ALKBH5 promotes cell aging mechanism and RNA epigenetic regulation cell aging preparation

Abstract

The application discloses an ALKBH5-promoted cell aging mechanism and an RNA epigenetic regulation cell aging preparation, relates to the technical field of biological medicine, and aims to solve the technical problem of lack of ALKBH5-regulated cell aging mechanism in the prior art. The mechanism is that retention of ALKBH5 in the cytoplasm promotes cell aging; retention of ALKBH5 in the cytoplasm leads to m6A disorder, and further causes cell aging; aggregation of ALKBH5 in the cytoplasm causes cytoplasm retention, leads to m6A disorder and m6A hypermethylation of Cdk2, and further causes Cdk2 RNA instability, and further causes cell aging; a key mediating factor for ALKBH5 entering the nucleus is Nup62; by transfecting an RNA probe containing m6A modification into cells, it is found that m6A-labeled RNA can effectively reverse cytoplasmic aggregation of ALKBH5; and an adeno-associated virus of NLS-labeled ALKBH5 is used for supplementing ALKBH5 to the nucleus in vivo, so as to inhibit cell aging.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the mechanism by which ALKBH5 promotes cell senescence and RNA epigenetic regulation of cell senescence preparations. Background Technology

[0002] Aging is a prominent risk factor for most chronic diseases. The underlying mechanisms of aging can manifest as genomic instability, progenitor cell dysfunction, telomere and epigenetic alterations, and protein homeostasis. Cellular senescence, as a key mechanism in the aging process, has attracted considerable attention. Abnormal tissues within the transcriptional apparatus associated with aging remain elusive. Recent studies have shown that post-transcriptional modifications of RNA play a crucial role in aging, primarily through specific RNA epigenetic modifications. This provides a new research perspective for exploring the mechanisms of cellular senescence and discovering new anti-aging targets. N6-methyladenosine (m6A) modification is the most abundant RNA modification in mammalian RNA. Currently, the role of m6A in aging remains unclear. Although a decrease in m6A has been found in prematurely aging human mesenchymal stem cells (hMSCs), a reduction or increase in m6A has been reported in aging bone marrow mesenchymal stem cells (MSCs). Similarly, in vivo studies have shown a reduction in m6A in primate skeletal muscle, and a reduction in specific brain regions involved in cognitive processes in aged mice and humans with Alzheimer's disease. In contrast, Shafik found significantly more m6A sites in aging brain tissue of mice and humans.

[0003] Emerging evidence suggests that ALKBH5 may be involved in cellular senescence, while the conclusions of two pioneering studies are contradictory and perplexing. One report showed higher ALKBH5 levels and lower m6A levels in bone marrow-derived MSCs of older adults. ALKBH5 deficiency alleviated MSC senescence. However, another study showed the opposite result: in senescent bone marrow mesenchymal stem cells induced by various stimuli, m6A levels were elevated and ALKBH5 expression was decreased, while ALKBH5 knockout promoted senescence. Therefore, a comprehensive study of the regulatory mechanisms of ALKBH5 in different senescent cells and tissues is needed to understand its role in aging. Previous studies have elucidated the mechanisms of protein aggregation involved in cellular senescence and age-related diseases. PKM2 aggregation has recently been shown to drive metabolic reprogramming during aging. Studies have shown that tau protein aggregation is associated with brain cell senescence. The prion-like domain protein FUS can form solid-like fibrous aggregates, similar to those found in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). FUS mutants in ALS patients accelerate the assembly of FUS aggregates. However, despite the growing epigenetic knowledge of m6A in the aging process, it remains unclear whether ALKBH5 regulates cellular senescence through solid-like aggregation. Summary of the Invention

[0004] This invention provides an ALKBH5-promoted cell senescence mechanism and an RNA epigenetic regulation of cell senescence formulation, aiming to solve the technical problem of the lack of ALKBH5-regulated cell senescence mechanism in the prior art.

[0005] To address the aforementioned technical problems, this invention provides an ALKBH5-mediated cell senescence mechanism, wherein the retention of ALKBH5 in the cytoplasm promotes cell senescence.

[0006] As some alternative embodiments of the present invention, the mechanism is that the retention of ALKBH5 in the cytoplasm leads to m6A dysregulation, thereby triggering cell senescence.

