Anti-aging transcription factor and use thereof

Abstract

The application discloses an anti-aging transcription factor and application thereof, and belongs to the technical field of biotechnology, and a technical scheme thereof is as follows: the transcription factor SIX5 or a coding nucleic acid thereof is used in preparation of a preparation for reducing a cell aging level, and the reduction of the cell aging level is at least embodied in one or more of the following effects: (1) reducing the expression level of an aging-related marker p21 in the cell; (2) reducing the expression level of SASP-related factors IL-6 and / or IL-8 in the cell; (3) reducing the proportion of SA-beta-Gal positive cells, the application provides the use of the transcription factor SIX5 in the anti-aging aspect, and it is found through test verification that when the SIX5 in the cell is silenced, the expression of cell aging-related genes (p21, IL-6, IL-8 and the like) increases, and when the SIX5 is overexpressed, the expression of the cell aging-related genes (p21, IL-6, IL-8 and the like) decreases, and the degree of cell aging is reduced.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to an anti-aging transcription factor and its applications. Background Technology

[0002] Cellular senescence is the core manifestation of aging in organisms, involving altered gene expression, disordered epigenetic modifications, and decreased cellular function. Transcription factors, as key proteins regulating gene expression, play a crucial role in cell fate determination, differentiation, and reprogramming. In recent years, cell reprogramming technology has become an important tool for studying cellular senescence and reversing aging. For example, by expressing a specific set of transcription factors, somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs). Further research has found that partial reprogramming (i.e., incomplete conversion into pluripotent stem cells) can achieve cell rejuvenation without completely losing the cell's specific functions.

[0003] Single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) are advanced technologies developed in recent years. The former is used to analyze the transcriptome level of a single cell, while the latter is used to detect chromatin accessibility in a single cell. These technologies can reveal subtle changes in gene expression and epigenetic regulation at the single-cell level, providing powerful tools for identifying key transcription factors associated with cellular senescence and rejuvenation. Furthermore, the GTEx (Genotype-TissueExpression) database provides a wealth of population cohort gene expression data, helping researchers understand trends in gene expression across different age groups. For example, GTEx data analysis can compare the expression levels of transcription factors in young, middle-aged, and elderly individuals, revealing age-related gene expression patterns.

[0004] The technical problem this application aims to solve is: how to provide a transcription factor with anti-aging capabilities. Summary of the Invention

[0005] The purpose of this application is to provide an application of transcription factor SIX5 in anti-aging. This application has verified through experiments that when SIX5 in cells is silenced, the expression of cellular senescence-related genes (p21, IL-6, IL-8, etc.) increases, while when SIX5 is overexpressed, the expression of cellular senescence-related genes (p21, IL-6, IL-8, etc.) decreases, and the degree of cellular senescence is reduced.

[0006] To address the aforementioned problems, this application provides the use of transcription factor SIX5 or its encoded nucleic acid in the preparation of formulations for reducing cellular senescence levels, wherein the reduction in cellular senescence levels is manifested in at least one or more of the following effects:

[0007] In one embodiment, the use of the above-mentioned transcription factor SIX5 or its encoded nucleic acid in the preparation of an agent for reducing cellular senescence levels, wherein reducing cellular senescence levels is manifested in reducing the expression level of the senescence-related marker p21 in cells;

[0008] In one embodiment, the use of the above-mentioned transcription factor SIX5 or its encoded nucleic acid in the preparation of an agent for reducing cellular senescence levels, wherein reducing cellular senescence levels is manifested in reducing the expression levels of SASP-related factors IL-6 and / or IL-8 in cells;

[0009] In one embodiment, the use of the above-mentioned transcription factor SIX5 or its encoded nucleic acid in the preparation of an agent for reducing cellular senescence levels, wherein reducing cellular senescence levels is manifested in reducing the proportion of SA-β-Gal positive cells.

[0010] In one embodiment, the cell is a somatic cell, and more specifically, the somatic cell includes, but is not limited to, fibroblasts, macrophages, erythrocytes, leukocytes, adipocytes, osteocytes, skeletal muscle cells, cardiomyocytes, smooth muscle cells, nerve cells, mesenchymal stem cells or their derivatives.

[0011] More preferably, the somatic cells are selected from fibroblasts, mesenchymal stem cells or their derivatives.

