Combination of three microRNAs associated with lipid metabolism disorders and their use in diagnosis and drug screening

Abstract

The present application relates to the field of biomedical and molecular biology, and relates to a combination of three microRNAs related to lipid metabolism disorder and application thereof in diagnosis and drug screening. By using an oleic acid-induced lipid accumulation model of human hepatoma cells, it is proved that lipid accumulation can cause the expression levels of miR-21-5p, miR-24-3p and miR-423-5p to increase significantly, and taurine can obviously inhibit the increase of the expression levels of the three miRNAs caused by oleic acid, including miR-21-5p, miR-24-3p and miR-423-5p. The present application discloses that miR-21-5p, miR-24-3p and miR-423-5p can be used as early molecular detection markers in abnormal and improved states of lipid metabolism, and can also be used as a method for evaluating the degree of lipid-lowering effect of taurine. The present application provides a new theoretical basis and application prospect for the discovery of molecular markers and intervention strategies of lipid metabolism disorder.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and molecular biology, specifically to a combination of three microRNAs associated with lipid metabolism disorders and their applications in diagnosis and drug screening. More particularly, it relates to the expression characteristics of specific microRNAs in lipid metabolism disorders and their applications in the diagnosis, drug screening, and treatment of lipid metabolism-related diseases. Background Technology

[0002] Lipid metabolism disorders are a key pathological feature of metabolic-related diseases, including obesity, atherosclerosis, and non-alcoholic fatty liver disease. Current research indicates that abnormally elevated levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C), and decreased levels of high-density lipoprotein cholesterol (HDL-C), are important markers of lipid metabolism abnormalities. Clinically, lipid-lowering drugs such as statins and fibrates are commonly used for intervention; however, these drugs have limited efficacy or side effects. Therefore, developing novel molecular biomarkers with high safety profiles, well-defined mechanisms of action, and applicability for lipid metabolism disorder intervention and drug screening is of significant research importance and application value.

[0003] Taurine is a sulfur-containing amino acid-like compound widely distributed in various tissues of the body, possessing multiple biological functions such as antioxidation, anti-inflammation, regulation of bile acid metabolism, and improvement of lipid metabolism. Existing studies suggest that taurine can, to some extent, reduce lipid accumulation, improve lipid metabolism-related indicators, and has a protective effect against hyperlipidemia-induced metabolic abnormalities. Compared with traditional lipid-lowering drugs, taurine has the advantages of wide availability, high safety, and good biocompatibility, thus it is considered a potential functional factor in intervention research on lipid metabolism disorders. However, current research mainly focuses on the effects of taurine on blood lipid indicators, oxidative stress, inflammatory responses, or the expression of lipid metabolism-related genes and proteins. Research on changes in miRNA expression during taurine intervention in hyperlipidemia models and its diagnostic, evaluation, and drug screening value remains relatively insufficient.

[0004] In recent years, microRNAs (miRNAs), as non-coding small RNAs, have gradually attracted attention in the regulation of lipid metabolism. Existing studies have shown that certain miRNAs are closely related to processes such as lipogenesis, lipid oxidation, and cholesterol metabolism. Hyperlipidemia-induced cell or animal models are important models for studying lipid metabolism disorders, effectively simulating excessive lipid accumulation and abnormal metabolic states. In these models, changes in miRNA expression profiles may reflect the occurrence and development of lipid metabolism disorders and may also serve as important molecular indicators for evaluating the effects of functionally active substance interventions. Especially in the context of taurine intervention in hyperlipidemia models, screening for miRNAs closely related to lipid accumulation improvement helps to reveal the potential molecular mechanisms by which taurine regulates lipid metabolism and provides a basis for subsequently establishing diagnostic biomarkers and drug screening models for lipid metabolism disorders.