[0007] As some optional embodiments of the present invention, the mechanism is that ALKBH5 accumulates in the cytoplasm, causing cytoplasmic retention, leading to m6A dysregulation and m6A hypermethylation of Cdk2, which in turn causes Cdk2 RNA instability and triggers cell senescence.

[0008] As some optional embodiments of the present invention, the key mediator of ALKBH5 entering the cell nucleus is Nup62.

[0009] As some alternative embodiments of the present invention, by synthesizing an RNA probe containing m6A modification and transfecting it into cells, it was found that m6A-labeled RNA can effectively reverse ALKBH5 cytoplasmic aggregation.

[0010] As some optional embodiments of the present invention, the mechanism is obtained through the following steps: obtaining a replicative senescent cell model from different cell lines through cell passage, preliminarily detecting changes in the subcellular localization distribution of ALKBH5 in the cell model, and finding that the retention of ALKBH5 cytoplasm promotes cell senescence.

[0011] As some optional embodiments of the present invention, the mechanism is obtained through the following steps: obtaining a replicative senescent cell model from different cell lines through cell passage, preliminarily detecting changes in m6A distribution in the cell model, and finding that the retention of ALKBH5 in the cytoplasm leads to m6A dysregulation, thereby inducing cell senescence.

[0012] As some optional embodiments of the present invention, the mechanism is obtained through the following steps: MeRIP-seq and RNA-Seq combined analysis of MEF and WI-38 cells revealed that Cdk2 is a key m6A modification target in senescent cells.

[0013] As some optional embodiments of the present invention, the mechanism is obtained by the following steps: detecting Nup62, a key mediator of ALKBH5 entry into the cell nucleus, by immunoprecipitation and immunofluorescence techniques.

[0014] Secondly, embodiments of the present invention also provide an RNA epigenetic regulatory cell senescence agent, including an adeno-associated virus labeled with ALKBH5 by nls, for use in vivo to inhibit cell senescence markers by supplementing the cell nucleus with ALKBH5; the anti-cellular senescence intervention target m6A is Cdk2.

[0015] Compared to existing technologies, the proposed solution of this invention achieves the following technological advancements: This invention successfully constructed a replicative senescence cell model using cell passage technology from two different cell lines: human embryonic lung cell line WI-38 and mouse embryonic MEF cells. Preliminary studies investigated the subcellular localization changes of ALKBH5 and alterations in m6A distribution within the cell model, revealing that ALKBH5 cytoplasmic retention promotes cell senescence, and that ALKBH5 retention leads to m6A imbalance, thus causing cell senescence. Based on the aforementioned replicative senescence cell model, this invention used MeRIP-seq technology to detect changes in m6A distribution within the senescent cell model, and combined this with RNA-seq analysis to explore changes in the expression of target genes dependent on m6A modification regulation, identifying Cdk2 as a key m6A target. Furthermore, this invention verified the regulatory role of ALKBH5 on the RNA expression of the m6A target Cdk2 through gene overexpression and intervention experiments. This invention transfects cells with synthesized RNA probes (with or without m6A modification) to observe and compare the effects of m6A modification on ALKBH5 protein distribution, revealing that m6A-labeled RNA effectively reverses ALKBH5 cytoplasmic aggregation, thereby slowing cellular senescence. This invention also utilizes immunoprecipitation and immunofluorescence techniques to detect Nup62, a key mediator of ALKBH5 entry into the nucleus. Furthermore, this invention artificially manipulates the ALKBH5 nuclear entry process by synthesizing NLS (nuclear entry signal sequence)-ALKBH5 and NES (nuclear exit signal sequence)-ALKBH5 from DNA, comparing and analyzing the effects of forced ALKBH5 nuclear entry / exit on cellular senescence phenotypes. Finally, this invention delivers NLS-ALKBH5 to a DOX-induced mouse aging model via AAV9 adeno-associated virus, demonstrating that promoting ALKBH5 entry into the nucleus significantly delays the aging process. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0017] Figure 1 This is a schematic diagram illustrating the ALKBH5 cytoplasm retention-promoting cell senescence method involved in this invention; wherein, Figure 1 A was used to characterize various morphological changes in replicative senescent cells in humans and mice (WI-38 and MEF); Figure 1 B is used to characterize β-gal staining positivity in various replicative senescent cells of humans and mice (WI-38 and MEF); Figure 1 C was used to characterize the subcellular localization of ALKBH5 protein in various replicative senescent cells of humans and mice (WI-38 and MEF) for immunofluorescence observation; Figure 1 D was used to characterize the expression changes of ALKBH5 protein after separation of nuclear and cytoplasmic components in Western blot analysis.

