Application of FAT10 gene in construction of inflammation-mediated pancreatic beta cell model and drug screening
By overexpressing the FAT10 gene in pancreatic β cells, an inflammation-mediated pancreatic β cell model was constructed, which solved the problem of lack of empirical evidence in the regulation of pancreatic β cell apoptosis by FAT10 in existing technologies. This provides a drug screening platform and enables the simulation of lipotoxicity-induced pancreatic β cell death and the screening of related drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-15
- Publication Date
- 2026-04-10
AI Technical Summary
Current technologies lack systematic and clear experimental evidence to show whether FAT10 is directly involved in the regulation of pancreatic β-cell apoptosis, and there is a lack of effective inflammation-mediated pancreatic β-cell models for drug screening.
An inflammation-mediated pancreatic β-cell model was constructed by overexpressing the FAT10 gene in pancreatic β-cells. The nucleotide sequence (SEQ ID No. 1) and amino acid sequence (SEQ ID No. 2) of the FAT10 gene were used to simulate inflammation-induced pancreatic β-cell apoptosis, and a drug screening platform was established.
An inflammation-mediated pancreatic β-cell model was successfully constructed, which significantly enhanced lipotoxicity-induced pancreatic β-cell death, providing an effective drug screening tool and mimicking the inflammation-inducing mechanism of the TLR4 pathway.
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Figure CN121825892A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and relates to application of FAT10 gene in construction of an inflammation-mediated pancreatic beta cell model and drug screening. BACKGROUND
[0002] Diabetes is mainly divided into type 1 diabetes (T1DM) and type 2 diabetes (T2DM), wherein T1DM mainly occurs in adolescents and is mainly caused by autoimmune-mediated destruction of pancreatic beta cells; and T2DM mainly occurs in the elderly and its pathogenesis involves insulin resistance and progressive loss of pancreatic beta cell function.
[0003] In the occurrence and development of diabetes, damage and loss of quantity of pancreatic beta cells are considered to be one of the core pathological mechanisms. Influenced by various harmful factors such as obesity, chronic inflammation, glucose-lipid toxicity and oxidative stress, pancreatic beta cells can undergo damage responses such as endoplasmic reticulum stress and mitochondrial dysfunction, thereby inducing biological processes such as cell apoptosis, aging and autophagy abnormalities, and ultimately leading to gradual reduction in the number of pancreatic beta cells and recession of insulin secretion function, thereby promoting the occurrence and aggravation of diabetes. Among the numerous pathogenic mechanisms, apoptosis of pancreatic beta cells is a key link in the occurrence of diabetes. Various inflammatory factors, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), can induce programmed death of pancreatic beta cells by activating death receptor signaling pathways or mitochondrial apoptosis pathways, thereby leading to decreased insulin synthesis and secretion capacity.
[0004] Ubiquitin-like protein FAT10 (also known as UBD protein) is a ubiquitin-like modified protein induced by interferon-γ and tumor necrosis factor-α, which can modify target proteins through covalent binding, thereby affecting the stability, subcellular localization and biological function of the target proteins. In recent years, the function of FAT10 in various cell types such as hepatocytes and tumor cells has been gradually revealed, and its role in apoptosis, inflammation amplification and metabolic disorders has attracted increasing attention. However, whether FAT10 is directly involved in the regulation of apoptosis of pancreatic beta cells is still lacking systematic and explicit experimental evidence. SUMMARY
[0005] The present application provides application of FAT10 gene in construction of an inflammation-mediated pancreatic beta cell model and drug screening. The method for promoting apoptosis of pancreatic beta cells by inducing expression of ubiquitin-like protein FAT10 through inflammation by the pancreatic beta cells overexpressing FAT10 can be used for constructing an inflammation-mediated pancreatic beta cell death model and screening of targeted drugs.
[0006] In a first aspect, the present application provides application of FAT10 gene or a protein encoded by FAT10 gene in construction of an inflammation-mediated pancreatic beta cell model.
[0007] Furthermore, the nucleotide sequence of the FAT10 gene is shown in SEQ ID No. 1.