[0005] However, the dynamic expression patterns and application value of miR-21-5p, miR-24-3p, and miR-423-5p in lipid accumulation and improvement processes have not yet been systematically reported. In particular, regarding the technical route of "taurine intervention—high-lipid-induced lipid metabolism disorder model—specific miRNA response changes," there is currently a lack of technical solutions to combine the effects of taurine intervention with miR-21-5p, miR-24-3p, and miR-423-5p for the diagnosis, intervention evaluation, and drug screening of lipid metabolism disorders. Therefore, it is urgent to explore the relationship between these miRNAs and lipid metabolism abnormalities in order to provide new molecular targets and application strategies for the diagnosis and intervention of lipid metabolism disorders. Summary of the Invention

[0006] The purpose of this invention is to reveal the expression characteristics of specific miRNAs in the process of lipid metabolism abnormalities and improvement, and based on this, to propose their application in the diagnosis, drug screening, and treatment of lipid metabolism disorders. To solve the above problems, the technical solution of this invention is as follows: a combination of three microRNAs related to lipid metabolism disorders, namely miR-21-5p, miR-24-3p, and miR-423-5p. The RNA sequence of miR-21-5p is 5′-UAGCUUAUCAGACUGAUGUUGA-3′; The RNA sequence of miR-24-3p is 5′-UGGCUCAGUUCAGCAGGAACAG-3′; The RNA sequence of miR-423-5p is 5′-UGAGGGGCAGAGAGCGAGACUUU-3′ A method for assessing lipid metabolism disorders includes detecting the expression levels of the aforementioned combination of microRNAs associated with lipid metabolism disorders in a cell or tissue sample.

[0007] In the aforementioned method for assessing lipid metabolism disorders, the expression of miR-21-5p, miR-24-3p, and miR-423-5p is increased under lipid accumulation conditions; and the expression of miR-21-5p, miR-24-3p, and miR-423-5p is downregulated under lipid improvement conditions.

[0008] A method for screening candidate drugs to improve lipid metabolism involves treating a cell model with the candidate drug and detecting changes in the combination of microRNAs mentioned above that are associated with lipid metabolism disorders.

[0009] The above method determines that the drug has the effect of improving lipid metabolism when it downregulates the expression of miR-21-5p, miR-24-3p and miR-423-5p.

[0010] A molecular diagnostic kit for lipid metabolism disorders, the kit comprising nucleic acid probes or primers targeting miR-21-5p, miR-24-3p and miR-423-5p.

[0011] Based on the above findings, this invention proposes: Diagnostic applications: By detecting the expression levels of the above miRNAs, it can be used to assess and diagnose the occurrence and improvement status of lipid metabolism disorders.

[0012] Drug screening applications: Changes in the expression of the above miRNAs can be used as indicators to screen candidate drugs or natural products with the potential to improve lipid metabolism.

[0013] Therapeutic applications: By regulating the expression of miR-21-5p, miR-24-3p and miR-423-5p, drugs can be prepared to improve lipid metabolism disorders or treat NAFLD and related diseases.

[0014] Application of detection tools: Based on the above miRNA probes or primers, molecular diagnostic kits can be prepared for the auxiliary diagnosis and efficacy monitoring of lipid metabolism-related diseases.

[0015] In summary, this invention reveals for the first time the characteristic expression patterns of miR-21-5p, miR-24-3p, and miR-423-5p in the process of lipid metabolism abnormality and improvement, expanding the application prospects of miRNA in the molecular diagnosis and treatment of metabolic diseases, and has important theoretical significance and practical application value. Attached Figure Description

[0016] Figure 1Taurine can alleviate oleic acid (OA)-induced lipid accumulation in HepG2 cells. (A) CCK-8 assay to assess the effect of taurine on cell viability. (B) Measurement of intracellular TG levels. (C) Measurement of TC levels. (D) Detection of LDL-C. (E) Detection of HDL-C. (F) Calculation of the LDL-C / HDL-C ratio. (G, H) Oil Red O to assess lipid droplet accumulation.

[0017] Figure 2 Taurine regulates the expression of genes related to lipid metabolism. (A) Srebp-1c mRNA expression. (B) FABP4 mRNA expression.

