Application of AGGF1 as a target in modification of ADSCs stemness and treatment of obesity-related diseases

Abstract

The application discloses application of AGGF1 as a target point in modification of ADSCs stemness and treatment of obesity-related diseases and belongs to the technical field of biological medicine, wherein AGGF1 activates a Hippo signal path and reduces ADSCs stemness and promotes fat differentiation by combining with its receptors integrin alpha7beta1 and alpha7beta2, so that AGGF1 knockout or knockdown can be used as a brand-new potential strategy for obesity treatment, and the effect of ADSCs-based regenerative medicine therapy can be optimized by maintaining ADSCs stemness.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of angiogenesis factor AGGF1 as a target in the modification of adipose-derived stem cells (ADSCs) and the treatment of obesity-related diseases. Background Technology

[0002] Obesity refers to the excessive accumulation of adipose tissue and is associated with an increased risk of diabetes, cardiovascular disease, hyperlipidemia, cancer, and other diseases. Currently, drug therapy is the primary treatment for obesity, but due to the side effects and long-term dependence associated with weight-loss drugs, there is a constant search for more effective solutions. In recent years, emerging stem cell therapy, which directly replaces damaged and non-functional cells, has been successfully applied to treat obesity and related metabolic diseases.

[0003] Adipose-derived stem cells (ADSCs) are adult stem cells that maintain the function and homeostasis of adipose tissue. Abnormal function of ADSCs can have harmful effects on human health and is associated with various diseases such as obesity and cancer. ADSCs belong to the pluripotent mesenchymal stem cell (MSC) family and can differentiate into various cell lineages, including adipocytes, chondrocytes, osteoblasts, neuron-like cells, cardiomyocytes, and endothelial cells. Based on this characteristic, ADSCs have been extensively explored in regenerative medicine research for various diseases, including osteoarthritis, congenital muscular dystrophy, Parkinson's disease, cardiovascular disease, chronic pancreatitis, and wound repair. Furthermore, ADSCs can secrete growth factors and extracellular vesicles, promoting the differentiation and phenotypic maturation of various cell populations, thereby achieving the repair of various damaged organs and tissues. Adipose-derived stem cell therapy has extremely high clinical application value, with advantages including convenient acquisition, efficient isolation from various tissues (e.g., subcutaneous liposuction), rapid in vitro expansion, safe and minimally invasive operation, multi-lineage differentiation potential, low immunogenicity, and fewer ethical constraints. Loss of stemness in ADSCs derived from subcutaneous white adipose tissue in obese individuals promotes the differentiation of these cells into adipocytes, while the expression levels of inflammation-related genes are upregulated with the loss of stemness. Therefore, maintaining the stemness function of ADSCs (maintaining their undifferentiated state) becomes particularly important.

[0004] Upon receiving specific signals, adipocyte lineage commitment occurs, irreversibly committing to differentiating into a specific cell lineage from a pluripotent "ready" state. Preadipocyte proliferation and further differentiation then produce mature adipocytes. This subsequent process is known as adipogenesis. Both lineage determination and adipogenesis can influence the formation and expansion of white adipose tissue (WAT), and obesity is caused by excessive WAT expansion. Currently, most research focuses on the adipogenesis process, but the molecular mechanisms behind lineage determination remain unclear. A deeper understanding of the mechanisms of adipocyte lineage determination may lay the foundation for developing novel regenerative therapeutic strategies, thereby alleviating obesity and other diseases.

[0005] The hippopotamus signaling pathway is an evolutionarily conserved pathway that plays a crucial role in organ size regulation, tissue regeneration, and tumor suppression. YES-associated protein (YAP) and transcriptional coactivator containing a PDZ-binding motif (TAZ) are the main downstream effector molecules of the hippopotamus signaling pathway. As key regulators of tissue regeneration and size, YAP / TAZ, as core components of the stem cell circuit, are fundamentally significant in regulating stem cell renewal, fate, and plasticity. Studies have shown that YAP and TAZ are indispensable for maintaining the pluripotency and self-renewal of embryonic stem cells.

[0006] AGGF1 is an angiogenesis factor, and mutations in it increase the risk of Klinefelter syndrome (KTS), a vascular disease. Previous research demonstrated that AGGF1 is the earliest regulator of mesodermal angiogenesis blast differentiation, participating in the differentiation of hematopoietic and endothelial lineages in zebrafish. Furthermore, AGGF1 promotes the proliferation, migration, and angiogenesis of endothelial progenitor cells in diabetic mice by activating the AKT / Fyn / Nrf2 pathway, thereby promoting vascular defect repair. However, the role of AGGF1 in the homeostasis regulation of ADSCs remains unclear. Summary of the Invention

[0007] Based on the novel discovery that AGGF1 can significantly regulate the homeostasis of adipose-derived stem cells, this invention provides a novel strategy for treating obesity by modifying the stemness of adipose-derived stem cells.

[0008] In a first aspect, the present invention provides the application of AGGF1 as a target in the preparation of reagents that modify the stemness of adipose-derived stem cells, wherein the reagents can maintain or inhibit the stemness of adipose-derived stem cells.

[0009] Specifically, when the reagent contains substances that inhibit AGGF1 expression (such as siRNA, shRNA, and / or antisense oligonucleotides that knock out or reduce AGGF1 expression), the reagent can be used to maintain the stemness of adipose-derived stem cells; when the reagent contains substances that upregulate AGGF1 expression (such as recombinant AGGF1 protein), the reagent can be used to inhibit the stemness of adipose-derived stem cells.

[0010] Secondly, the present invention provides the application of AGGF1 as a target in the preparation of a drug for treating obesity-related diseases, the drug containing at least a substance that inhibits the expression of AGGF1, and the drug achieves the treatment of obesity-related diseases by inhibiting the adipogenic differentiation of preadipocytes; wherein obesity-related diseases include, but are not limited to, obesity.

[0011] Preferably, in the above applications, the substance that inhibits AGGF1 expression is a siRNA, shRNA, and / or antisense oligonucleotide that knocks out or reduces AGGF1 expression.

