Application of miR-1260b in improving metabolism and inhibiting obesity

Abstract

The invention provides an adipocyte for overexpressing hsa-miR-1260b, and further discloses application of the hsa-miR-1260b or a simulant of the hsa-miR-1260b or the adipocyte, and the application comprises: A) application in inhibiting adipogenesis and differentiation, B) application in inhibiting adipogenesis and differentiation, C) application in inhibiting adipogenesis and differentiation, D) application in inhibiting adipogenesis and differentiation, and E) application in inhibiting adipogenesis and differentiation; the invention relates to application of hsa-miR-1260b in preparation of a medicine for treating fat metabolism diseases, in particular to application of hsa-miR-1260b in preparation of a medicine for treating fat metabolism diseases, in particular to application of hsa-miR-1260b in preparation of a medicine for treating fat metabolism diseases, in particular to application of hsa-miR-1260b in preparation of a medicine for treating fat metabolism diseases, in preparation of a medicine for treating fat metabolism diseases and application of hsa-miR-1260b in preparation of a medicine for treating fat metabolism diseases from differentiation to metabolism.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to miR-1260b for improving metabolism and inhibiting obesity. Background Technology

[0002] MicroRNAs (miRNAs) have become important epigenetic regulators, controlling various cellular processes including proliferation, apoptosis, stem cell self-renewal, and differentiation. In metabolism, miRNAs function as key regulators of energy balance and metabolic immune homeostasis, and their dysregulation is increasingly linked to obesity and related diseases. They play a crucial role in controlling the function of mature adipocytes (such as lipolysis, glucose uptake, and insulin sensitivity), and as key regulators of adipogenesis, they can exert both pro- and anti-adipogenic effects.

[0003] Furthermore, microRNA expression is typically controlled by upstream cis-regulatory elements. Super-enhancer (SE) are regulatory elements composed of clusters of enhancers with high transcriptional activity and significant cell type specificity. The crucial role of SE in cell fate has been widely established. Crucially, a landmark study (Suzuki et al., Super-Enhancer-Mediated RNA Processing Revealed by Integrative MicroRNA Network Analysis, Cell 168(6):1000-1014 e1015, 2017) revealed that SE regulates a cell type-specific miRNA network, demonstrating that SE-driven miRNAs (SE-miRNAs) are a major driver of cell characteristics. This concept provides a powerful framework for identifying key regulators of cell fate determination. By focusing on SE-miRNAs, the master regulators controlling adipogenesis can be precisely located, providing a novel and targeted approach for discovering interventions to treat obesity. Summary of the Invention

[0004] This invention screens a microRNA (miRNA) epigenetically regulated by a super enhancer (SE) that can act as a molecular switch to change adipocyte fate from differentiation to metabolism. This application employs a combination of epigenetics, functional biology, and proteomics to demonstrate, from multiple dimensions, the coordinated dual regulatory program of hsa-miR-1260b, which simultaneously inhibits adipogenesis and activates lipid catabolism, achieving a potent anti-adipogenic function. The specific scheme is as follows: In a first aspect, the present invention provides adipocytes overexpressing hsa-miR-1260b.

[0005] Adipocytes overexpressing hsa-miR-1260b were obtained by introducing an hsa-miR-1260b mimic into adipocytes.

[0006] The hsa-miR-1260b analogue includes a justice chain and an antisense chain; The justice chain includes 5'-AUCCCACCACUGCCACCAU-3' (SEQ ID NO: 1); The antisense strand includes 5'-GGUGGCAGGUGGGAUUU-3' (SEQ ID NO: 2).

[0007] The importation refers to transformation, transduction, or transfection.

[0008] In the adipocytes overexpressing hsa-miR-1260b, the expression level of hsa-miR-1260b was increased by at least 2-fold, at least 10-fold, and at least 10-fold compared to before overexpression. 2 times, at least 10 3 times, at least 10 4 times, at least 10 5 times, at least 10 6 times or at least 5×10 7 times.