[0018] Figure 3 Taurine regulates the expression of proteins related to lipid metabolism. (A) FABP4 protein expression. (B) SREBP-1c protein expression.

[0019] Figure 4 To illustrate the regulatory effect of taurine on oleic acid-induced miRNA expression in HepG2 cells. (A) qPCR analysis of miR-21-5p expression. (B) qPCR analysis of miR-24-3p expression. (C) qPCR analysis of miR-423-5p expression. Detailed Implementation

[0020] The technical solution of the present invention will be further described below through specific embodiments. However, the present invention is not limited to the following embodiments. The raw materials used in the present invention can be purchased on the market or synthesized using methods known in the art.

[0021] Experimental samples and reagents: This invention uses human liver cancer cells (HepG2 cells) as an in vitro experimental model. The HepG2 cells are in the exponential growth phase, grown at a rate of 8 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of [number] cells / mL in six-well plates and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, then incubated at 37°C in a 5% CO2 incubator. The experiment included a control group, an oleic acid model group, and a taurine intervention group. After 24 h of adherent culture, oleic acid (OA) was added to the model group to establish a lipid accumulation model. The intervention group received OA treatment along with different concentrations of taurine, and treatment continued for another 24 h.

[0022] The main reagents used in this invention include: TG detection kit (A110-1-1, Nanjing Jiancheng, China), TC detection kit (A111-1-1, Nanjing Jiancheng, China), LDL-C detection kit (A113-1-1, Nanjing Jiancheng, China), HDL-C detection kit (A112-1-1, Nanjing Jiancheng, China), RNA Easy Fast kit (DP451, TIANGEN, China), FastKing RT Kit (KR116, TIANGEN, China), first-strand cDNA synthesis kit (B532451, sangong, China), miRNA first-strand cDNA synthesis kit, Taq SYBR® Green qPCR Premix, and BCA protein quantification kit (A55861, Thermo Fisher). Scientific (Finland), SREBP-1c antibody (1:1000, 14088-1-AP, Proteintech, China), FABP4 antibody (1:1000, 12802-1-AP, Proteintech, China), GAPDH antibody (1:1000, BL006A, Biosharp, China), enzyme-labeled goat anti-rabbit secondary antibody (1:2000, BL023A, Biosharp, China), and ECL chemiluminescence kit (SQ201, Yamei, China).

[0023] The miRNAs involved in this invention include miR-21-5p, miR-24-3p, and miR-423-5p, with RNA sequences of 5′-UAGCUUAUCAGACUGAUGUUGA-3′, 5′-UGGCUCAGUUCAGCAGGAACAG-3′, and 5′-UGAGGGGCAGAGAGCGAGACUUU-3′, respectively. Furthermore, this invention also relates to the lipid metabolism-related genes SREBP-1c and FABP4, wherein the human mRNA reference sequence number for FABP4 is NM_001442.3, and SREBF1 is the protein-coding gene for SREBP-1, whose variable transcripts include SREBP-1c.

[0024] Table 1

[0025] All experimental results are expressed as mean ± standard deviation. Differences between groups were analyzed using statistical methods; p < 0.05 indicated statistical significance, and p < 0.01 indicated highly significant difference.

[0026] Example 1: The effect of taurine on improving oleic acid-induced lipid accumulation in HepG2 cells

[0027] This example illustrates that taurine can reduce oleic acid-induced lipid accumulation in HepG2 cells. Figure 1 .

[0028] HepG2 cells were seeded in culture plates and, after cell adhesion, were treated with different concentrations of taurine for 24 h. Cell viability was then assessed using the CCK-8 assay to evaluate the safe concentration range of taurine for HepG2 cells. The results showed that cell viability decreased in a dose-dependent manner with increasing taurine concentration. Specifically, 30 mM taurine had no significant effect on cell viability, with cell survival still greater than 90%; however, treatment with 50 mM and 100 mM taurine significantly reduced cell viability (p < 0.05). Therefore, subsequent experiments selected 0.1 mM, 1 mM, 10 mM, and 30 mM taurine as intervention concentrations.