[0012] In one embodiment, the use is for improving or delaying cellular replicative senescence and / or stress-induced cellular senescence;

[0013] It should be noted that the above-mentioned cellular replicative senescence should be understood as normal cells entering a state of slowed proliferation and growth arrest after a limited number of divisions, accompanied by irreversible senescence phenomena such as reduced stemness and loss of differentiation ability.

[0014] The stress-induced cellular senescence mentioned above should be understood as the premature entry of cells into an irreversible state of proliferation arrest after being subjected to external pressures (such as, but not limited to, DNA damage, oxidative stress, radiation, or chemical drugs).

[0015] In one embodiment, the SIX5-encoded nucleic acid is delivered in the form of RNA, which is either linear RNA or circular RNA.

[0016] More preferably, the SIX5-encoded nucleic acid is delivered in the form of circular RNA.

[0017] In one embodiment, SIX5 functions as a protein.

[0018] In addition, this application also discloses a circular RNA that can express the transcription factor SIX5 protein in eukaryotic cells and maintain stable expression of the SIX5 protein during continuous cell culture, thereby reducing the senescence level of the cells.

[0019] It should be noted that stable expression of SIX5 protein should be understood as stable expression of SIX5 protein in multiple dimensions, such as the concentration or content of SIX5 protein in cells not fluctuating significantly due to time or environmental changes at the expression level.

[0020] In terms of functional activity, the SIX5 protein maintains its native conformation, ensuring that its biological functions (such as regulating gene expression) are performed normally.

[0021] In one embodiment, the circular RNA includes: a 5' homologous arm, a circular element, a ribozyme sequence, an internal ribosome entry site (IRES), and a SIX5 protein-coding sequence.

[0022] In one embodiment, the nucleotide sequence encoding the SIX5 protein is as shown in SEQ ID NO:1, or is a sequence having at least 85% homology with it.

[0023] In addition, this application also discloses a recombinant virus carrying an expression cassette for expressing the transcription factor SIX5, which expresses the SIX5 protein in host cells, thereby reducing the senescence level of the host cells.

[0024] In one embodiment, the recombinant virus is adeno-associated virus (AAV).

[0025] It should be noted that the serotypes of adeno-associated virus (AAV) include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.

[0026] In a preferred embodiment, the AAV is AAV2, AAV6, AAV8, or AAV9.

[0027] In addition, this application also discloses a cell that has been treated with the above-mentioned circular RNA or recombinant virus and exhibits a reduced cellular senescence phenotype.

[0028] In one embodiment, the cells are fibroblasts or mesenchymal stem cells.

[0029] In addition, this application also discloses a composition comprising the above-described circular RNA or recombinant virus, and a pharmaceutically acceptable vector.

[0030] In addition, this application also discloses the use of the above-described composition in the preparation of formulations for reducing cellular senescence levels.

[0031] In this invention, the formulation includes, but is not limited to: drugs, health products, or cosmetics; the drugs are oral medications, topical medications, or intravenous or intramuscular injections; the cosmetics are cosmetics for use in contact with the skin, hair care products, or cosmetics administered via microneedles.

[0032] The beneficial effects of this application are:

[0033] This application provides the use of transcription factor SIX5 in anti-aging. Through experimental verification, this application found that when SIX5 in cells is silenced, the expression of cellular senescence-related genes (p21, IL-6, IL-8, etc.) increases, while when SIX5 is overexpressed, the expression of cellular senescence-related genes (p21, IL-6, IL-8, etc.) decreases, and the degree of cellular senescence is reduced. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the representation and analysis of GTEx data;

[0035] Figure 2 Box plot of gene expression differentials using scRNA-seq;

[0036] Figure 3 A stacked bar chart showing the proportions of cell cycle stages;

[0037] Figure 4 This is a clustering diagram of scRNA-seq.

[0038] Figure 5 This is the scATAC clustering diagram when clustering by sample;

[0039] Figure 6 This is the scATAC clustering diagram when clustering by cell group;

[0040] Figure 7 This is a diagram illustrating the Epitrace score.