[0009] The adipocytes mentioned are adipose-derived stem cells, preferably human adipose-derived stem cells.

[0010] The adipocytes mentioned can be autologous or allogeneic.

[0011] In a second aspect, the present invention provides a method for preparing adipocytes overexpressing hsa-miR-1260b, the method comprising introducing an hsa-miR-1260b mimic into adipocytes to obtain adipocytes overexpressing hsa-miR-1260b.

[0012] The preparation method includes introducing a vector containing hsa-miR-1260b into adipocytes.

[0013] The vector can be a viral vector or a non-viral vector.

[0014] The viral vectors include, for example, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, or cytomegalovirus vectors.

[0015] The non-viral carriers include, for example, liposomes or lipid nanoparticles.

[0016] A third aspect of the present invention provides an application of hsa-miR-1260b or its mimicry, or an adipocyte overexpressing hsa-miR-1260b, or the adipocytes described in the first aspect, said application comprising: A) Applications in inhibiting adipogenesis and differentiation; for example, reducing the expression or function of FABP4, PPARG, ADIPOQ, ME1 and / or ACLY, and / or inhibiting the KEAP1-NFE2L2 stress response pathway.

[0017] B) Applications in activating lipid catabolism; for example, activating or upregulating ABHD5, ATGL, HADHA and / or HADHB proteins.

[0018] C) Use in the preparation of health products for weight control or weight loss, preferably for weight control or weight loss in non-disease subjects, and / or D) Use in the preparation of medicines for the treatment and / or prevention of lipid metabolism disorders.

[0019] The hsa-miR-1260b analogue includes a justice chain and an antisense chain; The justice chain includes 5'-AUCCCACCACUGCCACCAU-3' (SEQ ID NO: 1); The antisense strand includes 5'-GGUGGCAGGUGGGAUUU-3' (SEQ ID NO: 2).

[0020] The lipid metabolism disorders mentioned include hyperlipidemia, atherosclerosis, fatty liver, diabetes (such as type 2 diabetes), or obesity.

[0021] A fourth aspect of the present invention provides a method for inhibiting adipogenesis and / or activating lipid metabolism, the method comprising introducing an hsa-miR-1260b mimic into adipocytes, or administering an effective amount of adipocytes overexpressing hsa-miR-1260b to a subject in need, or administering an effective amount of a reagent or carrier overexpressing hsa-miR-1260b to a subject in need.

[0022] A fifth aspect of the present invention provides a method for treating and / or preventing lipid metabolism disorders, the method comprising introducing an hsa-miR-1260b mimic into adipocytes, or administering an effective amount of adipocytes overexpressing hsa-miR-1260b to a subject in need, or administering an effective amount of a reagent or carrier overexpressing hsa-miR-1260b to a subject in need.

[0023] In a sixth aspect, the present invention provides a method for controlling weight or losing weight, the method comprising introducing an hsa-miR-1260b mimic into adipocytes, or administering an effective amount of adipocytes overexpressing hsa-miR-1260b to a subject in need, or administering an effective amount of a reagent or carrier overexpressing hsa-miR-1260b to a subject in need.

[0024] The miRNA "mimic" or "miRNA" described in this invention is a class of chemically synthesized double-stranded RNA molecules that mimic endogenous miRNAs. Its sequence is identical to the mature form of the target miRNA, and it can enhance the function of existing miRNAs within the cell. Generally, it enters the cell through transfection and participates in regulating target gene expression, with a mechanism of action similar to that of natural miRNAs.

[0025] The "obesity" described in this invention is a lipid metabolism disorder, specifically defined as a body fat percentage exceeding 20% ​​of the ideal body weight.

[0026] The "weight control or weight loss" described in this invention includes weight control or weight loss for obesity, as well as weight control or weight loss for healthy individuals who have not reached the level of obesity but are doing so for aesthetic reasons, etc. Preferably, it refers to weight control or weight loss for healthy individuals who have not reached the level of obesity but are doing so for aesthetic reasons, etc.

