ALKBH5 preparation for promoting cell senescence mechanism and RNA epigenetic regulation cell senescence

Abstract

The invention discloses an ALKBH5 preparation for promoting a cell senescence mechanism and RNA epigenetic regulation cell senescence, relates to the technical field of biological medicines, and aims to solve the technical problem that an ALKBH5 regulation cell senescence mechanism is lacked in the prior art. The mechanism is that the retention of ALKBH5 in cytoplasm can promote cell aging; and retention of ALKBH5 in cytoplasm can cause maladjustment of m6A and further cause cell aging. And ALKBH5 is gathered in cytoplasm to cause cytoplasm retention, m6A imbalance and m6A hypermethylation of Cdk2 are caused, further Cdk2 RNA instability is caused, and cell senescence is caused. The key mediating factor of ALKBH5 entering the cell nucleus is Nup62; by synthesizing an m6A-modified RNA probe and transfecting the m6A-modified RNA probe into cells, it is found that the m6A-labeled RNA can effectively reverse ALKBH5 cytoplasm aggregation; and an adeno-associated virus using ALKBH5 labeled by NLS for inhibiting cell senescence in vivo by supplementing ALKBH5 to a cell nucleus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the mechanism by which ALKBH5 promotes cell aging and a preparation for RNA epigenetic regulation of cell aging. Background Art

[0002] Aging is a prominent risk factor for most chronic diseases. The underlying mechanisms of aging can be manifested by hallmarks such as genomic instability, progenitor cell dysfunction, telomere and epigenetic alterations, and protein homeostasis. Cellular senescence has attracted considerable attention as a key mechanism of the aging process. The abnormal organization of the transcriptional machinery associated with aging and senescence remains elusive. Recent studies have demonstrated that posttranscriptional RNA modification plays 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 identifying novel anti-aging targets. N6-methyladenosine (m6A) 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 observed in prematurely aged human mesenchymal stem cells (hMSCs), both a decrease and an increase in m6A have been reported in aged bone marrow mesenchymal stem cells (MSCs). Similarly, in vivo studies have demonstrated a decrease in m6A in primate skeletal muscle and 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 aged brain tissue from mice and humans.

[0003] Emerging evidence suggests that ALKBH5 may be involved in cellular senescence, but the conclusions of two pioneering studies are contradictory and confusing. One report showed that ALKBH5 levels were elevated and m6A levels were low in bone marrow-derived MSCs from elderly individuals. ALKBH5 depletion attenuated MSC senescence. However, another study showed the opposite: m6A levels were elevated and ALKBH5 expression was decreased in senescent MSCs induced by various stimuli, while ALKBH5 knockout promoted senescence. Therefore, comprehensive studies of the regulatory mechanisms of ALKBH5 in different senescent cells and tissues are 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, fibrillar aggregates similar to those found in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). FUS mutations in ALS patients accelerate the assembly of FUS aggregates. However, despite increasing knowledge on the epigenetic role of m6A in aging, whether ALKBH5 regulates cellular senescence through solid-like aggregation remains unclear. Summary of the Invention

[0004] The present invention provides a mechanism for ALKBH5 to promote cell aging and a preparation for RNA epigenetic regulation of cell aging, aiming to solve the technical problem that the prior art lacks a mechanism for ALKBH5 to regulate cell aging.

[0005] To solve the above technical problems, embodiments of the present invention provide: a mechanism by which ALKBH5 regulates cell senescence, wherein the retention of ALKBH5 in the cytoplasm promotes cell senescence.

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

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

[0008] As some optional embodiments of the present invention, the key mediating factor for ALKBH5 to enter the cell nucleus is Nup62.

[0009] As some optional embodiments of the present invention, RNA probes containing m6A modification were synthesized and transfected into cells, and it was found that m6A-labeled RNA could effectively reverse the cytoplasmic aggregation of ALKBH5.

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

[0011] As some optional embodiments of the present invention, the mechanism is obtained by the following steps: obtaining a cell model of replicative aging from different cell lines by cell passaging, preliminarily detecting changes in the distribution of m6A in the cell model, and finding that the retention of ALKBH5 in the cytoplasm can lead to m6A imbalance, thereby inducing cell aging.