[0041] Figure 8 Box plot of Epitrace score;

[0042] Figure 9 This is a schematic diagram of the chromVAR bias score distribution;

[0043] Figure 10A SA-β-Gal staining image of third-generation SBK-M14 cells;

[0044] Figure 10BSA-β-Gal staining image of 10th generation SBK-M14 cells;

[0045] Figure 10C SA-β-Gal staining image of third-generation SBK-M35 cells;

[0046] Figure 10D SA-β-Gal staining image of 10th generation SBK-M35 cells;

[0047] Figure 10E SA-β-Gal staining image of third-generation SBK-M57 cells;

[0048] Figure 10F SA-β-Gal staining image of 10th generation SBK-M57 cells;

[0049] Figure 10G This is a schematic diagram showing the proportion of SA-β-Gal positive cells in each experimental group in Example 2;

[0050] Figure 11 This is a comparison chart of SIX5 expression levels in each experimental group in Example 2 (qPCR).

[0051] Figure 12A This is a comparison chart of IL-6 expression levels in each experimental group in Example 2;

[0052] Figure 12B This is a comparison chart of IL-8 expression levels in each experimental group in Example 2;

[0053] Figure 12C This is a Western blotting (WB) comparison of SIX5 expression levels in each experimental group in Example 2.

[0054] Figure 13 This is a Western blot (WB) test image of the SIX5 protein expression level detection in each experimental group in Example 2;

[0055] Figure 14A This is a Western blot (WB) test image of the P21 protein expression level detection in each experimental group in Example 2;

[0056] Figure 14B This is a comparison chart of P21 expression levels in each experimental group in Example 2;

[0057] Figure 15 This is a comparison chart of the silencing abilities of each siRNA on SIX5 in Example 3;

[0058] Figure 16A This is an SA-β-Gal staining image of the Blank group in Example 4;

[0059] Figure 16BThis is an SA-β-Gal staining image of the NC group in Example 4;

[0060] Figure 16C This is an SA-β-Gal staining image of the SIX5-siRNA group in Example 4;

[0061] Figure 16D This is a comparison of SA-β-Gal expression levels in the Blank group, NC group, and SIX5-siRNA group in Example 4.

[0062] Figure 17 This is a comparison of SIX5 expression levels between the Blank group and the SIX5-siRNA group in Example 4.

[0063] Figure 18 This is a comparison of the expression levels of aging genes in the Blank group and the SIX5-siRNA group in Example 4;

[0064] Figure 19A This is a Western blot (WB) test image showing the SIX5 protein expression level detection in the Blank group and the SIX5-siRNA group in Example 4.

[0065] Figure 19B This is a comparison of SIX5 expression levels between the Blank group and the SIX5-siRNA group in Example 4.

[0066] Figure 20A This is a Western blot (WB) test image showing the P21 protein expression level detection in the Blank group and the SIX5-siRNA group in Example 4.

[0067] Figure 20B This is a comparison of P21 expression levels between the Blank group and the SIX5-siRNA group in Example 4;

[0068] Figure 21 The image shows the Western blot (WB) test results for the expression levels of SIX5 protein in the NC group, the GFP virus control group, and the SIX5 group in Example 6.

[0069] Figure 22A This is an SA-β-Gal staining image of the low-generation group in Example 7;

[0070] Figure 22B This is an SA-β-Gal staining image of the high-generation group in Example 7;

[0071] Figure 22C This is an SA-β-Gal staining image of the high-generation group overexpressing SIX5 in Example 7;

[0072] Figure 22DThis is a comparison of SA-β-Gal expression levels of SIX5 overexpression in the low-generation group, high-generation group, and high-generation group in Example 7.

[0073] Figure 23A The image shows the Western blot (WB) test results for the expression levels of SIX5 protein in the high-generation group, the high-generation group overexpressing GFP, and the high-generation group overexpressing XIS5 in Example 7.

[0074] Figure 23B This is a comparison of the SIX5 protein expression levels in the high-generation group, the high-generation group overexpressing GFP, and the high-generation group overexpressing XIS5 in Example 7.

[0075] Figure 24A The image shows the Western blot (WB) test results of p21 protein expression levels overexpressing XIS5 in the low-generation group, high-generation group, and high-generation group in Example 7.

[0076] Figure 24B This is a comparison of the p21 protein expression levels of XIS5 overexpression in the low-generation group, high-generation group, and high-generation group in Example 7.

[0077] Figure 25 This is a comparison of the expression levels of the aging gene XIS5 overexpression in the low-generation group, high-generation group, and high-generation group in Example 7.

[0078] Figure 26 This is the sequence of the elements of the SIX5 circular RNA. Detailed Implementation

[0079] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0080] It should be noted that the codon-optimized nucleotide sequence of SIX5 in the circular RNA (SIX5 protein coding sequence) is shown in SEQ ID NO: 1;

[0081] In addition, the amino acid sequence of the SIX5 protein is shown in SEQ ID NO: 2;

[0082] In the viral vector, the codon-optimized nucleotide sequence of SIX5 is shown in SEQ ID NO: 3.