[0027] The "method" described in this invention can be used for the purpose of diagnosing or treating a disease, or for the purpose of diagnosing or treating a non-disease.

[0028] The term "treatment" as used in this invention refers to slowing down, interrupting, preventing, controlling, stopping, alleviating, reducing, or reversing a sign, symptom, disorder, condition, or progression or severity of a disease after it has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0029] The term "effective amount" as used in this invention refers to the amount or dose of hsa-miR-1260b or adipocytes of this invention that provides the desired treatment or prevention after being administered to a subject in one or more doses.

[0030] The term "prevention" as used in this invention refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in the body.

[0031] The "subject" described in this invention can be a human or a non-human mammal, or a cell, tissue, or organ of a human or non-human mammal. The non-human mammal can be a wild animal, a zoo animal, an economically important animal, a pet, a laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), or monkeys, etc. Attached Figure Description

[0032] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein: Figure 1 qRT-PCR analysis verified the transfection efficiency. The results showed that hsa-miR-1260b was significantly overexpressed in hASC compared with the normal control (NC). Unpaired two-tailed Student's t-test was used to confirm the significance, where **** represents P < 0.0001. Data are expressed as mean ± standard deviation.

[0033] Figure 2 Representative Oil Red O staining (ORO) images of hASCs transfected with NC, hsa-miR-1260b mimic, or hsa-miR-1260b inhibitor during adipogenesis. Scale bar represents 100 µm.

[0034] Figure 3 qRT-PCR analysis was performed on key adipogenesis markers (PPARG, ADIPOQ, and FABP4) in the three groups. Unpaired two-tailed Student's t-test was used to determine significance, where * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001. Data are expressed as mean ± standard deviation.

[0035] Figure 4 Western blot analysis was performed on key lipogenesis markers (PPARG, ADIPOQ, and FABP4) in the three groups. Actin was used as a normalized internal control.

[0036] Figure 5 hsa-miR-1260b mediates global proteomic reprogramming in adipocytes, specifically the total number of gene products identified in each group.

[0037] Figure 6 A quantitative proteomics data overview showing the dynamic changes in the intensity of proteins after logarithmic transformation in the NC group, hsa-miR-1260b mimic group, and hsa-miR-1260b inhibitor group.

[0038] Figure 7 Principal component analysis (PCA) showed significant differences among the three proteomic profiles, indicating that hsa-miR-1260b regulation significantly altered the overall protein expression profile.

[0039] Figure 8Volcano plots show differentially expressed proteins (DEPs) in the hsa-miR-1260b mimic compared to NC (n=3 per group). Red dots represent significantly upregulated proteins, and blue dots represent significantly downregulated proteins (P<0.05; fold change>1.5).

[0040] Figure 9 The volcano plot shows the DEP (differences per unit number) of the hsa-miR-1260b mimic compared to the inhibitor (n=3 per group). Red dots represent significantly upregulated proteins, and green dots represent significantly downregulated proteins (P<0.05; fold change>1.5).

[0041] Figure 10 The Venn diagram identified 423 DEPs that were co-regulated in both comparisons, representing the core proteome affected by hsa-miR-1260b.

[0042] Figure 11 The scatter plot shows the expression trends of these 423 common DEPs. Proteins with sustained upregulation of hsa-miR-1260b are shown in red, while proteins with sustained downregulation are shown in blue.

[0043] Figure 12 Enrichment analysis of the KEGG and GO pathways was performed on DEP, which is continuously regulated. Bar charts show the most significantly enriched pathways, with pathways associated with upregulated proteins shown in red and those associated with downregulated proteins shown in blue. Unpaired two-tailed Student's t-tests were used to determine significance.