[0008] SEQ ID NO:1
[0009] 1 ATGGCTCCCA ATGCCTCCTG CCTGTGCGTC CACGTGAGAA GTGAGGAGTG GGACCTCATG
[0010] 61 ACCTTCGATG CCAATCCTTA CGATTCCGTG AAAAAAATCA AGGAGCATGT GAGGTCTAAG
[0011] 121 ACGAAAGTGC CAGTGCAGGA TCAAGTTCTG TTGTTGGGCT CCAAAATCTT GAAACCCCGC
[0012] 181 CGATCCCTTT CTAGCTATGG GATCGATAAG GAGAAAACAA TTCACCTGAC TCTGAAAGTC
[0013] 241 GTGAAGCCAA GCGATGAAGA GCTGCCCCTG TTCCTGGTCG AGTCCGGCGA CGAGGCCAAG
[0014] 301 AGACATCTGC TTCAGGTTCG GAGGTCTTCC AGTGTAGCTC AGGTCAAGGC TATGATAGAG
[0015] 361 ACCAAAACCG GAATAATCCC CGAGACACAA ATCGTAACAT GTAATGGAAA GAGGCTGGAG
[0016] 421 GATGGCAAGA TGATGGCAGA TTACGGAATC CGAAAAGGAA ACCTCCTCTT TCTCGCATGC
[0017] 481 TATTGCATTG GGGGTTGA
[0018] Furthermore, the amino acid sequence of the protein encoded by the FAT10 gene is shown in SEQ ID No. 2.
[0019] SEQ ID NO:2
[0020] 1 MAPNASCLCV HVRSEEWDLM
[0021] 21 TFDANPYDSV KKIKEHVRSK
[0022] 41 TKVPVQDQVL LLGSKILKPR
[0023] 61 RSLSSYGIDK EKTIHLTLKV
[0024] 81 VKPSDEELPL FLVESGDEAK
[0025] 101 RHLLQVRRSS SVAQVKAMIE
[0026] 121 TKTGIIPETQ IVTCNGKRLE
[0027] 141 DGKMMADYGI RKGNLLFLAC
[0028] 161 YCIGG
[0029] Furthermore, the application is as follows: an inflammation-mediated pancreatic β-cell model is constructed by overexpressing the FAT10 gene in pancreatic β-cells; the nucleotide sequence of the FAT10 gene is shown in SEQ ID No. 1; and the amino acid sequence of the protein encoded by the FAT10 gene is shown in SEQ ID No. 2.
[0030] Furthermore, the inflammation-mediated pancreatic β-cell model is an inflammation-mediated pancreatic β-cell apoptosis model.
[0031] Furthermore, the inflammation is TLR4 pathway inflammation.
[0032] Furthermore, the pancreatic β-cell apoptosis is lipotoxic-induced pancreatic β-cell death.
[0033] Secondly, the present invention provides the application of the FAT10 gene or the protein encoded by the FAT10 gene in drug screening, wherein the application is as follows:
[0034] An inflammation-mediated pancreatic β-cell model was constructed by overexpressing the FAT10 gene in pancreatic β-cells, and then drug screening was performed using the pancreatic β-cell model; the drug is a pancreatic β-cell apoptosis antagonist.
[0035] Furthermore, the pancreatic β-cell apoptosis is lipotoxic-induced pancreatic β-cell death.
[0036] Thirdly, the present invention provides a method for constructing an inflammation-mediated pancreatic β-cell model, wherein the method comprises overexpressing the FAT10 gene in pancreatic β-cells, and the nucleotide sequence of the FAT10 gene is shown in SEQ ID No. 1;
[0037] The construction method includes the following steps:
[0038] S1. The protein coding region sequence of the human FAT10 gene was cloned into the pLKO-CMV-puro vector to obtain the recombinant plasmid pLKO-CMV-puro-FAT10;
[0039] S2. Recombinant plasmid pLKO-CMV-puro-FAT10, packaging plasmid psPAX2 and envelope protein plasmid pMD2.G were co-transfected into packaging cells to obtain FAT10 overexpressing lentivirus solution;
[0040] S3. Infect pancreatic β cells with the FAT10-overexpressing lentiviral solution, and then screen for puromycin resistance to obtain a cell line that stably overexpresses FAT10 protein as an inflammation-mediated pancreatic β cell model.
