Circrna as cellular senescence marker and regulatory target, and use thereof

Abstract

Disclosed are use of a circRNA molecule as a diagnostic marker in a cell senescence diagnosis kit, and use thereof as a treatment target for cell senescence. The present invention finds that circHERC1 is lowly expressed in both cell models of natural senescence and induced senescence. Overexpression of circHERC1 in endothelial cells can improve the proliferation capacity of the endothelial cells, effectively extend the telomere length of the cells, activate telomerase, reduce the β-galactosidase positive rate of the cells, and reduce the expression of all senescence-associated proteins. Introduction of exogenous circHERC1 into mice can effectively delay the senescence of various organs and the shortening of telomeres in aged mice and reduce the β-galactosidase positive rates of various organs of aged mice.

Description

A circRNA as a marker and regulatory target of cellular senescence and its applications Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a circRNA associated with telomerase activity and cellular senescence and its applications. Background Technology

[0002] Aging is a complex process influenced by both genetic and environmental factors, closely related to the permanent and progressive decline of physiological cells, and is one of the reasons for the increasingly serious aging of the population. Studies have shown that aging can significantly increase the body's morbidity, namely age-related diseases such as diabetes, Alzheimer's disease, cardiovascular disease, and neurodegenerative diseases.

[0003] circRNAs are a class of non-coding RNA molecules found in living organisms that lack a 5' cap and a 3' poly(A) tail and form a circular structure through covalent bonds. With the development of high-throughput sequencing and bioinformatics analysis techniques, research on circRNAs has become quite extensive, and in recent years, circRNAs have received widespread attention due to their unique structure and function. circRNAs can act as circRNA "sponges," transcriptional regulators, and play important roles in various biological functions through binding to RNA-binding proteins. Compared to other non-coding RNAs, circRNAs are more stable due to their circular structure and exhibit stronger tissue and disease specificity, making them of significant research value in various diseases and physiological processes. Technical issues

[0004] The purpose of this invention is to provide a circRNA that can extend the length of cellular telomeres by activating telomerase, thereby inhibiting cellular and organismal aging. Technical solutions

[0005] Based on the above objectives, and based on the circular RNA (circ_0035796) disclosed in CN115948548A that is associated with the development and progression of non-small cell lung cancer, this invention first provides the application of the circRNA molecule as a diagnostic biomarker in a cell senescence diagnostic kit. The sequence of the circRNA molecule is shown as bases 21-1163 of SEQ ID NO. 1. In this invention, the circRNA molecule is named "circHERC1" or "circ_0035796". The kit contains reagents for detecting the content or expression level of the circRNA molecule in the sample to be tested. According to the disclosure of this invention, the degree of cell senescence can be determined by detecting the content or expression level of circRNA molecules in cells using the kit; that is, the lower the content or expression level of circRNA molecules, the higher the degree of cell senescence, and vice versa. Therefore, any reagent that can detect the amount of circRNA present in cells, or the amount of its precursors, metabolic derivatives, modification and conversion products, or the reporter gene products regulated by it, falls under the definition of a reagent for detecting the content or expression level of the circRNA molecule in the sample to be tested as defined in this invention. The test samples described in this invention can be blood, body fluids, lymph, secretions, excrement, biopsy specimens, etc.

[0006] In a preferred embodiment, the cell senescence diagnostic kit is a gene detection kit. The gene detection kit refers to a kit that detects the presence of the circRNA molecule in a sample at the nucleic acid level. The nucleic acid refers to DNA or RNA.

[0007] In a more preferred embodiment, the gene detection kit is one of the following: a gene amplification kit, a gene hybridization kit, and a gene sequencing kit, or a combination of any two or all three. The gene amplification may include polymerase chain reaction (PCR) amplification, isothermal amplification, etc.; the gene hybridization may be the detection of complementary pairing between DNA molecules, RNA molecules, or DNA and RNA molecules; and the gene sequencing refers to the sequencing of DNA or RNA molecules. The gene detection kit of the present invention also includes any combination of the above technologies, such as gene amplification and gene hybridization, or a combination of gene amplification and gene sequencing to detect the circRNA molecule.