[0012] As some optional embodiments of the present invention, the mechanism is obtained by 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 of senescent cells.

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

[0014] In a second aspect, an embodiment of the present invention further provides an RNA epigenetic regulation cell aging preparation, including an adeno-associated virus containing nls-labeled ALKBH5, which is used to inhibit cell aging markers in vivo by supplementing ALKBH5 to the cell nucleus; the anti-cell aging intervention m6A target is Cdk2.

[0015] Compared to the existing technology, the proposed solution achieves the following technical advances: Using cell passage technology, the present invention successfully constructed a cell model of replicative senescence from two different cell lines: the human embryonic lung cell line WI-38 and mouse embryonic MEF cells. Preliminary studies investigated the subcellular localization and distribution of ALKBH5 and alterations in the distribution of m6A in these cell models, revealing that cytoplasmic retention of ALKBH5 promotes cellular senescence and that ALKBH5 retention leads to an imbalance in m6A, thus causing cellular senescence. Based on the aforementioned replicative senescence cell model, the present invention employed MeRIP-seq technology to detect changes in m6A distribution in the senescent cell model. Combined with RNA-seq analysis, the present invention explored changes in the expression of target genes that are regulated by m6A modification, identifying Cdk2 as a key m6A target. Furthermore, through gene overexpression and intervention experiments, the present invention validated the regulatory effect of ALKBH5 on the RNA expression of the m6A target Cdk2. This study transfected synthetic RNA probes (with or without the m6A modification) into cells to observe and compare the effects of the presence or absence of the m6A modification on ALKBH5 protein distribution. The study found that m6A-labeled RNA effectively reversed ALKBH5 cytoplasmic accumulation, thereby slowing cellular aging. Using co-immunoprecipitation and immunofluorescence techniques, the study detected Nup62, a key mediator of ALKBH5 nuclear entry. The study manipulated ALKBH5 nuclear entry by synthesizing NLS (nuclear import signal sequence)-ALKBH5 and NES (nuclear export signal sequence)-ALKBH5 from DNA, and compared the effects of forced ALKBH5 nuclear entry and exit on cellular aging phenotypes. The study used AAV9 adeno-associated virus to deliver the NLS (nuclear import signal sequence)-ALKBH5 into a DOX-induced aging mouse model. Experiments demonstrated that promoting ALKBH5 nuclear entry significantly slowed aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.

