7; Step 2: Quantify the relative value of miR-150-5p in peripheral blood mononuclear cells. (-ΔCt) The MASLD state to be identified was included in the variables to construct the ROC curve (Receiver Operating Characteristic curve). The point corresponding to the maximum sum of sensitivity and specificity in the coordinate correspondence table was selected as the cut-off point of the ROC curve, which is used to determine whether the sample is in the first or second state. The MASLD state to be identified was obtained based on liver pathological staining (H&E staining, Sirius red staining) and NAS score (nonalcoholic fatty liver disease activity score) and fibrosis stage (METAVIR score). Step 3: Compare the status information of healthy and simple MASL with relative quantitative values ​​2. (-ΔCt) The cutoff value obtained from the constructed ROC curve is denoted as the first critical value (Ca); the state information of simple MASL and MASH is compared with the relative quantitative value 2. (-ΔCt)The cut-off value obtained from the constructed ROC curve is denoted as the second critical value (C-b); the status information of whether there is liver fibrosis and the relative quantitative value 2 (-ΔCt) The cut-off value obtained from the constructed ROC curve is denoted as the third critical value (C-c); Step 4: Denote the miR-150 relative quantitative value 2 obtained in Step 1 (-ΔCt) as X, and compare it with the first critical value (C-a), the second critical value (C-b), and the third critical value (C-c) described in Step 3, so as to output a grading diagnosis result: If X ≥ C-a, it is judged as "healthy"; If C-b ≤ X < C-a, it is judged as "simple fatty liver (MASL)"; If X < C-b, it is judged as "metabolic associated steatohepatitis (MASH)"; If X < C-c, it is judged as "there is liver fibrosis".

[0013] Compared with the prior art, the advantages of the present invention are as follows: 1. At present, the screening work of fatty liver mainly relies on abdominal B-ultrasound in routine physical examination items. This screening method has a large workload and its accuracy is doubtful. Liver tissue pathological biopsy, which is the "gold standard" for diagnosing MASH and evaluating the degree of fibrosis, has the limitations of being invasive and having high risks. The scheme developed by us for grading the different stages of MASLD based on detecting the level of miR-150-5p in peripheral blood monocytes has low experimental requirements, is simple and convenient, has relatively high accuracy, can be applied to the early screening and risk stratification of MASLD, quickly identify patients who have progressed from simple fatty liver to MASH, and is easy to be developed into a detection kit or expression chip, which is easy to popularize.

[0014] 2. The advantage of the present invention also lies in its broad application prospects. In the later stage, this scheme can be applied to the function development of intelligent Internet of Things home medical devices, intelligent wearable devices, etc., to dynamically monitor the changes in the level of miR-150-5p before and after treatment (such as lifestyle intervention, drug therapy), and evaluate the treatment efficacy.

[0015] To sum up, the present invention provides a diagnostic method with the advantages of non-invasiveness, simple operation, repeatable monitoring, etc. around the important significance of MASLD, which is applicable to the dynamic assessment of different stages of MASLD, and can provide a new technical means and judgment basis for the grading diagnosis, disease progression monitoring and intervention effect evaluation of MASLD. In addition, this method can also be used in combination with existing biochemical indexes, imaging examinations or histological evaluation results to improve the accuracy and comprehensiveness of the grading diagnosis of MASLD. Brief Description of the Drawings

[0016] Figure 1 H&E staining for pathological examination of liver specimens from healthy cynomolgus monkeys and MASLD model cynomolgus monkeys.

[0017] Figure 2 Sirius red staining for pathological examination of liver specimens from healthy cynomolgus monkeys and MASLD model cynomolgus monkeys.

[0018] Figure 3 NAS scores for liver specimens from healthy cynomolgus monkeys and MASLD model cynomolgus monkeys.

[0019] Figure 4 This is the melting curve of the internal reference gene snRNA U6 in qRT-PCR.

[0020] Figure 5 The melting curve of miR-150-5p in qRT-PCR is shown.

[0021] Figure 6 To detect the 2% concentration of peripheral blood mononuclear cell miR-150-5p in healthy, unilateral MASL and MASH cynomolgus monkeys (-ΔCt) Differences in relative expression levels.

[0022] Figure 7 ROC curves for identifying healthy and MASLD-affected peripheral blood mononuclear cells miR-150-5p in cynomolgus monkeys.

[0023] Figure 8 ROC curves of miR-150-5p from peripheral blood mononuclear cells of cynomolgus monkeys recognizing healthy and simple MASL.

[0024] Figure 9 ROC curves of miR-150-5p from peripheral blood mononuclear cells of cynomolgus monkeys recognizing simple MASL and MASH.