[0041] This application reveals that the TLR4 pathway can significantly induce FAT10 expression in pancreatic β cells, and that FAT10 expression can significantly enhance lipotoxicity-induced pancreatic β cell death (the process of pancreatic β cell death caused by lipotoxicity). This method can construct FAT10-overexpressing pancreatic β cells to mimic the inflammation-induced pancreatic β cell death mechanism, providing a good in vitro cell model for inflammation-mediated pancreatic β cell injury and death, which can be applied to related drug library screening and mechanism studies. Attached Figure Description
[0042] These and / or other aspects and advantages of the present invention will become apparent and readily understood from the following description of preferred embodiments taken in conjunction with the accompanying drawings, in which:
[0043] Figure 1 This study demonstrates the promoting effect of the lipopolysaccharide (LPS)-mediated TLR4 pathway on FAT10 protein expression in pancreatic β cells;
[0044] Figure 2 The PCR identification results of the FAT10 overexpression vector in Example 2 are shown;
[0045] Figure 3 The validation results of the FAT10 overexpression cell model constructed in Example 3 are shown;
[0046] Figure 4The apoptosis levels of pancreatic β cells under palmitic acid (PA) stimulation in the control group, FAT10 overexpression group, and LPS treatment group in Example 4 are shown ((ns, P > 0.05; ***, P < 0.001)). Detailed Implementation
[0047] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Some embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the invention will be thorough and complete.
[0048] 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 in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
[0049] Example 1: The LPS / TLR4 immune pathway can induce FAT10 expression in pancreatic β cells.
[0050] To elucidate the induction of FAT10 expression by inflammation, we analyzed the promoting effect of the lipopolysaccharide (LPS)-mediated TLR4 pathway on FAT10 protein expression in pancreatic β cells.
[0051] Human pancreatic β-cell line EndoC-βH1 was used as a cell model and cultured routinely in DMEM medium containing 15% fetal bovine serum. Cells were treated with 10 ng / mL LPS for 0, 6, and 24 hours to establish a dose-time effect. Cells were collected after treatment, and total protein was extracted using RIPA lysis buffer containing a protease inhibitor. After quantification using the BCA method, an equal volume of protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk, incubated overnight at 4°C with specific rabbit anti-FAT10 primary antibody, washed with TBST, and incubated at room temperature for 1 hour with goat anti-rabbit HRP-labeled secondary antibody. Finally, the membrane was visualized using ECL chemiluminescence reagent. The results are shown below. Figure 1 As shown.
[0052] Figure 1 The results showed that LPS treatment significantly induced the expression of FAT10 protein in EndoC-βH1 cells, and this induction effect was significantly dose- and time-dependent. The internal control protein GAPDH was observed to have consistent total protein levels across all groups. Figure 1 ).
[0053] These results suggest that the TLR4 inflammatory pathway can significantly promote the abnormal expression of FAT10 in human pancreatic β cells, implying that FAT10 may play a potential role in TLR4 inflammatory pathway-mediated pancreatic β cell pathology.
[0054] Example 2: Construction and validation of FAT10 overexpression vector
[0055] To obtain a gene tool that can be used to stably express FAT10, this embodiment describes the specific process of cloning the protein coding region sequence of the human FAT10 gene into the pLKO-CMV-puro vector.
[0056] 1. Vector Construction: First, a DNA fragment containing the complete human FAT10 gene protein-coding region (synthesized by Shanghai Sangon Biotech Co., Ltd., sequence information SEQ ID NO:2) was obtained through chemical synthesis, and EcoRI and Age I restriction endonuclease recognition sites were introduced at both ends of the fragment. Simultaneously, the pLKO-CMV-puro empty vector plasmid was double-digested using the same restriction endonucleases EcoRI and Age I to linearize the vector and generate compatible sticky ends. The digestion products were then separated by agarose gel electrophoresis, and the target DNA fragment and linearized vector were purified using a gel extraction kit. Subsequently, the FAT10 gene fragment at a molar ratio of 3:1 was ligated to the pLKO-CMV-puro vector backbone using T4 DNA ligase, and the ligation was incubated overnight at 16°C. The ligation product was transformed into competent Escherichia coli DH5α by heat shock, spread on LB agar plates containing 100 μg / mL ampicillin, and incubated upside down in a 37°C incubator for 12-16 hours until single colonies appeared.
[0057] 2. Screening and Verification of Positive Clones: Multiple single colonies were randomly picked from the transformation plate and inoculated into LB broth containing antibiotics for small-scale amplification. Plasmid DNA was extracted from the bacterial culture using a plasmid mini-prep kit. Preliminary PCR identification was performed using universal vector primers, and the results are as follows: Figure 2 As shown in the figure. The results showed that, compared with the empty vector control group, the PCR product of the positive clone showed a specific amplification band at approximately 500 bp, consistent with the theoretical size of the CDS of the FAT10 gene. Figure 2 The above vector was verified by Sanger sequencing and sequence alignment, confirming that the reading frame was correct and there were no unexpected mutations. The recombinant plasmid was named pLKO-CMV-puro-FAT10.