[0083] Example 1: Single-cell multi-omics combined with population cohort data mining and transcription factor screening

[0084] GTEx data analysis: Gene expression data from 698 non-exposed skin sites, covering ages 20-79, were loaded from the GTEx database. Age groups were simplified into three categories: youth (20-39 years), middle-aged (40-59 years), and elderly (60-79 years). By comparing the expression levels of transcription factors in different age groups (in terms of expression levels per million transcripts / TPM), transcription factors whose expression decreases with age were identified. For example, Figure 1 The results showed that SIX5 expression was significantly lower in the older group than in the younger group, suggesting that it is associated with a youthful state.

[0085] scRNA-seq analysis: 10X expression matrices of three samples (blank control "BC", positive control "OSK", and experimental group "IPR") were loaded. Objects were created using Seurat, filtering cells (gene count 1000-6000, UMI count 2000-20000, mitochondrial proportion <15%, gene complexity >0.8), retaining approximately 80% of the cells (16706 in the BC group, 20938 in the OSK group, and 18871 in the IPR group). Log normalization was performed, 2000 highly variable genes were selected, data were centered, the top 30 principal components (PCA) were calculated, UMAP embeddings were generated, and clustering was performed based on dimensions 1-30. The expression of aging-related genes and the proportion of cell cycle stages (e.g., ...) were compared. Figure 2 , Figure 3 As shown in the figure, cells are divided into three groups: aging, stable, and rejuvenated.

[0086] like Figure 4 As shown, the rejuvenated cell population (red) accounts for approximately 35%, the stable cell population (blue) accounts for 50%, and the senescent cell population (yellow) accounts for 15%. It can be seen that the proportion of the rejuvenated cell population is significantly higher in the positive control group (OSK) and the experimental group (IPR) compared to the blank control group, while the proportion of the senescent cell population is reduced.

[0087] In addition, it should be noted that, Figure 2 The figure includes the expression changes of nine genes across different cell state groups. The horizontal axis represents the sample groups: Rejuvenation, Stable, and Aging; the vertical axis represents the normalized gene expression levels (Log2-normalized counts). Figure 2It contains nine subplots, corresponding to the expression distribution of genes COL1A1, MMP2, LMNA, PRDX1, CCNB1, NFKB1, SQSTM1, CDKN1A, and DDIT3, respectively. The box plots in each subplot show the median, quartiles, and outliers of the expression levels, with the rejuvenated group represented in red, the stable group in blue, and the senescent group in yellow.

[0088] Figure 3 The figure shows the distribution of cell cycle stages in different cell state groups. The horizontal axis represents cell state groups: rejuvenation, stable, and aging; the vertical axis represents the proportion of cells. Figure 3 It contains three stacked bar charts, each using three colors to represent cell cycle stages: G1 phase in red, G2M phase in green, and S phase in blue. A legend (phase) is located on the right, labeling the cell cycle stage corresponding to each color.

[0089] scATAC-seq analysis: Fragment files from three samples, totaling approximately 5 million fragments, were loaded. The human genome hg38 was loaded using ArchR to generate Arrow files. A minimum TSS enrichment threshold of 5 was set, and the fragment number range was 3000-40000. Mitochondrial and Y chromosome data were excluded. Cells were filtered based on nucleosome ratio <1, blacklist ratio <0.05, and FRIP >0.15, retaining approximately 80% of the cells. Iterative Latent Semantic Indexing (IterativeLSI) dimensionality reduction was performed, with 4 iterations, 20000 variable features, and dimensions 1-30. Batch effects were corrected using addHarmony, generating UMAP embeddings (30 neighbors, minimum distance 0.5). Cells were divided into three groups—rejuvenated, stable, and senescent—based on epitrace scores. Figure 5-8 As shown. Figure 8 The median Epitrace score was 0.033 in the rejuvenation group, 0.12 in the stable group, and 0.362 in the aging group, with significant differences among the three groups (p<0.001).

[0090] Motif deviation analysis: such as Figure 9 As shown, the motif bias distribution of SIX5 (Z:SIX5_540) was analyzed. The density map showed that the motif enrichment peak of SIX5 transcription factor was high in the rejuvenated cell population, suggesting that its activity is related to the rejuvenated state.