[0025] Figure 10 ROC curve of peripheral blood mononuclear cells miR-150-5p in cynomolgus monkeys to identify the presence of liver fibrosis. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments, but these are not intended to limit the scope of the invention.

[0027] Example 1: Constructing a core hierarchical interpretation module for cynomolgus monkeys using the MASLD / MASH model. (1) Constructing the MASLD / MASH model of the cynomolgus monkey All experimental protocols involving non-human primates were approved by the Experimental Animal Use and Management Committee of Xi'an Jiaotong University (Approval No.: 20191278) and the Institutional Animal Care and Use Committee of Guangdong Landao Biotechnology Co., Ltd. (Approval No.: 201901). Thirty cynomolgus monkeys (aged ≥ 9 years, body mass index > 30) were selected from the Guangdong Landao Biotechnology base and housed individually with free access to food and water. The rearing environment was a temperature-controlled room (22℃ to 26℃) with a 12-hour light-dark cycle.

[0028] Prior to induction, liver biopsies were performed under ultrasound guidance using a Bard Magnum biopsy gun equipped with a 17G biopsy needle. The collected liver tissue was preserved in formalin for pathological analysis. Simultaneously, peripheral blood mononuclear cells were extracted from blood samples, and the expression level of miR-150-5p was detected through standardized sample processing.

[0029] After pathological confirmation of the absence of MASLD, animals were paired and enrolled in a healthy control group and a MASH diet group based on age and body mass index, with 10 animals selected in each group. The healthy control group was fed a normal diet, while the MASH diet group was fed a high-fat, high-fructose, and high-cholesterol MASH diet (40% fat, 20% fructose, and 2% cholesterol). Peripheral blood mononuclear cells were extracted from blood samples collected after 10 and 15 months of feeding the normal diet and MASH diet, respectively. The expression level of miR-150-5p was detected through standardized sample processing. Liver biopsies were performed to obtain tissue for pathological analysis.

[0030] (2) Blood sample collection and peripheral blood mononuclear cell extraction Blood was collected from cynomolgus monkeys via venous blood collection. A portion of the blood sample was taken, and an equal volume of calcium- and magnesium-free PBS buffer was added. The diluted blood sample was then slowly added at a 45° angle to the top layer of Ficoll-Paque PREMIUM (Cytiva, 17544202) solution. The centrifuge tube was symmetrically placed in a horizontal rotor and centrifuged at 18-20°C, 400×g for 40 minutes. After centrifugation, the liquid in the tube separated into four layers from top to bottom: the top layer was diluted plasma / platelets; the white membrane layer consisted of monocytes (target cells) in a cloudy ring; the clear layer was Ficoll medium; and the bottom layer consisted of erythrocytes and granulocytes. The top plasma / platelet layer was carefully aspirated with a sterile pipette until approximately 0.5 cm from the white membrane layer. Using a new sterile pipette, all the monocytes in the white membrane layer were aspirated and transferred to a new 15 mL centrifuge tube. Add 10–12 mL of pre-chilled, calcium- and magnesium-free PBS buffer to a centrifuge tube containing mononuclear cells. Gently pipette or invert to mix, resuspend the cells, and centrifuge at 250 × g for 10 minutes at 4°C. After centrifugation, carefully and thoroughly discard the supernatant and repeat the washing once. Resuspend the cell pellet in 1 mL of pre-chilled PBS and transfer it to a 1.5 mL RNase-free centrifuge tube. Centrifuge at 500 × g for 5 minutes at 4°C, discarding the supernatant completely to obtain a pure cell pellet for subsequent RNA extraction.

[0031] (3): RNA was extracted and the expression level of miR-150-5p was detected by RT-qPCR. The miR-150-5p described herein is derived from peripheral blood and is continuously expressed in peripheral blood mononuclear cells. It is unaffected by RNase, environmental pH, and low temperature, and has the characteristics of reproducibility, non-invasive acquisition, and high sensitivity and specificity. It is an ideal material for distinguishing different disease states.