[0017] Figure 1 Schematic diagram of ALKBH5 cytoplasmic retention promoting cell senescence involved in the present invention; wherein, Figure 1 A is used to characterize various morphological changes of replicative senescence cells in humans and mice (WI-38 and MEF); Figure 1 B is used to characterize various replicative senescent cells from humans and mice (WI-38 and MEF) by positive β-gal staining; 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) by immunofluorescence observation; Figure 1 D was used to characterize the expression changes of ALKBH5 protein after separation of nuclear and cytoplasmic fractions by Western blot analysis; Figure 2 Schematic diagram of how the retention of ALKBH5 in the cytoplasm leads to an imbalance of m6A and thus to aging; wherein, Figure 2 A is used to characterize the total amount of m6A in various replicative senescent cells of humans and mice (WI-38 and MEF) by semi-quantitative analysis of Dot-blot; Figure 2 B was used to characterize the total amount of m6A in various replicative senescent cells from humans and mice (WI-38 and MEF) by LC-MS / MS analysis; Figure 2 C is used to characterize the total amount of m6A in different cellular components of various replicative senescent cells of humans and mice (WI-38 and MEF) by semi-quantitative analysis by Dot-blot; Figure 2 D was used to characterize the total amount of m6A in different cellular fractions of various replicative senescent cells from humans and mice (WI-38 and MEFs) by LC-MS / MS analysis; Figure 3 Schematic diagram of Cdk2 involved in the present invention as a key m6A regulatory target for cell senescence; wherein, Figure 3 A is used to characterize the distribution of m6A sites in the transcriptome; Figure 3 B is used to characterize the gene loci and expression patterns of m6A changes in various replicative senescent cells of humans and mice (WI-38 and MEF); Figure 3 C was used to characterize the changes in m6A modification of Cdk2 in various replicative senescent cells of humans and mice (WI-38 and MEF) by MeRIP-qPCR detection; Figure 3D is used to characterize the IGV map showing the location and changes of Cdk2 m6A sites in the transcriptome; Figure 3 E was used to characterize the changes in cell morphology and β-gal staining after knocking out CDK2; Figure 3 F was used to characterize the effect of ALKBH5 on the half-life of Cdk2; Figure 4 Schematic diagram of m6A-labeled RNA involved in the present invention effectively reversing ALKBH5 cytoplasmic aggregation and restoring its nuclear entry to alleviate cell aging; wherein, Figure 4 A is used to characterize the binding of ALKBH5 recombinant protein to RNA probes with or without m6A modification; Figure 4 B is used to characterize the co-localization of ALKBH5 and aggregates (Aggresome) observed by immunofluorescence; Figure 4 C is used to characterize the changes in molecular phenotypes of aging after cells were transfected with RNA probes containing or not containing m6A modification by immunoblotting; Figure 4 D is used to characterize the changes in senescence phenotype after cells were transfected with RNA probes containing or not containing m6A modification by β-gal staining; Figure 5 Schematic diagram of the ALKBH5 nuclear translocation involved in the present invention requiring the formation of ALKBH5 droplet phase by binding to nucleoporin p62 (Nup62); wherein, Figure 5 A is used to characterize the ALKBH5 nuclear binding protein detected by co-immunoprecipitation; Figure 5 B is used to characterize the co-immunoprecipitation detection of Nup62 nuclear binding protein; Figure 5 C is used to characterize the distribution expression of ALKBH5 after knockdown of Nup62 by immunofluorescence observation; Figure 5 D is used to characterize the key protein regions of the interaction between ALKBH5 and Nup62 detected by segmented immunoprecipitation; Figure 5 E is used to characterize the co-localization of ALKBH5 segmented protein region and Nup62 by immunofluorescence observation; Figure 6 This is a schematic diagram of the NLS-ALKBH5 involved in the present invention forcibly entering the cell nucleus and having anti-aging effects both in vitro and in vivo; wherein, Figure 6 A is used to characterize the abnormal cytoplasmic localization of ALKBH5 in DOX-treated mouse tissues; Figure 6 B is used to characterize the increase of cell 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 6 D was used to characterize the significant suppression of senescence markers p53 and p16 at day 60; Figure 7Schematic diagram of the research process involved in the present invention.

[0018] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0020] Cellular senescence, a key hallmark of aging and related diseases and a potential therapeutic target, has garnered significant attention for its mechanisms. The role of ALKBH5, an m6A demethylase, in cellular senescence remains controversial. This study reveals that reversible cytoplasmic accumulation of ALKBH5 promotes cellular senescence. Mechanistically, ALKBH5 accumulation leads to its retention in the cytoplasm, which in turn causes dysregulated m6A modification and hypermethylation of Cdk2, further leading to Cdk2 RNA instability and driving cellular senescence. Furthermore, this imbalance in m6A modification exacerbates ALKBH5 accumulation in the cytoplasm in a feedback loop. This study further demonstrates that ALKBH5 nuclear translocation depends on its association with the nucleoporin p62 (Nup62), forming ALKBH5 droplet phases. Cytoplasmic accumulation of ALKBH5 is associated with Nup62. Reduced Nup62 impedes ALKBH5 nuclear translocation, leading to cellular senescence. Notably, administration of m6A-labeled RNA effectively reversed ALKBH5 accumulation in the cytoplasm and restored its nuclear translocation, thereby alleviating cellular senescence. Forced nuclear translocation of NLS-ALKBH5 demonstrated anti-aging effects both in vitro and in vivo. Taken together, these findings reveal a novel mechanism of RNA epigenetic regulation in cellular senescence and provide potential therapeutic targets and strategies for intervention in aging and age-related diseases.