[0044] Figure 13 : Targeted analysis of differentially expressed proteins involved in lipid metabolism to elucidate the specific metabolic pathways regulated by hsa-miR-1260b. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] 1. Materials and steps used in cell culture and adipogenesis differentiation in the examples: hASCs were cultured in OriCell™ complete medium (Cyagen Biosciences, Guangzhou, China; catalog number: HUXMD-90011). The complete medium was prepared by mixing OriCell™ basal cell culture medium (catalog number: BHDM-03011) and OriCell™ fetal bovine serum + medium supplement (catalog number: HUXMD-05001) according to the manufacturer's instructions. In short, the supplement was thawed at 4°C for at least 6 hours, added to the basal medium, and then the mixture was gently homogenized. The prepared complete medium was stored at 4°C and used within one month. Cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0047] For adipogenesis differentiation, hASC was 2×10 4 cells / cm 2 The cells were inoculated at a density in culture vessels pre-coated with 0.1% gelatin (OriCell™, catalog number: GLT-11301) and grown to 100% confluence. The growth medium was then replaced with adipogenic differentiation medium, which consisted of two solutions used alternately. Solution A (induction medium) was prepared by mixing OriCell™ basal medium (catalog number: BLDM-03011), OriCell™ advanced fetal bovine serum (catalog number: FBSSR-01021), and OriCell™ supplements (catalog number: HUXMD-04031-a1) and A-II (catalog number: HUXMD-04031-a2) for adipogenic differentiation AI of human adipose-derived MSCs. Solution B (maintenance medium) was prepared by mixing OriCell™ basal medium (catalog number: BLDM-03011), OriCell™ advanced fetal bovine serum (catalog number: FBSSR-01021), and OriCell™ supplement for adipogenic differentiation B of human adipose-derived MSCs (catalog number: HUXMD-04031-b). All reagents were from Cyagen Biosciences. The differentiation protocol involved culturing cells in solution A for 3 days, followed by culturing in solution B for 1 day. This cycle was repeated for 7 to 10 days, changing the medium every 2 to 3 days, until sufficient lipid droplet formation was observed.

[0048] The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Review Committee of the Capital Institute of Pediatrics (Program No. SHERLLM2023051).

[0049] 2. RNA extraction and quantitative real-time PCR (qRT-PCR) To analyze hsa-miR-1260b, total RNA was extracted from hASCs using Trizol reagent (catalog number: 15596018, Invitrogen, USA) according to the manufacturer's instructions. hsa-miR-1260b cDNA synthesis was performed using the Super Script II Reverse Transcription Kit (catalog number: 18064014, Invitrogen, USA), with the reverse transcription reaction specifically performed using Bulge-Loop™ miRNA qRT-PCR primers (RiboBio, China). The relative expression level of hsa-miR-1260b was quantified using U6 (RiboBio, China) as an internal control. Quantitative real-time PCR was performed using SYBR Green Master Mix (catalog number: RR036A, Takara, Dalian, China) on an ABI-7900HT real-time PCR system (Applied Biosystems), with each sample repeated three times.

[0050] To analyze mRNA expression, total RNA was isolated using TRIzol reagent, and cDNA was synthesized using the SuperScript II Reverse Transcription Kit (catalog number: 18064014, Invitrogen, USA) according to the manufacturer's instructions. qRT-PCR reactions were performed using a DNA-free kit (Ambion, Austin, Texas, USA) and SYBR® Premix Ex Taq™ (Takara, Japan), with each sample repeated three times. The following primers were used to amplify the target gene: PPARG (forward primer: 5'-AGCCCTTCACTACTGTTGACTTCT-3' (SEQ ID NO: 3), reverse primer: 5'-TCCACTTTGATTGCACTTTGG-3' (SEQ ID NO: 4)); ADIPOQ (forward primer: 5'-CTGGCTGAGTTGTGTGCCTGT-3' (SEQ ID NO: 5); reverse primer: 5'-GTCCCAATTTTGCTTTAGGGAA-3' (SEQ ID NO: 6)); FABP4 (forward primer: 5'-CTTCCACGAGAGTTTATGAGAGAGC-3' (SEQ ID NO: 7); reverse primer: 5'-CAGAATGTTGTAGAGTTCAATGCGA-3' (SEQ ID NO: 8)); and ACTB1 (forward primer: 5'-TCAAGATCATTGCTCCTCCTGAG-3' (SEQ ID NO: 4)). NO: 9); reverse primer: 5'-ACATCTGCTGGAAGGTGGACA-3' (SEQ ID NO: 10) was used as an internal reference gene. The relative expression level of the target gene was calculated using the 2-ΔΔCt method.