[0018] Figure 2 This is a schematic diagram illustrating how the retention of ALKBH5 in the cytoplasm leads to m6A imbalance and thus senescence, as described in this invention. Figure 2 A was used to characterize the total m6A content in various replicative senescent cells of humans and mice (WI-38 and MEF) using a Dot-blot semi-quantitative analysis. Figure 2 B was used to characterize the total m6A content in various replicative senescent cells of humans and mice (WI-38 and MEF) for LC-MS / MS analysis; Figure 2 C was used to characterize the total m6A content of different cellular components in various replicative senescent cells of humans and mice (WI-38 and MEF) using a Dot-blot semi-quantitative analysis. Figure 2 D was used to characterize the total m6A content of different cellular components in various replicative senescent cells of humans and mice (WI-38 and MEF) in LC-MS / MS analysis.

[0019] Figure 3 This is a schematic diagram illustrating that Cdk2, involved in this invention, is a key m6A regulatory target for cell senescence; wherein, Figure 3 A is used to characterize the distribution of the m6A site in the transcriptome; Figure 3 B was used to characterize the gene loci and their expression in different cells of various replicative senescent cells in humans and mice (WI-38 and MEF); Figure 3 C was used to characterize the changes in m6A modification of Cdk2 in different cells of various replicating senescent cells in humans and mice (WI-38 and MEF) detected by MeRIP-qPCR. Figure 3D is used to characterize the IGV diagram, which shows the location and changes of the Cdk2 m6A site in the transcriptome. Figure 3 E was used to characterize changes in cell morphology and β-gal staining after CDK2 knockout; Figure 3 F was used to characterize the effect of ALKBH5 on the half-life of Cdk2;

[0020] Figure 4 This is a schematic diagram illustrating how m6A-labeled RNA, as described in this invention, effectively reverses ALKBH5 cytoplasmic aggregation and restores its nuclear entry to alleviate cellular senescence; wherein, Figure 4 A is used to characterize the binding of the recombinant protein ALKBH5 to RNA probes with or without m6A modification; Figure 4 B is used to characterize the colocalization of ALKBH5 and aggregates observed by immunofluorescence; Figure 4 C is used to characterize changes in senescence molecular phenotypes after cells are transfected with RNA probes containing or without m6A modification, as detected by Western blotting. Figure 4 D is used to characterize changes in senescence phenotypes after cells are transfected with RNA probes containing or without m6A modification, as detected by β-gal staining.

[0021] Figure 5 This is a schematic diagram illustrating the ALKBH5 nuclear translocation involved in this invention, which requires the formation of an ALKBH5 droplet phase through the binding of nuclear porin p62 (Nup62); wherein, Figure 5 A was used to characterize the ALKBH5 nuclear binding protein detected by immunoprecipitation. Figure 5 B was used to characterize the Nup62 nuclear-binding protein detected by immunoprecipitation. Figure 5 C was used to characterize the distribution and expression of ALKBH5 after Nup62 knockdown, as observed by immunofluorescence. Figure 5 D is used to characterize the key protein regions involved in the ALKBH5-Nup62 interaction in segmented immunoprecipitation detection. Figure 5 E was used to characterize the co-localization of the ALKBH5 segmented protein region and Nup62 observed by immunofluorescence.

[0022] Figure 6 This is a schematic diagram illustrating the NLS-ALKBH5 component involved in this invention forcibly entering the cell nucleus and exhibiting anti-aging effects both in vitro and in vivo; wherein, Figure 6 A was used to characterize the abnormal cytoplasmic localization of ALKBH5 in DOX-treated mouse tissues. Figure 6 B was used to characterize the increase of cellular senescence markers in DOX-treated mouse tissues; Figure 6 C was used to characterize that mice receiving AAV9 expressing NLS-ALKBH5 showed a significant reduction in DOX-induced p16-positive senescent cells; Figure 6D was used to characterize the significant inhibition of aging biomarkers p53 and p16 on day 60;

[0023] Figure 7 This is a schematic diagram of the research process involved in this invention.