[0008] In one specific embodiment of the present invention, the gene amplification kit is a reverse transcription PCR kit, and the sequences of the upstream primer and the downstream primer of the PCR are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0009] Secondly, the present invention provides the application of the circRNA molecule as a therapeutic target for cellular senescence, wherein the application is to prepare a drug for regulating cellular senescence by using substances that promote or reduce the expression of the circRNA molecule.

[0010] In a preferred embodiment, the application involves preparing a vector that promotes the overexpression of the circRNA molecule into a drug for delaying cellular senescence. The delay in senescence can be evaluated using conventional methods in the art, such as extending telomere length, activating telomerase activity, enhancing endothelial cell proliferation, reducing β-galactosidase positivity, and decreasing the gene, transcription, and / or expression levels of senescence markers such as P53, P21, and DJ-1. This delay in cellular senescence can be used for therapeutic purposes to delay or prevent the senescence of target cells, restore, maintain, and / or enhance the physiological functions of target cells, or for drug evaluation and screening purposes to establish cell or animal models for delaying cellular senescence.

[0011] In a more preferred embodiment, the vector promoting the overexpression of the circRNA molecule contains a DNA molecule with the sequence shown in positions 9-1175 of SEQ ID NO. 1. In one specific embodiment of the invention, the vector is pLC5-ciR, but other gene expression vectors in the art can also be used in this invention.

[0012] In another preferred embodiment, the application involves preparing a vector that silences the expression of the circRNA molecule into a drug that promotes cellular senescence. The promotion of senescence can be evaluated using conventional methods in the art, such as shortening telomere length, weakening telomerase activity, reducing endothelial cell proliferation, increasing β-galactosidase positivity, and increasing the gene, transcription, and / or expression levels of senescence markers such as P53, P21, and DJ-1. This promotion of cellular senescence can be for therapeutic purposes, inducing senescence, death, or apoptosis of target cells, or for drug evaluation and screening purposes, establishing cell or animal models that promote cellular senescence.

[0013] In a more preferred embodiment, the vector silencing the expression of the circRNA molecule contains a DNA molecule with the sequence shown in SEQ ID NO. 6.

[0014] Finally, this invention provides an ex vivo cell containing a DNA molecule with the sequence shown in positions 9-1175 of SEQ ID NO. 1. The ex vivo cell described in this invention refers to a cell isolated from a parent patient and independently cultured and viable in vivo.

[0015] In a more preferred embodiment, the ex vivo cells are peripheral blood mononuclear cells.

[0016] This invention provides peripheral blood mononuclear cells transformed with DNA molecules whose sequence is shown in positions 9-1175 of SEQ ID NO.1, and the senescence level of these cells can be significantly improved. In practical applications, the significantly improved senescence level of these ex vivo cells can be reinfused into the parental patient, allowing the cells to survive in the parental patient and thus perform their inherent physiological functions. Therefore, this invention provides the application of the above-mentioned ex vivo cells in the preparation of drugs that delay cell senescence. Beneficial effects

[0017] This invention reveals that circHERC1 is expressed at low levels in both natural and induced aging cell models. Furthermore, circHERC1 expression levels in peripheral blood mononuclear cells (PBMCs) from healthy individuals gradually decrease with age. circHERC1 in PBMCs from healthy individuals at different age groups shows a negative correlation with both age and telomere length. During the evaluation process, shortening of telomere length in human PBMCs was accompanied by a significant decrease in circHERC1 levels. Overexpression or knockout experiments with circHERC1 demonstrated its effective ability to prolong telomere length and activate telomerase, suggesting that circHERC1 is closely related to cellular and organismal aging. circHERC1 holds promise as a novel biomarker of aging and an effective molecular target for delaying cellular aging. The degree of aging in target cells can be evaluated by detecting the circHERC1 content in target cells. Overexpression of circHERC1 in target cells can also yield target cells with improved aging levels. Moreover, when target cells are in vitro, reinfusion of these cells can achieve therapeutic effects in delaying cellular and organismal aging. Attached Figure Description