[0012] Preferably, in the above applications, the drug further includes one or more of the following substances: Substances that knock down integrin α7, β1 and / or β2; Substances that increase TAZ expression and / or promote TAZ nuclear localization.

[0013] Preferably, in the above applications, the drug also includes pharmaceutically acceptable excipients.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a novel treatment strategy for obesity, namely, using AGGF1 as a target to modify the stem cells of adipose-derived stem cells to treat obesity and other related diseases. Furthermore, this invention elucidates in detail the relationship between AGGF1 and TAZ in the development of obesity, providing a theoretical basis for precise intervention. Simultaneously, the embodiments of this invention effectively demonstrate that targeting the AGGF1-TAZ signaling pathway has a significant therapeutic effect on obesity, thus laying the foundation for the subsequent development of commercial therapeutic drugs based on the AGGF1-TAZ signaling pathway. Attached Figure Description

[0015] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0016] Figure 1 The target disclosed in this invention AGGF1 A mechanistic diagram that could serve as a potential strategy for obesity treatment; Figure 2 Under the normal diet (NCD) conditions in Example 1 Aggf1Results of subcutaneous adipose tissue weight and adipocyte size detection in knockout mice, where A shows a significant increase in AGGF1 in white adipose tissue (WAT) of obese patients, B shows the Western blot results of AGGF1 protein in subcutaneous adipose tissue (iWAT) of obese mice, and C shows... Aggf1 Western blot results of knockout mice, D is Aggf1 The subcutaneous fat weight / body weight ratio of knockout mice, E represents the hematoxylin-eosin (H&E) staining result of mouse subcutaneous adipose tissue, and F represents the value calculated based on the H&E staining result. Aggf1 Changes in the average area of ​​adipocytes in knockout mice, where G represents statistical analysis of adipocytes with different areas; Figure 3 Under the high-fat diet (HFD) conditions in Example 1 Aggf1 Results of subcutaneous adipose tissue weight and adipocyte size detection in knockout mice, where A represents... Aggf1 Changes in body weight in knockout mice, B being... Aggf1 The subcutaneous fat weight / body weight ratio of knockout mice, C is Aggf1 Changes in the average area of ​​subcutaneous adipocytes in knockout mice. D represents H&E staining of subcutaneous adipose tissue in mice, and E represents statistical analysis of adipocytes with different areas. Figure 4 In Example 2 Aggf1 Transcriptome sequencing results of subcutaneous adipose tissue from knockout mice, where AB represents GO enrichment analysis of differentially expressed genes (DEGs), C represents KEGG pathway analysis of downregulated DEGs, and D represents KEGG pathway analysis of upregulated DEGs. Figure 5 In Example 2 Aggf1 Validation results related to the dryness of ADSCs, where A is Aggf1 Gene expression analysis for maintaining stem cell stemness in ADSCs of knockout mice, B is... Aggf1 Analysis of stem cell stemness marker expression in ADSCs of knockout mice, C = Aggf1 Immunofluorescence analysis of CD90, a marker in ADSCs of knockout mice, with D representing the immunofluorescence analysis of DLK1, a marker of preadipocytes, in primary ADSCs after induction treatment. Figure 6 Fat-specific knockout in Example 3 Aggf1 The results of the detection of inhibition of adipogenic differentiation are shown in the following figures: A is the result of cell proliferation detected by CCK8, B is the result of cell proliferation detected by EdU, C is the result of Oil Red O staining 8 days after induction of differentiation, and DG are the results of protein expression detection of mature adipocyte markers C / EBPα, PPARγ and FABP4 at different days of adipogenic differentiation detected by Western blot. Figure 7 In Example 4 Aggf1 The results of knockdown inhibition of adipogenic differentiation of 3T3-L1 preadipocytes are shown in the following table: A is the cell proliferation result detected by CCK8, B is the cell proliferation result detected by EdU, C is the Oil Red O staining result 8 days after adipogenic differentiation, and DG are the Western blot results of the expression of mature adipocyte markers C / EBPα, PPARγ and FABP4 in preadipocytes transfected with siAggf1. Figure 8 The results of the verification of the inhibition of ADSC stemness-related effects by recombinant human AGGF1 protein treatment in Example 5 are as follows: A is the qRT-PCR result of the expression level of the gene that maintains ADSC stemness after AGGF1 treatment; B is the qRT-PCR result of the expression level of stem cell marker after AGGF1 treatment; C is the immunofluorescence result of the stem cell marker CD90 after AGGF1 treatment; and D is the immunofluorescence result of the preadipocyte marker DLK1 after AGGF1 treatment. Figure 9 The results of the detection of adipogenic differentiation promoted by recombinant human AGGF1 protein treatment in Example 5 are shown in Figure 5. A is the result of cell proliferation detection by CCK8, B is the result of EdU proliferation detection, C is the result of Oil Red O staining, and DG are the Western blot results of C / EBPα, PPARγ and FABP4 expression during the adipogenic differentiation of ADSCs induced by AGGF1 treatment. Figure 10 The results of the detection of the promotion of 3T3-L1 