[0091] Data integration: scRNA-seq expression data were integrated into the scATAC-seq project using addGeneIntegrationMatrix. Cell identities were matched based on IterativeLSI dimensionality reduction results, and cells were annotated as “Rejuvenation”, “Stable”, and “Aging” for subsequent trajectory analysis.

[0092] Example 2: Changes in SIX5 expression during skin fibroblast replication and senescence

[0093] 2.1 Experimental Design

[0094] Three primary skin fibroblast cell lines from donors of different ages (SBK-M14, SBK-M35, and SBK-M57, derived from dermal tissue of men aged 14, 35, and 57, respectively) were selected, cultured in complete medium (DMEM, 10% FBS), and passaged. Cells in good condition were selected for subsequent assays.

[0095] Three fibroblast cell lines were cultured to passage 2 and passage 9 in vitro, respectively, and seeded in 6-well plates, divided into 6 groups: Group 1 SBK-M14, passage 3 cells;

[0096] Group 2 SBK-M14, 10th generation cells;

[0097] Group 3 SBK-M35, third generation cells;

[0098] Group 4 SBK-M35, 10th generation cells;

[0099] Group 5 SBK-M57, third generation cells;

[0100] Group 6 SBK-M57, 10th generation cells.

[0101] Once the fusion rate reaches approximately 90%, cells from each group are collected for testing.

[0102] 2.2 Detection Indicators

[0103] SA-β-Gal staining (SA-β-Gal staining kit No.: C0602, Beyotime), qPCR and Western Blot methods were used to detect SIX5 expression, and qPCR and Western Blot methods were used to detect aging-related markers (IL-6, IL-8 and p21) (p21 antibody No.: 10355-1-AP, Proteintech).

[0104] 2.3 Results Analysis: Reference Figure 10A-14BAs shown (where **: P < 0.01, ***: P < 0.001, ****: P < 0.0001), the expression of SA-β-gal and senescence-related genes (p21, IL-6, IL-8) increased with cell passage number in three different donor skin fibroblast strains, indicating a greater degree of cellular senescence. Simultaneously, SIX5 expression decreased, showing a negative correlation with the degree of cellular senescence.

[0105] Example 3: Preparation, Validation, and Screening of SIX5siRNA

[0106] 3.1 Experimental Design

[0107] The desired SIX5siRNA was directly designed and synthesized by a third-party sequence synthesis company (Germ Gene, China).

[0108] After 4 passages of SBK-M35 cell line were cultured in vitro, they were seeded in 12-well plates with a passage confluence of about 70% and divided into 5 groups: the first group was the NC group, which simulated transfection;

[0109] Groups two through five were transfected with SIX5 siRNA (4 siRNAs, 953, 1015, 1949, and 2990 respectively).

[0110] Six hours later, the supernatant was discarded and replaced with high-glucose DMEM medium containing 10% fetal bovine serum. Forty-eight hours after transfection, cells from each group were collected for assays.

[0111] 3.2 Detection indicators: SIX5 expression was detected by qPCR.

[0112] 3.3 Results Analysis: Reference Figure 15 The synthesized siRNA interference fragments all showed good silencing effects on the corresponding genes. The fragment with the best silencing effect was selected for subsequent experiments: SIX5-siRNA-1015.

[0113] Example 4: Effect of SIX5 knockdown on skin fibroblast senescence

[0114] 4.1 Experimental Design

[0115] Primary skin fibroblast cell line (SBK-M35) was selected, cultured in complete medium (DMEM, 10% FBS), and passaged. Cells in good condition were selected for subsequent assays.

[0116] Fibroblasts were cultured in vitro for the second generation and seeded in 12-well plates. The confluence of the cells was about 70%. They were divided into three groups: the first group, the Blank group, did not add siRNA.

[0117] The second group (NC group) simulated transfection.

[0118] The third group, the SIX5 siRNA group, was transfected with SIX5 siRNA. Six hours later, the supernatant was discarded and replaced with high-glucose DMEM medium containing 10% fetal bovine serum. Forty-eight hours after transfection, cells from each group were collected for assays.

[0119] 4.2 Detection indicators: SA-β-Gal staining, qPCR and Western Blot methods were used to detect SIX5 expression, and qPCR and Western Blot methods were used to detect aging-related indicators (IL-6, IL-8 and p21, etc.).