[0051] 3. Oil Red O staining Following the adipogenesis induction phase, lipid droplet formation was assessed using Oil Red O staining. Cells were gently washed twice with 1×PBS (OriCell™, catalog number: PBS-10001) and fixed with 4% paraformaldehyde for 30 minutes at room temperature. Oil Red O working solution was prepared by mixing OriCell™ Oil Red O solution (catalog number: OILR-10001) with distilled water at a ratio of 3:2, centrifuging at 250×g for 4 minutes, collecting the supernatant, and freshly preparing the solution. Fixed cells were washed twice with PBS, incubated with Oil Red O working solution at room temperature for 30 minutes, and then gently washed 2 to 3 times with PBS to remove excess dye. The stained cells were observed under a microscope, and images were captured for recording.

[0052] 4. Protein separation and Western blot analysis Cultured cells were washed twice with ice-cold PBS and then lysed with 0.5% NP-40 buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Nonidet P-40, and a mixture of protease inhibitors (Sigma-Aldrich, St. Louis, Missouri, USA). After centrifugation at 12,000 rpm and 4°C for 15 minutes, the supernatant (lysate) was collected for Western blot analysis. Protein concentration in the lysate was determined using the BCA Protein Assay Kit (Catalog No.: P0012, Beyotime, Shanghai, China). Subsequently, proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a 0.45 μm diameter polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was then blocked at room temperature with 5% BSA in TBST solution for 1 hour and incubated overnight at 4°C with primary antibody. The primary antibodies used were: anti-PPARG (catalog number: #2430S, Cell Signaling), anti-ADIPOQ (catalog number: #2789S, Cell Signaling), anti-FABP4 (catalog number: #50699S, Cell Signaling), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number: #10494, Proteintech). After washing, the membrane was incubated with HRP-labeled secondary antibodies. Immunoblotting was visualized using an enhanced chemiluminescence (ECL) kit (catalog number: P0018S, Beyotime Biotechnology Co., Ltd., Shanghai, China) and a ChemiDoc XRS system equipped with Quantity One software (Bio-Rad, Hercules, CA). Finally, the bands on the immunoblot were quantitatively analyzed by density analysis.

[0053] 5. Protein extraction and digestion Samples were lysed at 95°C for 5 min in a buffer containing 1% sodium deoxycholate, 100 mM Tris hydrochloride (pH 8.8), 100 mM Tris (2-carboxyethyl)phosphine (TCEP), and 400 mM 2-chloroacetamide (CAA). The initial lysate was sonicated under appropriate conditions. The lysate was centrifuged at 16,000 × g for 10 min at 4°C, and the supernatant was retained as whole-cell extract (WCE). Protein concentration was determined using NanoDrop One. Approximately 40 μg of WCE was digested with sequencing-grade trypsin (V5280, Promega), which cleaves the C-terminus of Arg or Lys residues at 37°C.