[0032] Total RNA was extracted from peripheral blood mononuclear cells using the TRIzol method: cell samples were directly lysed, the lysate was extracted with chloroform, centrifuged to separate the aqueous phase, isopropanol was added to precipitate RNA, and the RNA was dissolved in RNase-free water after washing with 75% ethanol. The concentration and purity were then determined for later use. Subsequently, miRNA 1 was used... stThe Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, MR101) was used for miRNA-150-5p cDNA synthesis. The reaction process involved genomic DNA removal at 42°C, followed by incubation at 25°C for 5 minutes, 50°C for 15 minutes, and finally 85°C for 5 minutes. qPCR was performed using the SYBR Green assay on a CFX96 system. A 10 μL reaction mixture contained 2×SYBR Premix, forward and reverse primers, and cDNA template. The reaction program consisted of 95°C pre-denaturation for 30 seconds, followed by 40 cycles of amplification at 95°C for 5 seconds and 60°C for 30 seconds. Melting curve analysis was then performed. The relative expression level of the target gene compared to the internal control gene was calculated using the 2^(-ΔCt) method, or further, the relative expression level compared to the healthy control group was calculated using the 2^(-ΔΔCt) method.

[0033] (4): Liver pathological staining and scoring Cynomolgus monkey liver tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. For H&E staining, sections were dewaxed, hydrated, stained with hematoxylin for 5-8 minutes, differentiated with hydrochloric acid ethanol, and blued with ammonia, counterstained with eosin for 1-3 minutes, then dehydrated, cleared, and mounted with neutral resin. For Sirius red staining, sections dewaxed to water were stained with 0.1% Sirius red solution for 1 hour, rinsed with running water, dehydrated, cleared, mounted, and photographed. Nonalcoholic fatty liver disease activity score (NAS) is a semi-quantitative score used to assess disease activity. It consists of the sum of scores for the following three histological features: (1) hepatocellular steatosis (0-3 points); (2) intralobular inflammation (0-3 points); (3) hepatocellular ballooning degeneration (0-2 points). The total NAS score is 0-8 points. The METAVIR scoring system for liver fibrosis is as follows: Stage 0 (no fibrosis), Stage 1 (portal fibrosis, no fibrotic septa), Stage 2 (portal fibrosis, few fibrotic septa), Stage 3 (fibrotic septa), Stage 4 (cirrhosis). All histological sections were independently reviewed by at least two pathologists unaware of the grouping.

[0034] (5): Data Analysis In the cynomolgus monkey study, cynomolgus monkeys in the MASH diet group were divided into healthy, simple MASL, and MASH groups, as well as those with or without liver fibrosis, based on pathological staining, NAS scores, and liver fibrosis staging. Figure 1-3 At the same time, 2 (-ΔCt) The relative expression levels of miR-150-5p in MASL and MASH cynomolgus monkeys compared to healthy cynomolgus monkeys were calculated. Results showed that the melting curve of the internal reference gene snRNA U6 in qRT-PCR ( Figure 4Melting curves of miR-150-5p () Figure 5 All values ​​showed a single peak, indicating gene-specific amplification. The expression of miR-150-5p in the tested peripheral blood mononuclear cells differed significantly among the three states. Figure 6 ), p <0.05, which is negatively correlated with the severity of MASLD and is statistically significant.

[0035] ROC curves of miR-150-5p in peripheral blood mononuclear cells of cynomolgus monkeys, identifying healthy and MASLD cells, were analyzed using SPSS software. Figure 7 The area under the ROC curve (AUC) of miR-150-5p was 0.913, with a specificity of 70.0% and a sensitivity of 95.0%. p <0.001 is statistically significant. This indicates that the expression level of miR-150-5p in peripheral blood mononuclear cells can be used to distinguish between healthy individuals and those with MASLD. (Note: The reference line is the opportunity diagonal; the area under the reference line (AUC) is 0.5. Below this line, the diagnostic method has no diagnostic value whatsoever.) Furthermore, the ROC curves of miR-150-5p in peripheral blood mononuclear cells of cynomolgus monkeys recognizing healthy and simple MASL were analyzed using SPSS software. Figure 8 The area under the ROC curve (AUC) of miR-150-5p was 0.825, with a specificity of 70.0%, a sensitivity of 90.0%, and a cutoff value of 0.1170. p =0.014, which is statistically significant. This indicates that the expression level of miR-150-5p in peripheral blood mononuclear cells can be used to distinguish between healthy individuals and simple MASL. (Note: The reference line is the opportunity diagonal; the area under the reference line (AUC) is 0.5. Below this line, the diagnostic method has no diagnostic value.) Furthermore, the ROC curves of miR-150-5p in peripheral blood mononuclear cells of cynomolgus monkeys recognizing simple MASL and MASH were analyzed using SPSS software. Figure 9 The area under the ROC curve (AUC) of miR-150-5p was 0.970, with a specificity of 100%, a sensitivity of 90.0%, and a cutoff value of 0.0633. p A value <0.001 is statistically significant. This indicates that the expression level of miR-150-5p in peripheral blood mononuclear cells can be used to distinguish between simple MASL and MASH. (Note: The reference line is the opportunity diagonal; the area under the reference line (AUC) is 0.5. Below this value, the diagnostic method has no diagnostic value whatsoever.) Furthermore, the ROC curve of miR-150-5p in peripheral blood mononuclear cells of cynomolgus monkeys was analyzed using SPSS software to identify the presence of liver fibrosis. Figure 10The area under the ROC curve (AUC) of miR-150-5p was 0.994, with a specificity of 95.7%, a sensitivity of 100%, and a cutoff value of 0.0563. p <0.001 is statistically significant. This indicates that the expression level of miR-150-5p in peripheral blood mononuclear cells can be used to identify the presence of liver fibrosis. (Note: The reference line is the opportunity diagonal; the area under the reference line (AUC) is 0.5. Below this line, the diagnostic method has no diagnostic value.) Furthermore, as described in the invention, a core interpretation module is constructed through standardized sample processing and detection. This module compares the miR-150 relative quantitative value 2^(-ΔCt) (denoted as X) obtained from the standardized detection with preset first threshold (0.1170), second threshold (0.0633), and third threshold (0.0563), thereby outputting a graded diagnostic result. If X ≥ 0.1170, then it is interpreted as "healthy"; If 0.0633 ≤ X < 0.1170, then it is interpreted as "simple MASL"; If X < 0.0633, it is interpreted as "metabolic steatohepatitis (MASH)".