preadipocyte proliferation and differentiation by recombinant human AGGF1 protein treatment in Example 5 are shown in Figure 5. A is the result of cell proliferation detection by CCK8, B is the result of EdU proliferation detection, C is the result of Oil Red O staining, and DG are the Western blot results of C / EBPα, PPARγ and FABP4 expression after AGGF1 protein treatment, respectively. Figure 11 In Example 6 Aggf1 Knockout or knockdown of TAZ expression upregulates the detection results, where A and C are Aggf1, respectively. ako ADSCs isolated from mice and control mice Taz The qRT-PCR and Western blot results, B and D are respectively transfection... Aggf1 siRNA and negative control siRNA in 3T3-L1 preadipocytes Taz qRT-PCR results and Western blot results; Figure 12 In Example 6 Aggf1The results of knockout or knockdown of ADSCs and 3T3-L1 cell adipogenic differentiation were detected by upregulating TAZ, where A and B were Aggf1, respectively. ako mouse-derived ADSCs and Aggf1 Immunofluorescence staining results of TAZ in knocked-down 3T3-L1 preadipocytes. CD values ​​represent siRNA knockdown 8 days after induction of adipogenic differentiation. Taz Reversible Aggf1 ako mouse-derived ADSCs and Aggf1 Oil Red O staining results of reduced adipogenesis capacity in knocked-down 3T3-L1 preadipocytes, EF representing siRNA knockdown. Taz Reversible Aggf1 ako Mouse-derived ADSCs (day 8 of differentiation) and Aggf1 Western blot results showing reduced expression of PPARγ and FABP4 in knocked-down 3T3-L1 preadipocytes; Figure 13 After treating mouse primary ADSCs or 3T3-L1 preadipocytes with recombinant human AGGF1 protein as described in Example 7 Taz The results of expression and nuclear localization detection, where A and B represent ADSCs and 3T3-L1 preadipocytes, respectively. Taz The results of qRT-PCR for expression, CD and T are Western blot results of Taz in ADSCs and 3T3-L1 preadipocytes, respectively; Figure 14 In Example 7 Taz The results of ADSCs or 3T3-L1 preadipocytes treated with AGGF1 overexpression were shown. AB represents the immunofluorescence staining results of TAZ in ADSCs and 3T3-L1 preadipocytes after AGGF1 treatment, CD represents the Oil Red O staining results of ADSCs and 3T3-L1 preadipocytes under different conditions, and EF represents the Western blot results of ADSCs and 3T3-L1 preadipocytes under different conditions. Figure 15 In vivo knockdown in Example 8 Taz For Aggf1 ako The results of the detection of the effects of mouse iWAT are shown in the following table: A is the qRT-PCR result of gene expression for maintaining stem cell stemness and stem cell markers in mouse iWAT; B is the Western blot result of TAZ in mouse iWAT; C is the immunohistochemical staining result of TAZ in mouse iWAT; and D is the result of Aggf1. ako Changes in the iWAT / body weight ratio in mice and control mice 45 days after injection of AAV2 / 8-shTaz viral vector or control AAV2 / 8-shNC vector; Figure 16 In vivo knockdown in Example 8 Taz Reversible Aggf1 ako The results of detecting the mouse adipogenesis defect phenotype are shown in the following figures: A is the H&E staining result of mouse iWAT, B is the change in the average area of ​​mouse adipocytes, C is the change in the proportion of mouse adipocytes, and D is the Western blot result of PPARγ and FABP4 in mouse iWAT. Figure 17 The results of RNAi screening for integrins affecting adipocyte differentiation in 3T3-L1 preadipocytes in Example 9 are shown in A, where Oil Red O staining shows the effect of 23 integrin subunit gene siRNAs on the differentiation of 3T3-L1 preadipocytes into mature adipocytes (induced for 8 days), and BC are the results of quantitative analysis based on the image data in A. Figure 18 For the RNAi screening in Example 9 Aggf1 The results of integrins mediated by 3T3-L1 preadipocytes during differentiation into mature adipocytes, where A represents Oil Red O staining showing the effects of siRNAs on 23 different integrin subunit genes. Aggf1 The effect of 3T3-L1 preadipocytes on differentiation into mature adipocytes (induced for 8 days). B and C represent the results of quantitative analysis based on the image data in A, and DE represent the results of Western blot analysis to screen regulators. Aggf1 Results of TAZ-mediated downregulation of integrin subunit genes; Figure 19 In Example 10 Aggf1 The results of detecting ADSC stemness inhibition by combining integrin α7β1 and α7β2 are shown in the table below. A represents the immunostaining results of CD90, a stem cell marker in ADSCs; BC represents the qRT-PCR results of genes maintaining stem cell characteristics and stem cell markers in ADSCs, respectively; D represents the immunofluorescence staining results of DLK1, a preadipocyte marker; E represents the Western blot results of TAZ in ADSCs after treatment with AGGF1 and transfection with siRNA targeting integrin α7, β1, or β2; and F represents the results of co-precipitation (Co-IP) of integrin α7 (ITGA7) and integrin β2 (ITGB2) (left panel: anti-ITGB2 antibody was used for immunoprecipitation, and anti-ITGA7 antibody was used for immunoblotting analysis; right panel: anti-ITGA7 antibody was used for immunoprecipitation, and anti-ITGB2 antibody was used for immunoblotting analysis). Figure labeling: Data are expressed as mean ± standard error (SEM); statistical significance was determined using Student's t-test and one-way ANOVA; ns = no statistical significance; P<0.05; P<0.01; P<0.001; P<0.0001. Detailed Implementation