[0054] 6. Proteomics analysis 1) Mass spectrometry analysis MS samples were analyzed on a Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific), which was connected to an Easy-nLC 1200 nanofluid chromatography system (Thermo Fisher Scientific). Briefly, the sample was redissolved in 30 µl of solvent A (0.1% formic acid aqueous solution), and one-fifth of the reconstituted sample was loaded onto a self-made reversed-phase C18 column (1 cm × 150 μm; particle size 3 μm; pore size 300 Å). Separation was then performed using a 150 μm × 30 cm silica microcolumn (self-made; particle size 1.9 μm; pore size 120 Å). The mobile phase B (0.1% formic acid acetonitrile solution) had a linear gradient of 5% to 35%, a flow rate of 600 nl / min, and a separation time of 150 min. A data-dependent strategy was employed, measuring MS1 at a resolution of 120,000 in Orbitrap, followed by tandem MS scans of the first 30 precursors using higher-energy collisional dissociation with a normalized collision energy of 27% and a dynamic exclusion time of 30 seconds. Trypsin digests from 293T cells were used as quality control samples for routine analysis to ensure good sensitivity and reproducibility.

[0055] 2) Protein identification and quantification MS data files were processed using Proteome Discoverer version 2.1 (Thermo Fisher Scientific, Bremen, Germany). MS / MS spectra were searched using the MASCOT engine and compared with the NCBI Human Reference Proteome Database (containing 32,015 protein entries, updated July 1, 2013). Search parameters were as follows: enzyme specificity was set to trypsin, allowing for two missed digestions. Fixed modifications were carbamoyl methylation of cysteine; variable modifications were methionine oxidation and N-terminal acetylation of proteins. Precursor mass tolerance was set to 20 ppm; fragment mass tolerance was set to 50 mmu. The maximum false discovery rate (FDR) for protein and peptide identification was set to 1%. Label-free, intensity-based absolute quantification (iBAQ) was used, with protein quantification calculated based on the area under the curve (AUC) of the precursor ion. The proportion of total (FOT) was used to represent the normalized abundance of a protein in each experiment. FOT was defined as the iBAQ of a protein in a single experiment divided by the total iBAQ of all identified proteins. For ease of representation, FOT was multiplied by 10. 5 To obtain iFOT. Missing values ​​are replaced with zero.

[0056] 7. Statistical Analysis All other analyses were performed in R (version 4.0.5). Principal component analysis (PCA) was performed using the R package factoextra to determine spatial segregation of the samples. Gene ontology (GO) term enrichment analysis was conducted using the DAVID (Annotation, Visualization, and Synthetic Discovery Database) bioinformatics resource. Statistical significance of this enrichment was determined using Fisher's exact test, and pathways with an FDR threshold of 0.05 were considered to have significant regulatory effects. Differentially expressed genes were identified using unpaired two-tailed Student's t-tests. All quantitative data are expressed as mean ± standard deviation. P < 0.05 was considered statistically significant.

[0057] Example 1: hsa-miR-1260b as a negative regulator of adipogenesis and differentiation 1. Transfecting hsa-miR-1260b mimic, hsa-miR-1260b inhibitor and NC into hASC cells Subfusion-bound hASCs were washed with PBS and incubated with trypsin / EDTA. The cells were then resuspended, centrifuged at 500g for 5 minutes to collect the pellet, and resuspended in OriCell™ basal medium (Catalog No.: HUXMD-90011, Cyagen). For transfection, hASCs were incubated at 1.5 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of [number] cells / well in 6-well plates, and cell confluence reached approximately 60% after about 24 hours. Using Lipofectamine™ RNAiMAX transfection reagent (catalog number: 13778150, Invitrogen), hsa-miR-1260b mimic, hsa-miR-1260b inhibitor, and miRNA mimic normal control (NC) (RiboBio, Guangzhou, China) were transfected according to the manufacturer's instructions, with a final miRNA concentration of 50 nM. After transfection, cells were cultured in a humidified environment at 37°C and 5% CO2. RNA was extracted 36 hours post-transfection to assess transfection efficiency. The sequences used are as follows: hsa-miR-1260b mimic (sense strand: 5'-AUCCCACCACUGCCACCAU-3' (SEQ ID NO: 1), antisense strand: 5'-GGUGGCAGGUGGGAUUU-3' (SEQ ID NO: 2)), and hsa-miR-1260b inhibitor (sense strand: 5'-AUGGUGGCAGUGGUGGGAU-3' (SEQ ID NO: 11)). After the first transfection, adipogenesis was induced only after the cells reached a confluence state. A second transfection was performed 3 days after differentiation. After adipogenesis induction was completed, cells were collected for subsequent analysis of adipogenesis markers at the mRNA, protein, and proteomic levels.