[0058] Example 3: Construction and validation of a FAT10 overexpression cell model
[0059] 1. Lentiviral Packaging: 293T cells in good growth condition with 70%-80% confluence were cultured in antibiotic-free DMEM complete medium. Co-transfection was performed using cationic polymer liposomes: 1.2 μg of pLKO-CMV-puro-FAT10 overexpression plasmid, 1.2 μg of psPAX2 packaging plasmid, and 0.6 μg of pMD2.G envelope plasmid were precisely mixed in 100 μL of Opti-MEM serum-free medium and gently mixed (referred to as solution A). In another centrifuge tube, 10 μL of Lipofectamine 2000 transfection reagent was mixed with 100 μL of Opti-MEM serum-free medium and incubated at room temperature for 5 minutes (referred to as solution B). Then, solutions A and B were slowly mixed, gently pipetted until homogeneous, and incubated at room temperature for 20 minutes to form a stable DNA-liposome complex. Add this complex dropwise to 293T cells that have been replaced with fresh antibiotic-free medium, and gently shake the culture dish to distribute it evenly. Incubate the cells in a 37°C, 5% CO2 incubator.
[0060] Six to eight hours after transfection, the medium was replaced with fresh complete culture medium. After 48 and 72 hours of further culture, cell supernatants containing lentiviral particles were collected. The collected supernatants were filtered through a 0.45 μm filter to remove cell debris, and then concentrated by ultracentrifugation. The resulting viral pellet was resuspended in an appropriate amount of PBS, aliquoted, and stored at -80°C to obtain the FAT10 overexpression lentiviral solution.
[0061] 2. Cell Culture and Infection: The pancreatic β-cell line EndoC-βH1 was used as the experimental subject. Cells were cultured in DMEM complete medium containing 15% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2 using standard methods. When the cells were in good growth condition and in the logarithmic growth phase, they were digested with trypsin and resuspended, then seeded at an appropriate density in 6-well cell culture plates. To ensure transfection efficiency, the medium was replaced with antibiotic-free complete medium at seeding, and the initial cell confluence was controlled at 30-40%. 24 hours after seeding, when the cell confluence reached approximately 60-70%, the cells were transfected with empty lentivirus (negative control) and FAT10 overexpressing lentivirus, respectively.
[0062] 3. Screening of stable expression cell lines: 24-48 hours after transfection, the medium was replaced with complete selection medium containing 400 μg / mL G418 for continuous pressure screening. This screening process lasted 7 to 14 days, with the medium replaced with fresh selection medium every 2-3 days, until the untransfected control group cells died completely, while obvious resistant clones were observed in the experimental group.
[0063] 4. Protein Expression Validation (Western Blot): To confirm the stable expression of exogenous FAT10 in EndoC-βH1 cells, we collected cells from each group for Western blot analysis: First, the culture medium was discarded, and the cells were gently washed twice with pre-cooled PBS. RIPA lysis buffer containing 1 mM PMSF was added to each well and the cells were lysed on ice for 30 minutes to extract total protein. The protein concentration was then determined using the BCA method. An equal volume of total protein (20 μg) was mixed with 5× SDS-PAGE loading buffer and heated at 100℃ for 10 minutes to fully denature the protein. The denatured protein sample was then added to a 12% SDS-polyacrylamide gel and electrophoretically separated at constant voltages of 80V and 120V, followed by wet transfer at 300℃. Proteins were transferred to a PVDF membrane under a constant current of mA for 60-90 minutes. After transfer, the membrane was blocked in 5% skim milk-TBST at room temperature for 1 hour, followed by overnight incubation at 4°C with specific anti-FAT10 primary antibody (rabbit anti-FAT10 antibody, 1:1000). After washing with TBST, the membrane was incubated with HRP-labeled goat anti-rabbit secondary antibody (1:5000) at room temperature for 1 hour. Finally, the membrane was reacted with ECL chemiluminescence reagent for 1-2 minutes, exposed in a chemiluminescence imaging system, and images were acquired. The results are as follows: Figure 3 As shown in the figure, compared with the empty vector control group, the experimental group transfected with FAT10-overexpressing lentivirus showed a clear FAT10 protein-specific band at approximately 18 kDa, indicating that FAT10 was successfully and stably overexpressed in EndoC-βH1 cells. This result confirms the successful construction of the FAT10-overexpressing cell model, which can be used for subsequent functional studies. Figure 3 ).