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0018] Based on the role of AGGF1 in the homeostasis regulation of ADSCs, this invention provides a method for modifying the stemness of adipose-derived stem cells, namely, treating adipose-derived stem cells with a reagent containing substances that inhibit or promote the expression of AGGF1, thereby maintaining or inhibiting the stemness of adipose-derived stem cells.

[0019] Specifically, when the reagent contains substances that inhibit AGGF1 expression (such as siRNA, shRNA, and / or antisense oligonucleotides that knock out or down AGGF1 expression), the reagent can be used to maintain the stemness of adipose-derived stem cells; when the reagent contains substances that upregulate AGGF1 expression (such as recombinant AGGF1 protein), the reagent can be used to inhibit the stemness of adipose-derived stem cells. As in some embodiments of the present invention, adipose-specific knockout... Aggf1 In mice, treatment significantly upregulated the expression of genes maintaining stem cell stemness and stem cell markers in ADSCs, while inhibiting the proliferation of preadipocytes derived from ADSCs, resulting in lower differentiation potential of ADSCs. In other embodiments of the present invention, treatment of ADSCs cells or preadipocytes with recombinant AGGF1 protein effectively inhibited ADSC stemness and promoted the proliferation and differentiation of preadipocytes, ultimately increasing adipogenesis.

[0020] This invention also provides the application of AGGF1 as a target in the preparation of drugs for treating obesity-related diseases, wherein the drug contains at least a substance that inhibits AGGF1 expression. The substance that inhibits AGGF1 expression is a siRNA, shRNA, and / or antisense oligonucleotide that knocks out or down AGGF1 expression. As in some embodiments of this invention, by constructing an adipose tissue-specific... Aggf1 Knockout mice demonstrate that adipose tissue-specific knockout occurs regardless of whether the mice are fed a normal diet or a high-fat diet. Aggf1All of these methods can reduce body weight, adipose tissue weight, adipocyte size, and the proportion of large adipocytes. In other embodiments of the invention, specific siRNA is used to knock down... Aggf1 The expression of AGGF1 was significantly inhibited, thus suppressing the adipogenic capacity of preadipocytes. These examples all suggest the potential application of substances that inhibit AGGF1 expression as drugs for treating obesity-related diseases.

[0021] Furthermore, the drug also includes one or more of the following substances: Substances that knock down integrin α7, β1 and / or β2; Substances that increase TAZ expression and / or promote TAZ nuclear localization; Pharmaceutically acceptable excipients.

[0022] This invention elucidates in detail the mechanism of action of AGGF1 in intervening in the development of obesity, pointing out that AGGF1 activates the Hippo signaling pathway through integrin α7β1 and α7β2, reduces TAZ levels, thereby inhibiting cell stemness and enhancing adipogenesis. Therefore, based on the above mechanism of action, to enhance the therapeutic effect of the drug, one or more of the following can be selected: substances that knock down integrin α7, β1 and / or β2, substances that increase TAZ expression, and / or substances that promote TAZ nuclear localization.

[0023] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0024] Example 1 This example demonstrates the construction of adipose tissue-specific... Aggf1 The knockout model was validated. Aggf1 The correlation with body weight, adipose tissue weight, and adipocyte volume is illustrated by the following experiments and results: Mining of the GEO database (GSE9624) revealed that AGGF1 was significantly increased in the white adipose tissue of some obese patients. Figure 2 A). Subsequently, after feeding mice with a high-fat diet for 20 weeks, the inventors found that compared to mice on a normal diet, the AGGF1 levels in the subcutaneous adipose tissue of obese mice fed a high-fat diet were also significantly increased. Figure 2 B).

[0025] To further explore Aggf1 The inventors have developed a series of adipose tissue-specific adipocyte homeostasis regulators to investigate the role of adipocytes in ADSCs and their role in obesity. Aggf1Knockout (KO) mice (hereinafter referred to as Aggf1) ako (mice), the specific construction method of which is: in C57B / L6 wild-type mice. Aggf1 Two flox sites were inserted into the first and second introns of the gene, respectively. The gene was then crossed with FABP4-Cre mice on a C57B / L6 background to obtain Aggf1. ako Mice. Western blot analysis showed that Aggf1 ako AGGF1 expression was significantly reduced in mouse white adipose tissue. Figure 2 C), indicating Aggf1 Fat-specific knockout mice were successfully constructed. These mice were compared with the control group Aggf1 flox / flox mice (hereinafter referred to as Aggf1). fl / fl Mice, in C57B / L6 wild-type mice Aggf1 Compared to Aggf1, which has two flux sites inserted in both intron 1 and intron 2, Aggf1... ako The weight of subcutaneous adipose tissue in mice was significantly reduced. Figure 2 D). H&E staining showed that Aggf1 ako The white adipocytes of mice were compared with those of the control Aggf1. fl / fl Mouse fat cells are smaller ( Figure 2 E). Consistent with the data above, Aggf1 ako The average area or volume of mouse adipocytes was significantly reduced ( Figure 2 F). Given the heterogeneity and varying sizes of adipocytes, the inventors divided them into four groups based on cell area: 0-600 μm², 600-1200 μm², 1200-2400 μm², and >2400 μm². Statistical analysis showed that Aggf1... ako In mice, the proportion of adipocytes in the 0-300 μm² and 600-1200 μm² groups was significantly increased, while the number of adipocytes in the >2400 μm² groups was significantly decreased. Figure 2 G).

[0026] This case also tracked Aggf1 cells on a high-fat diet (60% fat energy ratio, HFD) for 20 weeks. fl / fl Mice and Aggf1 ako Mouse weight changes showed that, starting from week 15, Aggf1... ako The mice weighed less than the control group Aggf1 fl / fl The mice showed significant relief ( Figure 3 A). Aggf1 ako The weight of subcutaneous adipose tissue in mice was significantly reduced, and the proportion of fat weight to body weight was significantly decreased. Figure 3 B). H&E staining of adipose tissue showed that Agggf1ako Mouse fat cells are smaller ( Figure 3 C), and the high-fat diet group Aggf1 ako The average area of ​​mouse adipocytes was significantly reduced ( Figure 3 D). High-fat diet group (Aggf1) ako In mice, the proportion of adipocytes with an area of ​​0-3000 μm² was significantly increased, while the number of large adipocytes with an area of ​​12000-24000 or >24000 μm² was significantly decreased. Figure 3 E).

[0027] The above findings indicate that, regardless of whether the diet is normal or high-fat, Aggf1 Knockout mice showed reduced adipose tissue weight, adipocyte size, and the proportion of large adipocytes, and under high-fat diet conditions, Aggf1 Knockout mice experience a significant reduction in body weight.