[0058] 2. Results qRT-PCR confirmed that hsa-miR-1260b mimic transfection achieved high transfection efficiency, with significantly increased hsa-miR-1260b levels compared to the NC group. Figure 1 ).

[0059] The effect of hsa-miR-1260b on adipogenesis was assessed using Oil Red O staining. Significant morphological differences existed among the groups. Overexpression of hsa-miR-1260b resulted in a significant reduction in the number and size of lipid droplets. Conversely, inhibition of endogenous hsa-miR-1260b did not lead to a significant increase in lipid accumulation compared to the NC group. Figure 2 This phenotypic evidence strongly suggests that hsa-miR-1260b inhibits hASC differentiation into adipocytes, and that under these conditions, inhibiting hASC alone is insufficient to significantly promote adipogenesis.

[0060] Further molecular-level confirmation confirmed the consistency between qRT-PCR and Western blot analysis results: overexpression of hsa-miR-1260b significantly reduced the mRNA and protein levels of PPARG and ADIPOQ, and significantly reduced the mRNA level of FABP4. Consistent with phenotypic data, inhibition of hsa-miR-1260b did not lead to a significant upregulation of these same markers compared to the NC group. Figure 3-4 ).

[0061] In summary, these results indicate that hsa-miR-1260b can act as a potent negative regulator of adipogenesis and differentiation, playing a role at both the phenotypic and molecular levels.

[0062] Example 2: Proteomic analysis of the regulation of lipid metabolism by hsa-miR-1260b This example demonstrates unbiased quantitative proteomics analysis of hASCs transfected with hsa-miR-1260b mimics, inhibitors, or NC (as in Example 3) followed by adipogenesis induction. High-confidence identification was performed on 7,064 proteomes (GPs), each containing at least one unique peptide that was quantified. 5,088, 4,747, and 4,930 GPs were identified in the NC, mimic, and inhibitor groups, respectively. Figure 5 The relative abundance of these proteins spans approximately eight orders of magnitude, highlighting significant proteomic changes during hsa-miR-1260b-induced adipogenesis. Figure 6 ).

[0063] To assess the overall impact of hsa-miR-1260b regulation, PCA analysis was performed. This analysis revealed that, based on protein expression profiles, the NC group, the mimic group, and the inhibitor group formed three distinct and separate clusters. Figure 7The results showed that both overexpression and inhibition of hsa-miR-1260b caused significant, holistic changes in the adipocyte proteome.

[0064] Then, differentially expressed proteins (DEPs) were identified by comparing the mimic group with the NC group and with the inhibitor group. Compared with NC, hsa-miR-1260b overexpression led to the upregulation of 944 proteins and the downregulation of 980 proteins. Figure 8 Compared to the inhibitor group, overexpression resulted in the upregulation of 271 proteins and the downregulation of 413 proteins. Figure 9 To determine the most reliable core target of hsa-miR-1260b, the intersection of the two DEP groups was identified. The Venn diagram showed overlap in 423 proteins that exhibited persistent changes in both comparisons. Figure 10 The core proteome consists of 181 proteins that are continuously upregulated and 226 proteins that are continuously downregulated. Figure 11 This constitutes a central control network controlled by hsa-miR-1260b.

[0065] To elucidate the biological function of this core proteome, KEGG and GO pathway enrichment analyses were performed. These analyses revealed a clear dual regulatory role for hsa-miR-1260b. It strongly activates pathways related to lipid catabolism, including lipid metabolism, lysosomal metabolism, and cholesterol metabolism. Simultaneously, hsa-miR-1260b inhibited several cellular stress response pathways, most notably the KEAP1-NFE2L2 pathway. Inhibition of this key antioxidant pathway suggests that hsa-miR-1260b may disrupt cellular defense mechanisms required for adipogenesis differentiation, thereby leading to its overall inhibitory effect. Figure 12 ).