[0029] Subsequently, a lipid accumulation model was established by treating HepG2 cells with oleic acid, and different concentrations of taurine were added to the model for intervention. After cell collection, the cells were washed twice with PBS, lysed with 300 μL of RIPA lysis buffer, and incubated for 30 min with shaking every 10 min. Intracellular TG, TC, LDL-C, and HDL-C levels were detected using a microplate reader (Multiskan GO, Thermo Fisher Scientific, Finland) according to the kit instructions, and the LDL-C / HDL-C ratio was calculated.

[0030] The results showed that, compared with the control group, the intracellular TG and TC levels in the oleic acid model group were significantly increased (p < 0.01), suggesting that oleic acid can induce significant lipid accumulation in HepG2 cells. Compared with the oleic acid model group, the intracellular TG and TC levels decreased in a concentration-dependent manner after taurine treatment, with 30 mM taurine significantly reducing the intracellular TG and TC levels in the high-lipid model cells (p < 0.01). Meanwhile, compared with the control group, the LDL-C level and LDL-C / HDL-C ratio were significantly increased in the oleic acid model group (p < 0.01), while the HDL-C level was significantly decreased (p < 0.01); however, after taurine intervention, the intracellular LDL-C level and LDL-C / HDL-C ratio decreased, while the HDL-C level increased, indicating that taurine can improve oleic acid-induced lipid metabolism abnormalities.

[0031] Oil Red O staining was used to further observe the accumulation of intracellular lipid droplets. HepG2 cells were cultured at 5 × 10⁶ cells / year. 5Lipid droplet accumulation was seeded at a density of [number] cells / mL in 24-well plates, with three replicates for each treatment condition. After treatment, the cells were fixed with 4% paraformaldehyde at room temperature for 2 hours. After discarding the fixative, the cells were washed twice with PBS, treated with 60% isopropanol for 10 minutes, and then stained with Oil Red O staining solution at room temperature for 30 minutes. After rinsing with PBS for 20 seconds, lipid droplet accumulation was observed under a microscope (DMI4000B, Leica, Germany). Subsequently, intracellular Oil Red O was dissolved in 100% isopropanol, and the absorbance was measured at 490 nm using a microplate reader for quantitative analysis.

[0032] Oil Red O staining results showed that oleic acid treatment significantly increased the number of lipid droplets in HepG2 cells (p < 0.01), indicating successful model establishment. Taurine treatment significantly reduced intracellular lipid droplet accumulation in a concentration-dependent manner, with the 30 mM taurine treatment group showing the most significant decrease in lipid accumulation (p < 0.01). These results indicate that taurine can effectively alleviate oleic acid-induced lipid accumulation in HepG2 cells.

[0033] Example 2: Taurine's effect on lipid metabolism-related genes SREBP-1c and FABP4 Regulation of mRNA expression

[0034] This example illustrates how taurine can regulate lipid metabolism-related genes. SREBP-1c and FABP4 mRNA expression, corresponding Figure 2 .

[0035] HepG2 cells from different treatment groups were collected, and total RNA was extracted using the RNA Easy Fast kit. mRNA reverse transcription was performed using the FastKing RT Kit and a first-strand cDNA synthesis kit. The resulting cDNA was used for subsequent real-time quantitative PCR analysis. The qPCR reaction volume was 20 μL, including 10 μL Taq SYBR® Green qPCR Premix, 0.4 μL each of forward and reverse primers, 2 μL cDNA, and 7.2 μL RNase-free H2O. The qPCR reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles. After amplification, melting curve analysis was performed to exclude non-specific amplification. 2 -ΔΔCt The relative expression levels of the target gene were calculated using a method with GAPDH as an internal reference gene.

[0036] This embodiment selects lipid metabolism-related genes. SREBP-1c and FABP4 As the object of detection. Among them, SREBP-1c Closely related to lipid synthesis process, FABP4 It is associated with fatty acid binding, transport, and lipid accumulation. The target gene sequence information can be retrieved from the NCBI database. The detection primers were designed using Primer Premier 6.0 software and synthesized by Shanghai Sangon Biotech Co., Ltd.