[0021] The technical solution of the present invention is described in detail below through specific embodiments: Example 1: This study aimed to verify whether the retention of ALKBH5 in the cytoplasm promotes the process of cellular aging. To this end, the following experiments were designed: First, we established multiple replicative senescence cell models derived from humans and mice (WI-38 and MEF), which are characterized by a significant enlargement of cell morphology ( Figure 1 A), and positive reaction of β-gal staining ( Figure 1B). It is worth noting that a striking phenomenon was observed by immunofluorescence technology: in senescent WI-38 and MEF cells, part of the ALKBH5 protein was transferred from the nucleus to the cytoplasm ( Figure 1 C).

[0022] Based on this, this study further prepared cytoplasmic and nuclear fractions and tested them using Western blot analysis. The results showed that in different aging cell lines, the level of ALKBH5 protein in the cell nucleus showed a downward trend, while the level in the cytoplasm was relatively increased ( Figure 1 D). This result suggests that the retention of ALKBH5 in the cytoplasm may play an important role in the regulation of cellular senescence.

[0023] 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: Given the m6A demethylase activity of ALKBH5, this study hypothesized that abnormal localization of ALKBH5 in cells may be a key factor leading to m6A modification disorders in senescent cells. Using semi-quantitative Dot-blot analysis, this study found that m6A levels in senescent cells were significantly lower than those in non-senescent control cells (see Figure 2 To obtain more accurate quantitative results, this study used liquid chromatography-tandem mass spectrometry (LC-MS / MS) to quantitatively analyze the m6A content.

[0024] The results showed that the absolute content of m6A in senescent cells was significantly reduced (see Figure 2 B). Based on this finding, this study further performed experiments to separate the nucleus and cytoplasm and measured the m6A content in the cytoplasm and nucleus respectively. Through dot-blot and LC-MS / MS analysis, this study observed that the m6A content in the cytoplasm of aging cells decreased, while the m6A content in the nucleus showed an increasing trend (see Figure 2 C and 2D).

[0025] Example 3: This study aimed to explore the key m6A modification regulatory targets during cellular senescence. To this end, the following experiments were designed: Given the close connection between m6A modification and transcriptional regulation, this study conducted a combined MeRIP-seq and RNA-seq analysis on MEF cells and WI-38 cells in non-senescent and senescent states. The results revealed significant differences in the distribution of m6A modification sites in the transcriptome. In particular, during aging, m6A modification in the coding region of mRNA from the start codon to the stop codon decreased significantly, while m6A modification in the 3' untranslated region (3' UTR) increased significantly ( Figure 3 A). In senescent MEF and WI-38 cells, a total of 4631 genes showed significant changes in m6A modification peaks ( Figure 3 B). Of particular note, the level of single-nucleotide m6A modification of cyclin-dependent kinase 2 (Cdk2) was increased 6-8 fold in senescent cells ( Figure 3 C). This study further confirmed the hypermethylation status of Cdk2 in various senescent cell types (including WI-38 and MSC) by MeRIP-QPCR analysis ( Figure 3 D).

[0026] To evaluate the function of Cdk2, this study knocked out the Cdk2 gene and observed a significant enlargement of both cell and nuclear morphology, accompanied by a significant increase in β-gal staining levels ( Figure 3 E), which supports the view that insufficient Cdk2 expression can promote cellular senescence. These data suggest that hypermethylation of Cdk2 in senescent cells may lead to its mRNA degradation, thereby reducing protein levels and accelerating the cellular senescence process.

[0027] To investigate whether ALKBH5 mislocalization affects cellular senescence by affecting the stability of Cdk2 mRNA, this study used actinomycin D to treat ALKBH5 or NES (nuclear export signal sequence) / NLS (nuclear import signal sequence)-tagged ALKBH5 cells to block transcription. Subsequently, Cdk2 levels were assessed at 0, 2, 4, 6, and 8 hours after treatment. The results showed that NLS-ALKBH5 significantly delayed the degradation of Cdk2 mRNA ( Figure 3 F).

[0028] Based on the above results, this study revealed that the abnormal distribution pattern of ALKBH5 has a direct impact on RNA methylation levels and stability, thereby accelerating the process of cell aging.