[0066] To further elucidate the specific metabolic pathways regulated by hsa-miR-1260b, a targeted analysis of differentially expressed proteins involved in lipid metabolism was performed. The results revealed a clear and coordinated reprogramming of the cellular lipid profile. Specifically, hsa-miR-1260b overexpression led to a significant downregulation of key proteins essential for adipogenesis and lipogenesis. These included FABP4 and FABP3 (crucial for intracellular fatty acid transport and hallmarks of mature adipocytes) and core lipogenic enzymes such as ACLY (ATP-citrate lyase) and ME1 (malate 1), which provide necessary structural units and reducing power for fatty acid synthesis. Conversely, key proteins in lipid catabolism were also significantly upregulated. Notably, ABHD5 (α / β hydrolase domain protein 5)—a potent coactivator of triglyceride lipase (ATGL), which initiates triglyceride breakdown—was strongly induced. Furthermore, key enzymes in mitochondrial fatty acid β-oxidation (including HADHA and HADHB) were also upregulated (see [link to study]). Figure 13 ).

[0067] In summary, proteomics analysis not only confirmed the profound reprogramming of the adipocyte proteome by hsa-miR-1260b, but also revealed the molecular mechanism of its dual function: inhibiting adipogenesis and differentiation by downregulating related proteins, while promoting lipid catabolism by upregulating metabolic pathways.

[0068] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0069] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A fat cell overexpressing hsa-miR-1260b.

2. The adipocytes according to claim 1, characterized in that, The fat cell overexpressing hsa-miR-1260b is obtained by introducing a hsa-miR-1260b mimic into a fat cell.

3. The adipocytes according to claim 2, characterized in that, The hsa-miR-1260b mimic comprises a sense strand and an antisense strand; The sense strand comprises 5'-AUCCCACCACUGCCACCAU-3' (SEQ ID NO: 1); The antisense strand comprises 5'-GGUGGCAGGUGGGAUUU-3' (SEQ ID NO: 2).

4. The adipocytes of claim 2, wherein, The introduction is transformation, transduction or transfection.

5. The adipocytes of claim 1, wherein, The expression amount of hsa-miR-1260b in the adipocyte overexpressing hsa-miR-1260b is increased by at least 2 times, at least 10 times, at least 10 2 times, at least 10 3 times, at least 10 4 times, at least 10 5 times, at least 10 6 times, or at least 5 x 10 7 times, compared to before overexpression.

6. The fat cell according to any one of claims 1 to 5, characterized in that, The fat cell is a fat stem cell, preferably a human fat stem cell.

7. Use of hsa-miR-1260b or a mimic thereof or of fat cells overexpressing hsa-miR-1260b or of the fat cells according to any one of claims 1 to 6, characterized in that, The use comprises: A) use in inhibiting lipogenic differentiation; B) use in activating lipolytic metabolism; C) use in preparing a health care product for controlling body weight or losing weight, and / or D) use in preparing a medicament for treating a lipid metabolism disease.

8. Use according to claim 7, characterized in that, The hsa-miR-1260b mimic comprises a sense strand and an antisense strand; The sense strand comprises 5'-AUCCCACCACUGCCACCAU-3' (SEQ ID NO: 1); The antisense strand comprises 5'-GGUGGCAGGUGGGAUUU-3' (SEQ ID NO: 2).

9. Use according to claim 7, characterized in that, The lipid metabolism disease comprises hyperlipidemia, atherosclerosis, fatty liver, diabetes (e.g., type 2 diabetes) or obesity.

10. A method of inhibiting adipogenic differentiation and / or activating lipolytic metabolism, characterized in that, The method comprises introducing a hsa-miR-1260b mimic into a fat cell.

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