[0036] If X < 0.0563, it is interpreted as "liver fibrosis exists". Example 2: Verification of the core hierarchical interpretation module Twenty cynomolgus monkeys, excluding the healthy control group and the MASH diet group, were randomly selected from the Guangdong Landao Biotechnology Base as the validation group. Two researchers conducted standardized sample processing and detected the relative expression level of miR-150-5p in peripheral blood mononuclear cells. (-ΔCt) Meanwhile, two other pathologists, unaware of the group assignments, independently reviewed the liver pathologies of the 20 cynomolgus monkeys in the validation group. The results (Table 1) show that the core interpretation module achieved a 95% accuracy rate in identifying whether MASLD was diseased, a 100% accuracy rate in identifying healthy individuals, an 86% accuracy rate in identifying simple MASL (6 / 7, only 1 case of MASL was misdiagnosed as healthy), and a 100% accuracy rate in identifying MASH. The accuracy rate in identifying the presence of liver fibrosis was 90%.

[0037] Table 1 shows the interpretation of the cynomolgus monkeys in the verification group through the core grading interpretation module. The results in parentheses are the pathological results determined by pathology experts based on liver biopsy.

[0038] serial number 2 (-ΔCt) MASLD status Liver fibrosis is present. NO.1 0.092782723 MASL (MASL) No (No) NO.2 0.122656308 Health (MASL) No (No) NO.3 0.140632311 Health No (No) NO.4 0.140337875 Health No (No) NO.5 0.071297732 MASL (MASL) No (Yes) NO.6 0.041333866 MASH (MASH) whether) NO.7 0.119575715 Health No (No) NO.8 0.053813258 MASH (MASH) Yes (Yes) NO.9 0.154077922 Health No (No) NO.10 0.034915223 MASH (MASH) Yes (Yes) NO.11 0.099456821 MASL (MASL) No (No) NO.12 0.075884794 MASL (MASL) No (No) NO.13 0.038864554 MASH (MASH) Yes (Yes) NO.14 0.133483616 Health No (No) NO.15 0.029456468 MASH (MASH) Yes (Yes) NO.16 0.089765413 MASL (MASL) No (No) NO.17 0.065784762 MASL (MASL) No (No) NO.18 0.158945602 Health No (No) NO.19 0.044781231 MASH (MASH) Yes (Yes) NO.20 0.025848873 MASH (MASH) Yes (Yes) In this embodiment, 20 cynomolgus monkeys were randomly selected from outside the control group. The relative quantitative values ​​of miR-150-5p in peripheral blood mononuclear cells were obtained through standardized sample processing and detection.(-ΔCt) The condition of each cynomolgus macaque was determined using the core grading interpretation module. Additionally, liver biopsies were performed on 20 cynomolgus macaques, and liver tissue samples were obtained for pathological staining and pathological diagnosis based on NAS scores and fibrosis stages. The cynomolgus macaque conditions obtained through the core grading interpretation module were compared with the pathological diagnoses to verify the accuracy of the proposed method.