[0024] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Cellular senescence, as a key marker of aging and related diseases and a potential therapeutic target, has attracted much attention for its mechanistic study. Among numerous studies, the role of ALKBH5, one of the m6A demethylases, in cellular senescence remains controversial. This study reveals the promoting effect of reversible ALKBH5 accumulation in the cytoplasm on cellular senescence. Mechanistically, ALKBH5 accumulation leads to its retention in the cytoplasm, which in turn causes dysregulation of m6A modification and m6A hypermethylation of Cdk2. This further leads to Cdk2 RNA instability, thereby promoting the cellular senescence process. Furthermore, the imbalance of m6A modification exacerbates ALKBH5 accumulation in the cytoplasm in a feedback loop. This study further confirms that the nuclear translocation of ALKBH5 depends on binding to nucleoporin p62 (Nup62), forming an ALKBH5 droplet phase, and that ALKBH5 accumulation in the cytoplasm is associated with Nup62. Reduction of Nup62 hinders the nuclear translocation of ALKBH5, thus leading to cellular senescence. Notably, the application of m6A-labeled RNA effectively reversed the accumulation of ALKBH5 in the cytoplasm and restored its nuclear translocation, thereby alleviating cellular senescence. Forced nuclear translocation of NLS-ALKBH5 demonstrated anti-aging effects in both in vitro and in vivo experiments. In summary, this study reveals a novel mechanism of RNA epigenetic regulation in cellular senescence and provides potential therapeutic targets and strategies for intervening in aging and age-related diseases.

[0027] The technical solution of the present invention will be described in detail below through specific embodiments:

[0028] Example 1: This study aimed to verify whether the retention of ALKBH5 in the cytoplasm promotes the cellular senescence process. The following experiment was designed for this purpose:

[0029] First, we constructed several replicative senescent cell models derived from humans and mice (WI-38 and MEF), which were characterized by a significant increase in cell morphology. Figure 1 A), and a positive reaction to β-gal staining ( Figure 1 B). Notably, a striking phenomenon was observed using immunofluorescence: in senescent WI-38 and MEF cells, a portion of the ALKBH5 protein was translocated from the nucleus to the cytoplasm. Figure 1 C).

[0030] Based on this, this study further prepared cytoplasmic and nuclear components and detected them using Western blot analysis. The results showed that in different senescent cell lines, the level of ALKBH5 protein in the nucleus decreased, while the level in the cytoplasm relatively increased. Figure 1 (D). This result suggests that ALKBH5 retention in the cytoplasm may play an important role in the regulation of cellular senescence.

[0031] Example 2: This study aimed to investigate how the retention of ALKBH5 in the cytoplasm leads to an imbalance in m6A modification, thereby inducing cellular senescence. To this end, the following experiments were designed and implemented:

[0032] Given the m6A demethylase activity of ALKBH5, this study hypothesizes that the aberrant localization of ALKBH5 in cells may be a key factor leading to dysregulation of m6A modification in senescent cells. Through semi-quantitative Dot-blot analysis, this study found that the m6A level in senescent cells was significantly lower than that in non-senescent control cells (see...). Figure 2 A). To obtain more accurate quantitative results, this study used liquid chromatography-tandem mass spectrometry (LC-MS / MS) to quantitatively analyze the m6A content.

[0033] The results showed that the absolute content of m6A in senescent cells was significantly reduced (see...). Figure 2 Based on this finding, this study further performed a separation experiment of the cell nucleus and cytoplasm, and measured the m6A content in the cytoplasm and nucleus separately. Through dot-blot and LC-MS / MS analysis, this study observed a decrease in the m6A content in the cytoplasm of senescent cells, while the m6A content in the nucleus showed an increasing trend (see B). Figure 2 (C and 2D).