[0037] qPCR results showed that, compared with the control group, the oleic acid model group of HepG2 cells... SREBP-1c and FABP4 The mRNA expression level was significantly increased, indicating that oleic acid-induced activation of genes related to lipid synthesis, fatty acid accumulation, and transport. Compared with the oleic acid model group, taurine treatment significantly increased the expression level of these genes. SREBP-1c and FABP4 The mRNA expression levels of taurine and oleic acid decreased, suggesting that taurine can inhibit the abnormal expression of oleic acid-induced lipid metabolism disorder-related genes at the transcriptional level. This result indicates that... SREBP-1c and FABP4 It can serve as an important molecular indicator for evaluating lipid metabolism disorders and the effectiveness of taurine intervention.

[0038] Example 3: Regulatory effect of taurine on the expression of lipid metabolism-related proteins SREBP-1c and FABP4

[0039] This example illustrates that taurine can regulate the expression of lipid metabolism-related proteins SREBP-1c and FABP4, corresponding to Figure 3 .

[0040] HepG2 cells from each treatment group were collected, washed with PBS, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined using the BCA method. 30 μg of protein sample was added to each well, and after separation by SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane. Transfer conditions were 250 mA on ice for 2 h. After transfer, the membrane was blocked with 5% skim milk in TBST at room temperature for 2 h. Rabbit anti-human SREBP-1c antibody, FABP4 antibody, and GAPDH antibody were then added, and the membrane was incubated overnight at 4°C on a shaker.

[0041] The following day, the membrane was washed three times with TBST for 5 min each time; then enzyme-labeled goat anti-rabbit secondary antibody was added, and the membrane was incubated at room temperature for 1 h. The membrane was then washed three more times with TBST for 5 min each time. Finally, the protein bands were developed using an ECL chemiluminescence assay kit, and the gray values ​​of the protein bands were quantitatively analyzed using ImageJ Gel analysis. GAPDH was used as an internal control protein, and the results are expressed as fold changes relative to the control group.

[0042] Western blot results showed that, compared with the control group, the expression levels of FABP4 and SREBP-1c proteins in HepG2 cells of the oleic acid model group were significantly increased, indicating that oleic acid treatment promoted lipid synthesis and fatty acid accumulation, as well as the expression of transport-related proteins. Compared with the oleic acid model group, the expression levels of FABP4 and SREBP-1c proteins decreased after taurine treatment, suggesting that taurine can not only regulate the expression of lipid metabolism-related genes at the mRNA level, but also inhibit oleic acid-induced lipid metabolism abnormalities at the protein level. These results indicate that taurine may improve oleic acid-induced lipid accumulation in HepG2 cells by regulating the SREBP-1c and FABP4-related pathways.

[0043] Example 4: The regulatory effect of taurine on oleic acid-induced miRNA expression in HepG2 cells

[0044] This example illustrates that taurine can regulate the expression of miR-21-5p, miR-24-3p, and miR-423-5p in oleic acid-induced HepG2 cells, corresponding to... Figure 4 .

[0045] HepG2 cells from the control group, oleic acid model group, and taurine intervention group were collected. Total RNA was extracted using the RNA Easy Fast kit, and miRNA first-strand cDNA synthesis was performed for reverse transcription. The expression levels of miR-21-5p, miR-24-3p, and miR-423-5p were then detected using the QuantStudio5 real-time quantitative PCR system. The qPCR amplification system consisted of 20 μL, including 10 μL Taq SYBR® Green qPCR Premix, 0.4 μL each of forward and reverse primers, 2 μL cDNA, and 7.2 μL RNase-free H2O. The qPCR reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles. Melting curve analysis was performed after amplification to avoid non-specific amplification. The relative expression levels of miRNAs were calculated using the 2^-ΔΔCt method.