[0120] 4.3 Results Analysis: Reference Figure 16A-20B Transfection with SIX5-siRNA resulted in decreased SIX5 expression, increased SA-β-gal expression in skin fibroblasts, and increased expression of aging-related genes (p21, IL-6, IL-8, etc.), indicating a greater degree of cellular senescence. This suggests that SIX5 is a factor contributing to the senescence of skin fibroblasts.

[0121] Example 5: Design of SIX5 circular RNA

[0122] refer to Figure 26 The nucleotide sequence of the SIX5 circular RNA (FlexCirc system) is shown in SEQ ID NO: 1. The order of the elements is as follows: 5' homologous arm, IG5, ribozyme, substrate E2 sequence, spacer sequence, IRES element, target protein CDS sequence, substrate E1 sequence, IG3, 3' homologous arm. The above FlexCirc system sequence, including the 5' T7 promoter sequence and the EcoRI sequence of the DNA template linearization restriction enzyme, is recombined into the pBlueScript plasmid. For the preparation method of circular RNA, please refer to the applicant's prior application (application number: 202311808436.9, invention title: A vector for preparing circular RNA and a method for its construction).

[0123] Example 6: Design, production, and validation of an AAV2 virus expressing SIX5

[0124] 6.1 Carrier Construction

[0125] The construction of the SIX5 AAV2 overexpression viral vector was based on the recombinant adeno-associated virus type 2 (AAV2) backbone, where the nucleotide sequence of SIX5 is shown in SEQ ID NO: 3. The core functional elements and their sequence are as follows: 5' inverted terminal repeat (5'ITR) → constitutive promoter (CMV) → Kozak sequence → complete CDS sequence of the target gene SIX5 → transcription termination signal (SV40 polyA) → selection marker gene (Neoᵣ, containing its own promoter SV40 Promoter) → 3' inverted terminal repeat (3'ITR). The above complete functional element cluster (5'ITR→3'ITR) was directly synthesized by a third-party sequence synthesis company and ligated with linearized pAAV2 empty vector using T4 DNA to construct the recombinant vector pAAV2-CMV-SIX5. The DNA sequence was confirmed to be correct by sequencing.

[0126] 6.2 Virus Packaging

[0127] Based on the recombinant vector pAAV2-CMV-SIX5 constructed above, high-titer, high-purity SIX5-overexpressing AAV2 viral particles were obtained using the HEK293T cell packaging system through a process of "helper plasmid co-transfection - viral particle release - gradient purification - quality control identification".

[0128] 6.3 Virus titer determination: Real-time quantitative PCR (qPCR) was used. Specific primers (upstream primer: SEQ ID NO.10; downstream primer: SEQ ID NO.11) were designed with the ITR sequence of the AAV2 vector as the target. A standard curve of the ITR sequence was constructed, and the genomic titer (vg / mL) of the virus stock solution was detected. The qualified standard was a titer ≥1×10¹² vg / mL.

[0129] 6.4 Purity identification: The viral capsid protein was separated by SDS-PAGE electrophoresis. After Coomassie brilliant blue staining, three characteristic bands, VP1 (87 kDa), VP2 (73 kDa), and VP3 (62 kDa), were observed, and the band ratio was consistent with 1:1:10.

[0130] 6.5 Results Analysis:

[0131] The SIX5-overexpressing adeno-associated virus was packaged and purified by a third-party virus preparation company. The AAV2-CMV-SIX5 virus titer was 5E+12 GC / mL; the purity identification results were as expected, and no visible impurities were observed; the endotoxin level was <10 EU / mL.

[0132] 6.6 Expression Validation

[0133] HEK293T cells were cultured in vitro for four passages and then seeded into 12-well plates with a confluence of approximately 70%. The cells were divided into three groups: Group 1 was a blank control group (not infected with AAV2 virus); Group 2 was a GFP control group; and Group 3 was a SIX5 overexpression group. Forty-eight hours after infection, cell protein lysates were collected from each group for Western blotting (WB) to detect SIX5 protein expression.

[0134] 6.7 Results Analysis (Expression Validation): Refer to Figure 21 As shown, after infection with AAV2-SIX5, the expression of SIX5 protein increased significantly, and the SIX5-overexpressing adeno-associated virus was successfully constructed.

[0135] Example 7: Validation of the effect of SIX5 overexpression on rejuvenating fibroblasts in aging skin.