[0029] 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 cell aging, this study designed the following experiments: To further explore the feedback regulation mechanism of the methylation status of RNA substrates on the function of ALKBH5 protein, this study synthesized RNA probes with or without m6A modification and conducted comparative analysis. First, in vitro experiments were conducted using ALKBH5 recombinant protein and RNA probes with or without m6A modification. The results showed that RNA probes without m6A modification promoted the aggregation of ALKBH5 protein, while RNA probes with m6A modification did not. Figure 4 A). Subsequently, RNA probes containing 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 (Aggresomes), while RNA probes containing m6A modification did not have this effect ( Figure 4 B). In addition, after transfection of RNA probes without m6A modification into young cells, upregulation of aging-related molecular markers p21 and p53, as well as cell damage marker γH2AX protein expression was observed, indicating that RNA probes without m6A modification can promote the aging process of young WI-38 cells; conversely, RNA probes containing m6A modification can help reduce the expression levels of aging-related molecules in aging cells ( Figure 4 C). Further β-galactosidase staining experiments also confirmed that RNA probes containing m6A modifications can significantly delay cell senescence ( Figure 4 D).

[0030] Example 5: To verify that ALKBH5 nuclear translocation requires binding to nucleoporin p62 (Nup62) to form ALKBH5 droplet phase, this study designed the following experiments: Given that abnormal ALKBH5 nuclear translocation promotes cellular aging, this study aims to further explore the key molecules that mediate ALKBH5 protein nuclear translocation in order to discover new anti-aging strategies.

[0031] Therefore, this study used immunoprecipitation (IP) technology to preliminarily confirm that nucleoporin p62 (Nup62) is the key mediating protein for nuclear entry ( Figure 5 AB). Further knockdown of Nup62 expression revealed that low Nup62 expression would affect the nuclear entry of ALKBH5 ( Figure 5 C).

[0032] To further explore the key protein domains of ALKBH5's interaction with Nup62, this study divided ALKBH5 into segment B (1-100aa) and segment C (101-394aa). Immunoprecipitation (IP) technology revealed that segment C (101-394aa) binds more significantly to Nup62. Figure 5 C).

[0033] Further immunofluorescence observation revealed that segment B of ALKBH5 enters the cell nucleus after binding to Nup62, while segment C of ALKBH5 aggregates in the cytoplasm and cannot promote its entry into the cell nucleus when binding to Nup62.

[0034] The above results indicate that ALKBH5 protein requires the assistance of Nup62 to enter the nucleus, but the aggregation state of ALKBH5 protein in the cell will also affect its nuclear entry efficiency.

[0035] 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, this study specifically designed the following experiments: At the cellular level, this study found that NLS-tagged ALKBH5 can effectively correct the imbalance of nuclear m6A, thereby preventing cellular senescence. Based on this finding, this study recommends using NLS-tagged ALKBH5 for in vivo intervention of cellular senescence.

[0036] Given that animal aging models require a long time to develop natural aging characteristics, this study evaluated the therapeutic efficacy of NLS-ALKBH5 in a doxorubicin (DOX)-induced aging mouse model. Cellular senescence was induced by intraperitoneal injection of 5 mg / kg DOX (four consecutive injections, four days apart). The study confirmed that abnormal cytoplasmic localization of ALKBH5 in DOX-treated mouse tissues coincided with an increase in cellular senescence markers ( Figure 6 A and 6B). This study evaluated the effect of adeno-associated virus (AAV) encoding NLS-ALKBH5 in vivo intervention. Given that the phenotype induced by DOX in kidney tissue is more significant, this study utilized an adeno-associated virus with an AAV9 promoter, which has a strong affinity for kidney and heart tissue in vivo. Cdkn2a-Ki-Luc-2A-tdTomato-2A-CreERT2-WPRE-pA reporter mice (NM-KI-18039) were used for in vivo imaging of p16-positive senescent cells. After AAV virus intervention, four intraperitoneal injections of DOX were performed to assess cell senescence. The results are shown in Figure 2. Figure 6 Finally, mice receiving AAV9 expressing NLS-ALKBH5 showed a significant reduction in DOX-induced p16-positive senescent cells compared to AAV9 controls ( Figure 6 C), and significantly inhibited the senescence markers p53 and p16 on day 60 ( Figure 6 D). In summary, NLS-tagged ALKBH5 can improve the senescence state of tissue cells, providing a potential strategy for anti-aging treatment.