[0034] Example 3: This study aims to explore key m6A modification regulatory targets during cellular senescence, and the following experiment was designed for this purpose:

[0035] Given the close relationship between m6A modification and transcriptional regulation, this study performed combined MeRIP-seq and RNA-Seq analyses on non-senescent and senescent MEF and WI-38 cells. The results revealed significant differences in the distribution of m6A modification sites within the transcriptome, particularly during senescence. M6A modification in the coding region of mRNA decreased significantly from the start codon to the stop codon, while m6A modification in the 3' untranslated region (3' UTR) significantly increased. Figure 3 A). In senescent MEF and WI-38 cells, the m6A modification peaks of a total of 4631 genes showed significant changes (A). Figure 3 B). Of particular note is that in senescent cells, the level of single nucleotide m6A modification of cyclin-dependent kinase 2 (Cdk2) is increased by 6-8 times (B). Figure 3 C). This study further confirmed the hypermethylation status of Cdk2 in various senescent cell types (including WI-38 and MSC) using MeRIP-qPCR analysis. Figure 3 D).

[0036] To assess the function of Cdk2, this study knocked out the Cdk2 gene, observing significant enlargement of both cell and nuclear morphology, accompanied by a significant increase in β-gal staining levels. Figure 3 This supports the idea that insufficient Cdk2 expression can promote cellular senescence. These data suggest that hypermethylation of Cdk2 in senescent cells may lead to its mRNA degradation, which in turn reduces protein levels and accelerates the cellular senescence process.

[0037] To investigate whether ALKBH5 mislocalization affects cellular senescence by influencing Cdk2 mRNA stability, this study treated ALKBH5 cells or NES (nuclear export signal sequence) / NLS (nuclear import signal sequence) labeled ALKBH5 cells with actinomycin D to block transcription. Cdk2 levels were then assessed at 0, 2, 4, 6, and 8 hours post-treatment. Results showed that NLS-ALKBH5 significantly delayed the degradation of Cdk2 mRNA. Figure 3 F).

[0038] In summary, this study reveals that the abnormal distribution pattern of ALKBH5 has a direct impact on RNA methylation levels and stability, thereby accelerating the process of cellular senescence.

[0039] Example 4: To verify the role of m6A-modified RNA in reversing the aggregation of ALKBH5 protein in the cytoplasm and restoring its nuclear localization to alleviate cellular senescence, the following experiment was designed in this study:

[0040] To further investigate the feedback regulation mechanism of RNA substrate methylation status on ALKBH5 protein function, this study conducted a comparative analysis using RNA probes with and without m6A modification. First, in vitro incubation experiments were performed using recombinant ALKBH5 protein and RNA probes with and without m6A modification. The results showed that RNA probes without m6A modification promoted ALKBH5 protein aggregation, while this phenomenon was not observed with RNA probes containing m6A modification. Figure 4 A). Subsequently, RNA probes with or without m6A modification were transfected into senescent cells. It was found that RNA probes without m6A modification promoted the formation of ALKBH5 protein aggregates, while RNA probes with m6A modification did not show this effect. Figure 4 B). Furthermore, transfection of young cells with RNA probes without m6A modification resulted in upregulation of the expression of aging-related molecular markers p21 and p53, as well as the cell damage marker γH2AX protein, indicating that RNA probes without m6A modification can promote the aging process of young WI-38 cells; conversely, RNA probes containing m6A modification help reduce the expression levels of aging-related molecules in senescent cells. Figure 4 C). Further β-galactosidase staining experiments also confirmed that RNA probes containing m6A modification can significantly delay cell senescence. Figure 4 D).

[0041] Example 5: To verify that ALKBH5 nuclear translocation requires binding to nuclear porin p62 (Nup62) to form an ALKBH5 droplet phase, the following experiment was designed in this study:

[0042] Given that abnormal nuclear translocation of ALKBH5 promotes cellular senescence, this study aims to further explore the key molecules that mediate the nuclear translocation of ALKBH5 protein in order to discover new anti-aging strategies.

[0043] Therefore, this study, through immunoprecipitation (IP) analysis, preliminarily confirmed that nucleoporin p62 (Nup62) is a key mediator of nuclear translocation. Figure 5 AB). Further knockdown of Nup62 expression revealed that low Nup62 expression affects ALKBH5 nuclear entry ( ). Figure 5 C).