[0028] Example 2 Based on the experimental results of Example 1, the inventors hypothesized that Aggf1 ako The reduction in subcutaneous adipose tissue weight in mice is associated with inhibited differentiation of adipocytes (ADSCs) into adipose lineages and adipogenesis, and this hypothesis was verified. Specific experiments and results are as follows: To verify this hypothesis and clarify Aggf1 The molecular mechanisms regulating ADSC function and adipogenesis, as illustrated in this case, involved 6-month-old AgGf1 cells. ako Mice and control group Aggf1 fl / fl Transcriptome sequencing analysis was performed on subcutaneous adipose tissue from mice. GO enrichment analysis of differentially expressed genes (DEGs) showed that... Aggf1 AKO is associated with genes related to cell differentiation, phylogeny, tissue development, and animal organ development. Figure 4 A-4B). KEGG pathway enrichment analysis showed that... Aggf1 AKO is associated with stem cell pluripotency signaling pathways and with signaling pathways related to MSC lineage differentiation, including Hippo, Wnt / β-catenin, AKT signaling, ECM, and focal adhesion pathways. Figure 4 C-4D).

[0029] Further analysis using qRT-PCR revealed that in Aggf1 fl / fl and Aggf1 ako RNA sequencing results were validated in isolated ADSCs from mice. The results showed that, compared to the control group Aggf1... fl / fl Compared to mice, in Aggf1 ako In mouse ADSCs, genes that maintain stem cell stemness (including Dvl1 , Hand1 , Pax6 , Zfhx3 , Jarid2 , Axin2 , Wnt10b and Lifr The expression level of ) was significantly increased ( Figure 5 A). Simultaneously, changes in the expression of stem cell marker genes were detected. qRT-PCR analysis showed that, compared to the control group Aggf1... fl / fl Compared to mice, in Aggf1 ako In mouse-isolated ADSCs, the expression of stem cell markers Cd29, Cd44, Cd73, Cd90, Cd105, and Cd166 was significantly increased. Figure 5 B).

[0030] To further investigate the effect of Aggf1 on ADSCs, this case study used immunofluorescence staining of the marker CD90 to discover that... Aggf1 Knockout significantly enhanced the CD90 signal strength and improved the dryness of ADSCs. Figure 5 C). Further testing was also conducted. Aggf1 The effect of knockout on the differentiation potential of ADSCs was investigated by treating primary ADSCs with induction medium 1 (containing 5 µg / mL insulin, 1 µM dexamethasone, 0.5 mM IBMX, and 1 µM rosiglitazone) for 12 hours, followed by immunofluorescence analysis of the preadipocyte marker DLK1. The results showed that after treatment with induction medium 1, [the knockout effect was reduced]. Aggf1 Knockout resulted in a significant decrease in DLK1 signal intensity, indicating a reduction in the proportion of preadipocytes. Figure 5 D).

[0031] The above results indicate that fat-specific knockout Aggf1 It can maintain the stem cell characteristics or pluripotency of ADSCs and inhibit the differentiation of ADSCs into adipocyte lineages.

[0032] Example 3 During adipogenesis, adipocytes (ADSCs) differentiate into preadipocytes, which further differentiate into mature adipocytes. Upon receiving specific signals, ADSCs undergo lineage commitment, meaning they irreversibly commit from a pluripotent "ready" state to differentiating into a specific cell lineage (such as adipocytes, osteoblasts, or chondrocytes). Therefore, this study investigated... Aggf1 The effects of AKO on the proliferation and differentiation of preadipocytes derived from ADSCs. Specific experiments and results are as follows: Get Aggf1 respectively ako Mouse and control group Aggf1 fl / flMouse-derived adipocytes (ADSCs) were induced to differentiate into preadipocytes, and their proliferative capacity was assessed. CCK8 assay showed that, compared to the control group, Aggf1... ako The proliferative capacity of preadipocytes induced from mouse-derived ADSCs was significantly reduced at 12, 24, 36, and 48 hours, indicating that... Aggf1 AKO significantly inhibited the proliferation of ADSC-derived preadipocytes. Figure 6 A), and this result was further verified by EdU detection (A). Figure 6 B).

[0033] Adipocytes were first cultured in induction medium 1 for 2 days, then in induction medium 2 (containing 5 µg / mL insulin) for 6 days to promote mature adipocyte differentiation. Oil Red O staining showed that after 8 days of induction differentiation, AgGf1 cells... ako The differentiation potential of mouse-derived ADSCs was significantly lower than that of the control group Aggf1. fl / fl mice ( Figure 6 C). Western blot analysis of the expression of C / EBPα, PPARγ, and FABP4, markers of mature adipocytes at different days of adipogenic differentiation, showed that... Aggf1 Knockout significantly reduced the expression levels of C / EBPα, PPARγ, and FABP4 on days 0-2, 4-8, and 4-8 of differentiation, respectively. Figure 6 D-6G).

[0034] These results indicate that Aggf1 Knockout inhibits adipogenesis by suppressing the proliferation of ADSCs-derived preadipocytes.

[0035] Example 4 To verify the third embodiment Aggf1 The discovery that AKO inhibits the proliferation and adipogenesis of ADSCs-derived preadipocytes was demonstrated in this study using Aggf1-specific siRNA (i.e., siAggf1, whose sequence is 5'-GGAGTCTCCTGGAGATGAT-3' (SEQ ID NO. 1)) to knock down AKO. Aggf1 The study investigated its effect on the proliferation of the most commonly used preadipocyte cell line, 3T3-L1.

[0036] CCK8 assay showed that, compared with the negative control siRNA (NC), the proliferation of 3T3-L1 preadipocytes transfected with siAggf1 was significantly reduced, indicating that... Aggf1 KD significantly reduced the proliferation ability of 3T3-L1 cells. Figure 7 A). The EdU experiment also confirmed... Aggf1 KD significantly reduced the number of proliferating 3T3-L1 cells (red fluorescent marker) Figure 7 B).

[0037] The differentiation potential of 3T3-L1 preadipocytes was then characterized. After in vitro induction of differentiation, Oil Red O staining showed that siAggf1 significantly reduced the differentiation potential of 3T3-L1 preadipocytes into mature adipocytes. Figure 7 C). Consistent with the above results, Western blot analysis showed that the expression of mature adipocyte markers C / EBPα, PPARγ, and FABP4 was significantly decreased on days 0, 2, and 4; days 2, 4, 6, and 8; and days 2, 4, 6, and 8, respectively. Figure 7 D-7G).