[0136] 7.1 Experimental Design

[0137] Primary skin fibroblast cell line (SBK-M57) was selected, cultured in complete medium (DMEM, 10% FBS), and passaged. Cells in good condition were selected for subsequent assays.

[0138] Fibroblasts were cultured in vitro to passage 9 and seeded in 12-well plates with approximately 70% confluence. They were divided into three groups: Group 1 (blank control); Group 2 (GFP control); and Group 3 (SIX5 overexpression group). Cells from each group were collected after 48 hours for analysis.

[0139] 7.2 Detection Indicators

[0140] SA-β-Gal staining, qPCR and Western Blot were used to detect SIX5 expression, and qPCR and Western Blot were used to detect aging-related markers (IL-6, IL-8 and p21, etc.).

[0141] 7.3 Results Analysis: For example... Figures 22A to 22D , Figure 23A and Figure 23B , Figure 24A and Figure 24B as well as Figure 25 As shown, overexpression of SIX5 increased SIX5 protein expression, decreased SA-β-gal expression in skin fibroblasts, decreased expression of aging-related genes (p21, IL-6, IL-8, etc.), and reduced cellular senescence. This indicates that overexpression of SIX5 can reverse skin fibroblast senescence.

[0142] Summarize:

[0143] The above experiments fully demonstrate that transcription factor SIX5 and its related forms, such as nucleic acids and proteins, have significant advantages in reducing cellular senescence levels and can play a role in multiple fields such as pharmaceuticals, health products, and new daily chemical materials.

[0144] The embodiments presented herein are merely selected implementations based on combinations of all possible embodiments. The appended claims should not be limited to the embodiments described herein. Some numerical ranges used in the claims include sub-ranges within them, and variations within these ranges should also be covered by the appended claims.

Claims

1. The use of transcription factor SIX5 or its encoded nucleic acid in the preparation of formulations for reducing cellular senescence levels, characterized in that, The reduction in cellular senescence levels is manifested in at least one or more of the following effects: (1) Reduce the expression level of p21, a aging-related marker in cells; (2) Reduce the expression levels of SASP-related factors IL-6 and / or IL-8 in cells; (3) Reduce the proportion of SA-β-Gal positive cells.

2. The use according to claim 1, characterized in that, The cells are somatic cells, preferably fibroblasts, mesenchymal stem cells or their derivatives.

3. The use according to claim 1 or 2, characterized in that, The stated purpose is to improve or delay cellular replicative senescence and / or stress-induced cellular senescence.

4. The use according to any one of claims 1 to 3, characterized in that, The SIX5-encoded nucleic acid is delivered in the form of RNA, which is either linear RNA or circular RNA.

5. The use according to any one of claims 1 to 3, characterized in that, The SIX5 functions as a protein.

6. A circular RNA, characterized in that, The circular RNA can express the transcription factor SIX5 protein in eukaryotic cells and maintain stable expression of the SIX5 protein during continuous cell culture, thereby reducing the senescence level of the cells.

7. The circular RNA according to claim 6, characterized in that, The circular RNA includes: a 5' homologous arm, a circular element, a ribozyme sequence, an internal ribosome entry site (IRES), and a SIX5 protein coding sequence.

8. The circular RNA according to claim 6 or 7, characterized in that, The nucleotide sequence encoding the SIX5 protein is as shown in SEQ ID NO:1, or is a sequence that has at least 85% homology with it.

9. A recombinant virus, characterized in that, The recombinant virus carries an expression cassette for expressing the transcription factor SIX5, which expresses the SIX5 protein in the host cell, thereby reducing the senescence level of the host cell. The nucleotide sequence encoding the SIX5 protein is shown in SEQ ID NO:3, or is a sequence having at least 85% homology with it.

10. The recombinant virus according to claim 9, characterized in that, The recombinant virus is adeno-associated virus (AAV).

11. The recombinant virus according to claim 10, characterized in that, The AAV is AAV2, AAV6, AAV8, or AAV9.

12. A cell, characterized in that, The cells were treated with circular RNA or recombinant virus as described in any one of claims 6 to 11 and exhibited a reduced cellular senescence phenotype.

13. The cell according to claim 12, characterized in that, The cells are fibroblasts or mesenchymal stem cells.

14. A composition, characterized in that, Includes the circular RNA or recombinant virus as described in any one of claims 6 to 11, and pharmaceutically acceptable vectors.

15. Use of the composition of claim 14 in the preparation of an agent for reducing the level of cellular senescence.

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