[0046] The miRNAs detected in this invention include miR-21-5p, miR-24-3p, and miR-423-5p, with the following RNA sequences: miR-21-5p: 5′-UAGCUUAUCAGACUGAUGUUGA-3′; miR-24-3p: 5′-UGGCUCAGUUCAGCAGGAACAG-3′; miR-423-5p: 5′-UGAGGGGCAGAGAGCGAGACUUU-3′.

[0047] The results showed that in the oleic acid-induced lipid accumulation model of HepG2 cells, the expression levels of miR-21-5p, miR-24-3p, and miR-423-5p were significantly upregulated compared with the control group, suggesting that these miRNAs may be involved in the oleic acid-induced lipid metabolism disorder. After taurine intervention, the expression levels of miR-21-5p, miR-24-3p, and miR-423-5p were significantly decreased (p < 0.05), indicating that taurine can regulate abnormally expressed miRNAs under hyperlipidemia. These results suggest that miR-21-5p, miR-24-3p, and miR-423-5p can serve as potential molecular markers reflecting lipid metabolism disorders and evaluating the effectiveness of taurine intervention, and can be further used for the diagnosis and drug screening of lipid metabolism disorders.

[0048] In summary, this invention establishes an in vitro lipid metabolism disorder model in HepG2 cells induced by oleic acid and then intervenes with taurine. The results show that taurine can improve lipid metabolism-related indicators in the hyperlipidemic model cells, reducing TG, TC, LDL-C, and the LDL-C / HDL-C level, increasing HDL-C level, and decreasing intracellular lipid droplet accumulation. Simultaneously, taurine can downregulate the abnormal expression of oleic acid-induced lipid metabolism-related genes and proteins SREBP-1c and FABP4.

[0049] Furthermore, this invention found that miR-21-5p, miR-24-3p, and miR-423-5p were upregulated in an oleic acid-induced lipid accumulation model in HepG2 cells, while their expression decreased after taurine intervention. This indicates that miR-21-5p, miR-24-3p, and miR-423-5p are closely related to lipid metabolism disorders and their improvement process. Therefore, miR-21-5p, miR-24-3p, and miR-423-5p can serve as potential molecular markers for the diagnosis, intervention efficacy evaluation, and drug screening of lipid metabolism disorders, providing a basis for developing diagnostic and screening methods related to lipid metabolism disorders.

[0050] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection defined by the claims of the present invention.

Claims

1. A combination of three microRNAs associated with disorders of lipid metabolism, characterized in that, The microRNA combination consists of miR-21-5p, miR-24-3p, and miR-423-5p. The RNA sequence of miR-21-5p is 5′-UAGCUUAUCAGACUGAUGUUGA-3′; The RNA sequence of miR-24-3p is 5′-UGGCUCAGUUCAGCAGGAACAG-3′; The RNA sequence of miR-423-5p is 5′-UGAGGGGCAGAGAGCGAGACUUU-3′.

2. A method for assessing disorders of lipid metabolism, characterized in that, This includes detecting the expression levels of the combination of microRNAs associated with lipid metabolism disorders as described in claim 1 in cell or tissue samples.

3. A method for assessing disorders of lipid metabolism according to claim 2, characterized in that, Under lipid accumulation conditions, the expression of miR-21-5p, miR-24-3p, and miR-423-5p is increased; under lipid improvement conditions, the expression of miR-21-5p, miR-24-3p, and miR-423-5p is downregulated.

4. A method for screening a candidate drug for improving lipid metabolism, characterized by, The candidate drug was used to treat cell models, and changes in the combination of microRNAs associated with lipid metabolism disorders as described in claim 1 were detected.

5. The method of claim 4, wherein, When the drug downregulates the expression of miR-21-5p, miR-24-3p, and miR-423-5p, the drug is deemed to have the effect of improving lipid metabolism.

6. A molecular diagnostic kit for preparing a disorder of lipid metabolism, characterized by, The kit includes nucleic acid probes or primers for miR-21-5p, miR-24-3p, and miR-423-5p.

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