[0064] Example 4: FAT10 overexpression significantly promotes lipotoxicity-induced apoptosis of pancreatic β cells.
[0065] The TUNEL assay demonstrated changes in apoptosis levels: Cells were divided into three groups: a control group, a FAT10 overexpression group, and an LPS treatment group. Based on these groups, pancreatic β-cell apoptosis was simulated with or without palmitic acid (PA) stimulation, mimicking lipotoxicity-induced apoptosis.
[0066] Cells were prepared by centrifuging in 15 mL centrifuge tubes, discarding the culture medium, and resuspending in 1 mL of fresh culture medium. In 12-well plates, 100 μL of resuspended cells were added to each well, and the plates were incubated for 12 h until cell adhesion. Cells were washed once with PBS and fixed with 4% paraformaldehyde for 30 min. They were then washed once with PBS, resuspended in 0.3% Triton X-100 (diluted in PBS), and incubated at room temperature for 5 min. Cells were washed twice with PBS. TdT enzyme, fluorescent labeling solution, and TUNEL assay solution were prepared at a ratio of 1:9:10. 50 μL of TUNEL assay solution was added to each well, and the plates were incubated at 37 °C for 60 min. Cells were washed three times with PBS, mounted with anti-fluorescence quenching, and observed under a fluorescence microscope. Differences in cell apoptosis among the groups were statistically analyzed. Results Figure 4 As shown in the figure. The results showed that FAT10 overexpression significantly increased the apoptosis level of pancreatic β cells after PA exposure, suggesting that it effectively enhances lipotoxicity-mediated pancreatic β cell death, and the level of pancreatic β cell death induced by this method is similar to that of the LPS / TLR4 pathway (LPS stimulation), suggesting that it may potentially mimic the promoting effect of the TLR4 inflammatory pathway on pancreatic β cell death. Figure 4 ).
Claims
1. Application of the FAT10 gene or proteins encoded by the FAT10 gene in constructing an inflammation-mediated pancreatic β-cell model.
2. The application according to claim 1, characterized in that, The nucleotide sequence of the FAT10 gene is shown in SEQ ID No.
1.
3. The application according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the FAT10 gene is shown in SEQ ID No.
2.
4. The application according to claim 1, characterized in that, The application is as follows: An inflammation-mediated pancreatic β-cell model was constructed by overexpressing the FAT10 gene in pancreatic β-cells. The nucleotide sequence of the FAT10 gene is shown in SEQ ID No. 1; The amino acid sequence of the protein encoded by the FAT10 gene is shown in SEQ ID No.
2.
5. The application according to claim 1, characterized in that, The inflammation-mediated pancreatic β-cell model is an inflammation-mediated pancreatic β-cell apoptosis model.
6. The application according to claim 5, characterized in that, The inflammation described is TLR4 pathway inflammation.
7. The application according to claim 5, characterized in that, The apoptosis of pancreatic β cells mentioned is lipotoxicity-induced pancreatic β cell death.
8. The application of the FAT10 gene or the protein encoded by the FAT10 gene in drug screening, wherein the application is as follows: An inflammation-mediated pancreatic β-cell model was constructed by overexpressing the FAT10 gene in pancreatic β-cells, and then drug screening was performed using the pancreatic β-cell model. The drug is a pancreatic β-cell apoptosis antagonist.
9. The application according to claim 8, characterized in that, The apoptosis of pancreatic β cells mentioned is lipotoxicity-induced pancreatic β cell death.
10. A method for constructing an inflammation-mediated pancreatic β-cell model, characterized in that, The construction method involves overexpressing the FAT10 gene in pancreatic β cells. The nucleotide sequence of the FAT10 gene is shown in SEQ ID No.
1. The construction method includes the following steps: S1. The protein coding region sequence of the human FAT10 gene was cloned into the pLKO-CMV-puro vector to obtain the recombinant plasmid pLKO-CMV-puro-FAT10; S2. Recombinant plasmid pLKO-CMV-puro-FAT10, packaging plasmid psPAX2 and envelope protein plasmid pMD2.G were co-transfected into packaging cells to obtain FAT10 overexpressing lentivirus solution; S3. Infect pancreatic β cells with the FAT10-overexpressing lentiviral solution, and then screen for puromycin resistance to obtain a cell line that stably overexpresses FAT10 protein as an inflammation-mediated pancreatic β cell model.