[0038] The above results further confirm that Aggf1 Effects on the proliferation and adipogenesis of preadipocytes derived from ADSCs.

[0039] Example 5 In this study, primary ADSCs isolated from mice were treated with in vitro purified recombinant human AGGF1 protein (100 ng AGGF1 protein treatment for 24 h) for relevant assays to further explore the role of AGGF1 in maintaining ADSC stem cell characteristics and lineage differentiation. The specific assay results include the following: qRT-PCR analysis showed that AGGF1 protein treatment significantly reduced the expression of genes associated with the maintenance of ADSC pluripotency, including those identified in ADSCs by RNA sequencing. Dvl1 , Hand1 , Pax6 , Zfhx3 , Jarid2 , Axin2 , Wnt10b and Lifr ( Figure 8 A). Similar analyses showed that AGGF1 protein treatment significantly reduced stem cell marker genes (including...) in ADSCs. Cd29 , Cd44 , Cd73 , Cd90 , Cd105 and Cd166 The expression of () Figure 8 B). Immunofluorescence staining showed that AGGF1 protein treatment significantly reduced the signal intensity of the stem cell marker CD90 (B). Figure 8 C).

[0040] To investigate the effect of AGGF1 on cell lineage differentiation, ADSCs were treated with either AGGF1 protein or control buffer and then co-cultured with induction medium 1 for 12 hours to promote their differentiation into preadipocytes. Immunofluorescence staining analysis of the preadipocyte marker DLK1 showed that AGGF1 protein treatment significantly enhanced DLK1 signaling. Figure 8 D). CCK8 assay showed that AGGF1 protein treatment significantly promoted the proliferation of preadipocytes derived from ADSCs at 12, 24, 36, and 48 hours. Figure 9 A). EdU experiments further verified that AGGF1 protein treatment significantly enhanced the proliferative capacity of preadipocytes. Figure 9 B).

[0041] Subsequently, ADSCs were treated with either AGGF1 protein or control buffer, first cultured in induction medium 1 for 2 days, then in induction medium 2 for 6 days to promote mature adipocyte differentiation. Oil Red O staining showed that AGGF1 protein treatment significantly promoted the differentiation of ADSCs into mature adipocytes, and lipid accumulation was significantly increased. Figure 9 C). Western blot analysis showed that AGGF1 significantly upregulated C / EBPα expression on days 0 and 4. Figure 9 D and 9E), AGGF1 significantly enhanced PPARγ and FABP4 expression on days 6-8 (D and 9E). Figure 9 F and 9G).

[0042] In summary, the above data indicate that AGGF1 protein treatment inhibits the stem cell characteristics of ADSCs, promotes the proliferation and differentiation of preadipocytes, and ultimately increases adipogenesis by increasing the expression levels of mature adipocyte markers (including C / EBPα, FABP4, and PPARγ).

[0043] Similarly, a similar study was conducted on 3T3-L1 preadipocytes in this case. CCK8 and EdU proliferation assays showed that AGGF1 protein treatment significantly promoted the proliferation of 3T3-L1 preadipocytes. Figure 10 A-10B). Oil Red O staining showed that AGGF1 protein treatment significantly promoted adipogenesis, specifically promoting the differentiation of 3T3-L1 preadipocytes into mature adipocytes and increasing lipid accumulation. Figure 10 C). Western blot analysis showed that AGGF1 treatment significantly increased the expression levels of C / EBPα on days 0-4 of differentiation, PPARγ on days 0, 4, 6, and 8, and FABP4 on days 6-8. Figure 10 These data further demonstrate that AGGF1 protein treatment promotes the proliferation and differentiation of preadipocytes, thereby enhancing adipogenesis.

[0044] Example 6 In Example 2, the inventors analyzed the KEGG pathway, which is enriched by differentially expressed genes, using RNA sequencing data and found that the Hippo signaling pathway had the highest enrichment factor ( Figure 4C). The Hippo signaling pathway transmits signals through a kinase response (phosphorylation) cascade, prompting YAP / TAZ to be transported to the nucleus, ultimately regulating target gene expression at the transcriptional level. YAP / TAZ heterodimers are the main downstream effector molecules of this pathway. RNA sequencing analysis also showed that Aggf1... ako mouse white adipose tissue Taz The expression is upregulated. Therefore, this example utilizes Aggf1. ako Mouse and 3T3-L1 preadipocytes transfected with siAggf1 further validated Taz Changes in expression.

[0045] qRT-PCR analysis confirmed that Aggf1 ako In mouse-derived ADSCs Taz Significantly increased expression ( Figure 11 A). Western blot analysis showed that Aggf1 ako TAZ was significantly upregulated in mouse ADSCs ( Figure 11 C). With Aggf1 ako The ADSC results are similar. Aggf1 siRNA knockout leads to Taz mRNA ( ) in 3T3-L1 preadipocytes. Figure 11 B) and protein ( Figure 11 D) Expression significantly increased.

[0046] Given that TAZ only functions after being localized to the cell nucleus, this study used immunofluorescence staining to detect the localization of TAZ in ADSCs (the cell nucleus was stained blue with DAPI, and TAZ was stained red with anti-TAZ antibody). The results showed that Aggf1... ako TAZ nuclear localization was significantly increased in mouse-derived ADSCs. Figure 12 A). Similar results were also observed in 3T3-L1 preadipocytes treated with siAggf1. Figure 12 B).

[0047] To verify that the regulation of adipogenic differentiation by Aggf1 requires TAZ mediation, this study also tested siRNA knockdown. Taz For Aggf1 ako Effects on the differentiation potential of mouse ADSCs. Oil Red O staining after induced adipogenic differentiation showed that knockdown... Taz Reversible Aggf1 ako Adipogenesis defects in ADSCs ( Figure 12 C). Similar results were also observed in siTaz-treated 3T3-L1 preadipocytes. Figure 12D). Western blot analysis showed that 8 days after differentiation of Aggf1 knockout ADSCs or Aggf1 knockdown 3T3-L1 preadipocytes, the expression of PPARγ and FABP4 in mature adipocytes decreased, while the expression of knockdown FABP4 decreased. Taz This effect can be reversed. Figure 12 E-12F).