[0039] Based on Examples 1 and 2, the following conclusions can be drawn: A molecular marker for metabolic-associated fatty liver disease (MASLD) is proposed for use in peripheral blood mononuclear cells. The expression of the miR-150-5p molecular marker is negatively correlated with the pathological severity of MASLD.

[0040] An application of miR-150-5p in peripheral blood mononuclear cells as a molecular marker for metabolic-associated fatty liver disease (MASLD) is proposed. This application is used in the preparation of diagnostic kits or expression chips for MASLD. By quantitatively detecting the expression level of miR-150-5p in mononuclear cells of peripheral blood samples, subjects can be diagnosed as healthy, in simple MASLD, in metabolic-associated steatohepatitis (MASH), or as to whether fibrosis is present.

[0041] In summary, this invention provides a molecular marker for miR-150-5p in peripheral blood mononuclear cells (PBMCs) as a metabolic-related fatty liver disease and its application. Based on the quantitative detection of miR-150-5p in peripheral blood, it enables non-invasive grading of the liver disease status of subjects (healthy, simple MASL, MASH, fibrosis) in clinical applications, which has the advantages of being non-invasive, convenient, and highly reproducible.

Claims

1. miR-150-5p in peripheral blood mononuclear cells as a molecular marker for metabolic associated steatohepatitis.

2. The molecular marker according to claim 1, characterized in that, The nucleotide sequence of the gene fragment of the molecular marker miR-150-5p is SEQ ID NO.

1.

3. The molecular marker according to claim 1, characterized in that, The expression of the miR-150-5p molecular marker is negatively correlated with the pathological severity of metabolic associated steatohepatitis (MASLD).

4. The molecular marker according to claim 1, characterized in that, The nucleotide sequence of the reverse transcription primer of the miR-150-5p molecular marker is SEQ ID NO.2, the nucleotide sequence of the forward amplification primer is SEQ ID NO.3, and the reverse amplification primer is a universal primer with the sequence SEQ ID NO.

4.

5. The application of the molecular marker according to claim 1, characterized in that, When applied in the preparation of a diagnostic kit or expression chip for metabolic associated steatohepatitis (MASLD), by quantitatively detecting the expression level of miR-150-5p in monocytes in a subject's peripheral blood sample, the subject is diagnosed as being in a healthy state, simple MASL state, metabolic associated steatohepatitis (MASH), and whether there is fibrosis.

6. The application of the molecular marker according to claim 5, characterized in that, The specific application is as follows: Step 1: RNA was extracted from peripheral blood mononuclear cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to obtain the Ct values ​​of miR-150-5p and the internal reference gene snRNA U6. The Ct value of snRNA U6 was subtracted from the Ct value of miR-150-5p to obtain ΔCt, and the relative quantitative value of miR-150-5p was obtained. (-ΔCt) ; Step 2: Quantify the relative value of miR-150-5p in peripheral blood mononuclear cells. (-ΔCt) The MASLD state to be identified was included in the variables to construct the ROC curve. The point corresponding to the maximum sum of sensitivity and specificity in the coordinate correspondence table was selected as the cut-off point of the ROC curve, which is the critical point used to determine whether the sample is in the first or second state. The MASLD state to be identified was obtained based on liver pathological staining and NAS score and fibrosis stage. Step 3: Compare the status information of healthy and simple MASL with relative quantitative values ​​2. (-ΔCt) The cutoff value obtained from the constructed ROC curve is denoted as the first critical value (Ca); the state information of simple MASL and MASH is compared with the relative quantitative value 2. (-ΔCt) The cutoff value obtained from the constructed ROC curve is denoted as the second critical value (Cb); the status information of whether liver fibrosis exists is compared with the relative quantitative value 2. (-ΔCt) The cutoff value obtained from the constructed ROC curve is denoted as the third critical value (Cc). Step 4: Calculate the relative quantitative value of miR-150 obtained in Step 1. (-ΔCt) Let X be the critical value, and compare it with the first critical value (Ca), the second critical value (Cb), and the third critical value (Cc) in step three to output the graded diagnosis result: If X ≥ C-a, it is interpreted as "healthy"; If C-b ≤ X < C-a, it is interpreted as "simple steatohepatitis (MASL)"; If X < C-b, it is interpreted as "metabolic associated steatohepatitis (MASH)"; If X < C-c, it is interpreted as "there is liver fibrosis".

7. The application of the molecular marker according to claim 5, characterized in that, The nucleotide sequence of the reverse transcription primer of the internal reference gene snRNA U6 in step one is SEQ ID NO.5, the nucleotide sequence of the forward amplification primer is SEQ ID NO.6, and the nucleotide sequence of the reverse amplification primer is SEQ ID NO.7.

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