[0037] According to the above analysis, if Figure 7As shown in the figure, the technical solutions proposed in this study can be divided into the following five main aspects: First, this study used cell passage technology to culture replicative senescence cell models from multiple cell lines and conducted 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 process of cell senescence. Secondly, this study also used cell passaging technology to obtain replicative senescence cell models from different cell lines and conducted preliminary testing on the distribution of m6A modification in the models. The study found that the retention of ALKBH5 in the cytoplasm can lead to an imbalance in m6A modification, thereby inducing cellular senescence. Third, this study revealed Cdk2 as a key m6A modification target in senescent cells by combined MeRIP-seq and RNA-seq analysis of MEF and WI-38 cells; Fourth, this study used immunoprecipitation and immunofluorescence techniques to verify Nup62, a key mediator for ALKBH5 to enter the cell nucleus; Finally, this study used AAV9 adeno-associated virus to deliver NLS (nuclear entry signal sequence)-ALKBH5 to a DOX-induced mouse aging model in vivo, confirming the mandatory localization of NLS-ALKBH5 in the cell nucleus and its significant anti-aging effect, which was verified both in vitro and in vivo experiments.

[0038] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. ALKBH5 regulates the cell aging mechanism, characterized by The mechanism is that the retention of ALKBH5 in the cytoplasm promotes cellular senescence.

2. The mechanism of ALKBH5 regulating cell aging according to claim 1, characterized in that: The mechanism is that the retention of ALKBH5 in the cytoplasm leads to m6A dysregulation, thereby inducing cell senescence.

3. The mechanism of ALKBH5 regulating cell aging according to claim 1, characterized in that: The mechanism is that ALKBH5 accumulates in the cytoplasm and causes cytoplasmic retention, leading to m6A dysregulation and m6A hypermethylation of Cdk2, which in turn causes Cdk2 RNA instability and triggers cell aging.

4. The mechanism of ALKBH5 regulating cell aging according to claim 1, characterized in that The key mediating factor for ALKBH5 to enter the cell nucleus is Nup62.

5. The mechanism of ALKBH5 regulating cell aging according to claim 1, characterized in that By synthesizing RNA probes containing m6A modification and transfecting them into cells, we found that m6A-labeled RNA could effectively reverse the cytoplasmic aggregation of ALKBH5.

6. The mechanism of ALKBH5 regulating cell aging according to claim 1, characterized in that The mechanism was obtained through the following steps: obtaining a cell model of replicative aging from different cell lines through cell passaging, preliminarily detecting changes in the subcellular localization distribution of ALKBH5 in the cell model, and finding that the retention of ALKBH5 in the cytoplasm promotes cell aging.

7. The mechanism of ALKBH5 regulating cell aging according to claim 2, characterized in that: The mechanism was obtained through the following steps: obtaining a cell model of replicative aging through cell passaging from different cell lines, preliminarily detecting changes in the distribution of m6A in the cell model, and finding that the retention of ALKBH5 in the cytoplasm would lead to m6A imbalance, thereby inducing cell aging.

8. The mechanism of ALKBH5 regulating cell aging according to claim 3, characterized in that: The mechanism was obtained by the following steps: Combined MeRIP-seq and RNA-Seq analysis of MEF and WI-38 cells revealed that Cdk2 is a key m6A modification target in senescent cells.

9. The mechanism of ALKBH5 promoting cell aging according to claim 4, characterized in that: The mechanism was obtained through the following steps: Nup62, a key mediator of ALKBH5 entering the cell nucleus, was detected by co-immunoprecipitation and immunofluorescence techniques.

10. RNA epigenetic regulation of cell aging preparation, characterized in that An adeno-associated virus comprising nls-tagged ALKBH5 is used to inhibit cell aging markers in vivo by supplementing ALKBH5 to the cell nucleus; the anti-cell aging intervention m6A target is Cdk2.

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