[0048] The combined results of ADSCs and 3T3-L1 cells indicate that Aggf1 Both knockout and knockdown increased TAZ expression and its nuclear localization, thereby inhibiting adipogenic differentiation of ADSCs and 3T3-L1 cells.

[0049] Example 7 In this example, mouse primary ADSCs and 3T3-L1 preadipocytes were treated with in vitro purified recombinant human AGGF1 protein (treatment method as described in Example 5) to verify the role of the Aggf1-Taz axis in adipogenesis.

[0050] The results showed that AGGF1 protein treatment significantly reduced the levels of ADSCs. Taz mRNA ( Figure 13 A) and TAZ protein ( Figure 13 The expression level of C was reduced, and the nuclear localization of TAZ was suppressed. Figure 14 A, cell nuclei were stained blue with DAPI, and TAZ was stained red with anti-TAZ antibody; similar results were observed when AGGF1 protein was treated on 3T3-L1 preadipocytes. Figure 13 B Figure 13 D and Figure 14 B). Oil Red O staining 8 days after induced differentiation showed that AGGF1 protein treatment significantly promoted adipogenic differentiation of ADSCs, but this effect was significantly weakened after further TAZ overexpression. Figure 14 C); 3T3-L1 preadipocytes also showed similar results ( Figure 14 D). Western blot analysis showed that ADSCs treated with AGGF1 protein ( Figure 14 E) or 3T3-L1 preadipocytes ( Figure 14 F) Eight days after differentiation, the expression of PPARγ and FABP4 in mature adipocytes was upregulated, and TAZ overexpression could reverse this effect.

[0051] The above detection data indicate that AGGF1 protein promotes the differentiation of ADSCs and 3T3-L1 preadipocytes into mature adipocytes by reducing TAZ expression and nuclear localization.

[0052] Example 8 This case further validates, through in vivo experiments, that AGGF1 maintains ADSC stem cell characteristics and regulates adipogenesis by modulating TAZ expression. Specific experiments and data are as follows: qRT-PCR analysis of mouse subcutaneous adipose tissue showed a significant decrease in the expression levels of genes associated with the maintenance of ADSC pluripotency, including Dvl1 , Hand1 , Pax6 , Zfhx3 , Jarid2 , Axin2 , Wnt10b and Lifr and stem cell markers Cd29 , Cd44 , Cd73 , Cd90 , Cd105 and Cd166 The expression levels of all three were significantly increased ( Figure 15 A). Furthermore, Aggf1 ako TAZ protein expression level in mouse subcutaneous adipose tissue Figure 15 B) Significantly upregulated. Immunohistochemical results also showed Aggf1. ako The TAZ positive signal was significantly enhanced in the subcutaneous adipose tissue of mice. Figure 15 C).

[0053] Subsequently, in Aggf1 ako Mice and control Aggf1 fl / fl Mice were injected bilaterally into the inguinal fat pads with pAAV2 / 8-U6-shTaz-CMV-EGFP-WPRE to specifically silence TAZ expression in adipose tissue, along with its corresponding control, pAAV2 / 8-U6-shRNA(NC)-CMV-EGFP-WPRE (total viral dose per mouse: 0.5–1 × 10¹¹ vg). Tissue samples were collected one and a half months later. Compared with the control Aggf1... fl / fl Compared to mice, Agggf1 ako The weight of subcutaneous adipose tissue in mice was significantly reduced, but this effect was partially reversed by knocking down Taz in mice. Figure 15 D). H&E staining showed that Aggf1 ako The average volume of adipocytes in the subcutaneous adipose tissue of mice was significantly reduced; however, this phenotype could be significantly reversed and the adipocyte volume restored by injecting Taz knockdown. Figure 16 A-16B). Aggf1 ako In mice, the proportion of adipocytes with a size of 0-300 μm² was significantly increased, while the number of adipocytes with sizes of 300-600 μm² and 600-900 μm² was significantly decreased. However, knockdown of adipocytes in mice... Taz These effects can be reversed ( Figure 16 C). Western blot analysis of subcutaneous adipose tissue showed that Aggf1 ako The expression levels of PPARγ and FABP4 were decreased in mice, but knockdown in vivo in mice... Taz Its effects can be reversed ( Figure 16 D). These data indicate that injection of the shTaz viral vector can restore AgGf1. ako The ability of mice to produce fat.

[0054] Example 9 Previous studies have identified integrin α5β1 as the receptor for AGGF1 on endothelial cells and integrin α7 as the receptor for AGGF1 on vascular smooth muscle cells. Based on this, the inventors hypothesized that integrins may act as AGGF1 receptors to regulate the function of ADSCs and 3T3-L1 preadipocytes.

[0055] To verify this hypothesis, siRNA was used to knock down 23 integrin subunits in 3T3-L1 preadipocytes. After 8 days of differentiation induction, adipogenic capacity was assessed by Oil Red O staining, and the results are as follows. Figure 17 As shown in Figure A. Within the α-integrin (α1-α11) subunits, knockdown of α2, α5, α6, α7, and α9 significantly reduced adipogenic potential, while knockdown of α10 and α11 significantly enhanced the adipogenic capacity of 3T3-L1 preadipocytes. Figure 17 B). Meanwhile, screening of the remaining integrins (αV, αL, αE, αM, and β1-β8) showed that knockdown of αV, αL, αE, β1, β2, β4, β6, β7, and β8 also significantly impaired adipogenic differentiation capacity. Figure 17 C). These data indicate that integrins are widely involved in regulating adipogenesis in 3T3-L1 preadipocytes.