[0044] To further explore the key protein domains involved in the interaction between ALKBH5 and Nup62, this study divided ALKBH5 into a B segment (1-100 aa) and a C segment (101-394 aa). Immunoprecipitation (IP) revealed that the C segment (101-394 aa) binds more significantly to Nup62. Figure 5 C).

[0045] Further immunofluorescence observation revealed that the B segment of ALKBH5 enters the cell nucleus after binding with Nup62, while the C segment of ALKBH5 aggregates in the cytoplasm and does not promote its entry into the cell nucleus upon binding with Nup62.

[0046] The above results indicate that Nup62 is required for ALKBH5 protein to enter the nucleus, but the aggregation state of ALKBH5 protein in the cell also affects its nuclear entry efficiency.

[0047] Example 6: To verify that NLS-ALKBH5 has the ability to enter the cell nucleus and exert anti-aging effects in vitro and in vivo, the following experiment was specifically designed in this study:

[0048] At the cellular level, this study found that NLS-labeled ALKBH5 can effectively correct the imbalance of nuclear m6A, thereby preventing cellular senescence. Based on this finding, this study recommends using NLS-labeled ALKBH5 for in vivo intervention in cellular senescence.

[0049] Given that animal aging models require a long time to exhibit natural aging characteristics, this study used an doxorubicin (DOX)-induced aging mouse model to evaluate the therapeutic effect of NLS-ALKBH5. Cellular senescence was induced by intraperitoneal injection of 5 mg / kg DOX (4 consecutive injections, 4 days apart). The study confirmed that in DOX-treated mouse tissues, abnormal cytoplasmic localization of ALKBH5 occurred synchronously with an increase in cellular senescence markers. Figure 6 A and 6B). This study evaluated the effects of adeno-associated virus (AAV) encoding NLS-ALKBH5 in vivo intervention. Given the significant phenotype induced by DOX in kidney tissue, this study utilized an AAV with the AAV9 promoter, which exhibits strong affinity for both kidney and heart tissues in vivo. In vivo imaging of p16-positive senescent cells was performed using Cdkn2a-Ki-Luc-2A-tdTomato-2A-CreERT2-WPRE-pA reporter mice (NM-KI-18039). Following AAV viral intervention, four intraperitoneal injections of DOX were administered, and cellular senescence was assessed. The results are as follows: Figure 6 As shown in C. Ultimately, compared to the AAV9 control group, mice receiving AAV9 expressing NLS-ALKBH5 showed a significant reduction in DOX-induced p16-positive senescent cells ( ). Figure 6 C), and significantly inhibited aging markers p53 and p16 on day 60. Figure 6 D). In summary, NLS-labeled ALKBH5 can improve the senescent state of tissue cells, providing a potential strategy for anti-aging therapy.

[0050] Based on the foregoing analysis, such as Figure 7As shown, the technical solution proposed in this study can be divided into the following five main aspects:

[0051] First, this study cultured a replicative senescent cell model from multiple cell lines using cell passage technology, and conducted a preliminary detection of the subcellular localization distribution of ALKBH5 in the model. The results showed that the retention of ALKBH5 in the cytoplasm can promote the senescence process.

[0052] Secondly, this study also obtained replicative senescent cell models from different cell lines using cell passage technology, and conducted a preliminary detection of the distribution of m6A modification in the models. The study found that the retention of ALKBH5 in the cytoplasm leads to an imbalance of m6A modification, which in turn triggers cell senescence.

[0053] Third, this study revealed Cdk2 as a key m6A modification target in senescent cells through combined MeRIP-seq and RNA-Seq analysis of MEF and WI-38 cells;

[0054] Fourth, this study used techniques such as immunoprecipitation and immunofluorescence to verify Nup62, a key mediator of ALKBH5 entry into the cell nucleus.

[0055] Finally, this study demonstrated the forced localization of NLS-ALKBH5 in the cell nucleus and its significant anti-aging effect by delivering NLS (nuclear entry signal sequence)-ALKBH5 to a DOX-induced mouse aging model using AAV9 adeno-associated virus. This effect was verified in both in vitro and in vivo experiments.

[0056] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. Application of NLS-labeled ALKBH5 adeno-associated virus in the preparation of agents to inhibit cell senescence.

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