[0056] Based on the above results, the following experiments were conducted: 3T3-L1 cells were treated with recombinant human AGGF1 protein, and then knocked down using single siRNAs targeting the integrin subunits α1-α11, αv, αl, αe, αm, and β1-β8, respectively. The treated cells were induced to undergo adipogenesis and stained with Oil Red O. The Oil Red O staining results 8 days after differentiation are shown below. Figure 18 As shown in A-18C: Knockdown of α2, α3, α5, α7, and α9 integrins in the α1-α11 subunits significantly inhibited AGGF1-promoted adipogenesis in 3T3-L1 cells. Figure 18 B); Similarly, knockdown of integrins αV, αE, β1, β2, β4, β6, β7, and β8 also has a similar inhibitory effect on adipogenesis (B). Figure 18 C).

[0057] Subsequently, Western blot analysis was used to screen for integrin subunits that could reverse AGGF1 protein-mediated TAZ downregulation after knockdown. The results showed that knockdown of integrin subunits α7, β1, β2, and β3 significantly reversed AGGF1 protein-induced TAZ downregulation. Figure 18 D-18E).

[0058] Combining Oil Red O staining and TAZ expression data, the common integrins were α7, β1, and β2. Therefore, the data indicate that AGGF1 promotes adipogenesis by reducing TAZ expression through its action on integrins α7, β1, and β2 on the surface of 3T3-L1 preadipocytes.

[0059] Example 10 Given that integrins α7, β1, and β2 function as AGGF1 receptors in 3T3-L1 preadipocytes, this study further verified whether AGGF1 regulates ADSC stem cell characteristics by binding to these three receptors. Specific experiments and results are as follows: Using isolated primary mouse ADSCs cells, recombinant human AGGF1 protein was first treated, followed by transfection with siRNAs targeting integrin α7, β1, and β2, and immunofluorescence staining for the stem cell marker CD90 was performed. The siRNA sequence for integrin α7 (siItga7) is 5'-ACCGACAGCAGTTCAAGGA-3' (SEQ ID NO. 2); the siRNA sequence for integrin β1 (siItgb1) is 5'-GCACGATGTGATGATTTAGAA-3' (SEQ ID NO. 3); and the siRNA sequence for integrin β2 (siItgb2) is 5'-CCAGGAATGCACCAAGTACAA-3' (SEQ ID NO. 4). Analysis showed that AGGF1 significantly reduced CD90 expression, and this effect could be reversed by knocking down integrin α7, β1, and β2. Figure 19 (A) indicates that AGGF1 inhibits ADSC stem cell characteristics through integrin α7, β1, and β2. This conclusion was also verified by qRT-PCR analysis. AGGF1 protein treatment significantly reduced stem cell characteristic genes. Dvl1 , Hand1 , Pax6 , Zfhx3 , Jarid2 , Axin2 , Wnt10b and Lifr Expression of stem cells and stem cell biomarkers Cd29 , Cd44 , Cd73 , Cd90 , Cd105 and Cd166The above effects can all be reversed by knocking down α7, β1, and β2. Figure 19 These findings further demonstrate that AGGF1 inhibits the stem cell properties of ADSCs through integrins α7, β1, and β2.

[0060] ADSCs cells treated with AGGF1 protein and transfected with siRNAs targeting integrin α7, β1, or β2 were treated with induction medium 1 for 12 hours, and the expression of the preadipocyte marker DLK1 was detected. Immunofluorescence staining showed that AGGF1 significantly upregulated DLK1 expression, and this effect could be reversed by knockdown of integrin α7, β1, and β2. Figure 19 D). Data indicate that AGGF1 promotes ADSC preadipocyte differentiation through integrin α7, β1, and β2. Western blot analysis showed that AGGF1 significantly reduced TAZ expression, and this effect could be reversed by knockdown of integrin α7, β1, and β2. Figure 19 E). Data indicate that AGGF1 reduces TAZ levels in ADSCs via integrin α7, β1, and β2 pathways.

[0061] In addition, Co-IP analysis was performed on HeLa cells 48 hours after co-transfection with ITGA7 and ITGB2 expression plasmids. The results showed that the anti-integrin β2 antibody could successfully precipitate integrin α7 ( Figure 19 F). Reverse Co-IP analysis showed that the anti-integrin α7 antibody could successfully precipitate integrin β2 (F). Figure 19 F). These data indicate that integrin α7 interacts with β2, which is consistent with the concept that integrins function by forming heterodimeric receptor proteins composed of α and β subunits. Interactions between integrin α7 and β1 have been reported.

[0062] These results indicate that AGGF1 activates the Hippo signaling pathway through integrin α7β1 and α7β2, thereby reducing the stemness of ADSCs cells.

[0063] In summary, this invention demonstrates that targeting the AGGF1-TAZ signaling pathway has a significant therapeutic effect on obesity and elucidates the relationship between AGGF1 and TAZ in the process of obesity development, laying a theoretical and experimental foundation for the development of novel treatments and drugs for obesity.

[0064] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. Application of AGGF1 as a target in the preparation of reagents for modifying the stemness of adipose-derived stem cells.

2. The application according to claim 1, characterized in that, The reagent contains a substance that inhibits AGGF1 expression, and the reagent is used to maintain the stemness of adipose-derived stem cells.

3. The application according to claim 1, characterized in that, The reagent contains AGGF1 protein and is used to inhibit the stemness of adipose-derived stem cells.

4. Application of AGGF1 as a target in the preparation of drugs for treating obesity-related diseases.

5. The application according to claim 4, characterized in that, The drug contains a substance that inhibits AGGF1 expression.

6. The application according to claim 5, characterized in that, The substance that inhibits AGGF1 expression is a siRNA, shRNA, and / or antisense oligonucleotide that knocks out or reduces AGGF1 expression.

7. The application according to claim 5, characterized in that, The drug treats obesity-related diseases by inhibiting adipogenic differentiation of preadipocytes.

8. The application according to claim 7, characterized in that, The drug also includes substances that knock down integrin α7, β1 and / or β2.

9. The application according to claim 7, characterized in that, The drug also includes substances that increase TAZ expression and / or promote TAZ nuclear localization.

10. The application according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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