[0018] Figure 1. Expression of circHERC1 in PBMCs of normal individuals of different ages;

[0019] Figure 2. Correlation analysis of circHERC1 expression levels with age in normal individuals' PBMCs at different ages;

[0020] Figure 3. Expression of circHERC1 in naturally aging human umbilical vein endothelial cells;

[0021] Figure 4. Expression of circHERC1 in doxorubicin-induced senescent HeLa cells;

[0022] Figure 5. Expression of circHERC1 in hydrogen peroxide-induced senescent HeLa cells;

[0023] Figure 6. Map of plasmids used for overexpression of circHERC1;

[0024] Figure 7. Expression of shRNA in HUVEC cells, verifying its knockdown efficiency;

[0025] Figure 8. Expression of the circHERC1 overexpression vector in HUVEC cells, verifying its overexpression efficiency;

[0026] Figure 9. Growth curves measured using CCK8 assays show that high expression of circ0035796 significantly enhances the proliferation of HUVEC cells.

[0027] Figure 10. Growth curves measured using CCK8 assays show that low expression of circ0035796 significantly inhibited HUVEC cell proliferation.

[0028] Figure 11. Proliferation assay using edu showed that high expression of circ0035796 significantly enhanced the proliferation of HUVEC cells, while low expression of circ0035796 significantly inhibited the proliferation of HUVEC cells.

[0029] Figure 12. Results showing that high expression of circHERC1 significantly prolongs the telomere length of HUVEC cells, while low expression of circ0035796 significantly shortens the telomere length of HUVEC cells;

[0030] Figure 13. The results show that high expression of circHERC1 significantly activates telomerase activity in HUVEC cells, while low expression of circHERC1 significantly inhibits telomerase activity in HUVEC cells.

[0031] Figure 14. Senescence staining experiment using β-galactosidase showed that high expression of circHERC1 significantly inhibited the senescence of HUVEC cells, while low expression of circHERC1 promoted the senescence of HUVEC cells.

[0032] Figure 15. The results show that high expression of circ_0035696 inhibits the expression of aging-related marker proteins P53, P21, and DJ-1, while low expression of circ_0035696 promotes the expression of aging-related marker proteins P53, P21, and DJ-1.

[0033] Figure 16. High expression of circ_0035696 promotes the expression of telomerase reverse transcriptase TERT, while low expression of circ_0035696 inhibits the expression of telomerase reverse transcriptase TERT.

[0034] Figure 17. Ageed mice injected with AVV circHERC1 Subsequently, the expression of circHERC1 in various organs;

[0035] Figure 18. AAV circHERC1 Telomere shortening rate measured in the tails of treated male (left) and female (right) mice and control group;

[0036] Figure 19. Aged mice injected with AVV circHERC1 Subsequently, changes in telomere length in various organs of male mice;

[0037] Figure 20. Ageed mice injected with AVV circHERC1 Subsequently, changes in telomere length in various organs of female mice;

[0038] Figure 21. Ageed mice injected with AVV circHERC1 Afterwards, the appearance and morphology of the mice;

[0039] Figure 22. Senescence staining experiment using β-galactosidase shows that: aging mice injected with AVV circHERC Subsequently, the proportion of β-galactosidase senescence staining positive in various organs of male mice;

[0040] Figure 23. Senescence staining experiment using β-galactosidase showing: AVV injection in mice circHERC1 Subsequently, the proportion of β-galactosidase senescence staining positive in various organs of female mice;

[0041] Figure 24. Aged mice injected with AVV circHERC1 Subsequently, the expression of p16, a aging-related marker protein, in various organs of male mice;

[0042] Figure 25. Aged mice injected with AVV circHERC1 Subsequently, the expression of p16, a aging-related marker protein, in various organs of female mice;

[0043] Figure 26. Aged mice injected with AVV circHERC1 Subsequently, the expression of p-H2A, a protein associated with DNA damage, in various organs of male mice;

[0044] Figure 27. Ageed mice injected with AVV circHERC1 Subsequently, the expression of p-H2A, a protein associated with DNA damage, in various organs of female mice;

[0045] Figure 28. Western blot experiment showing that mice injected with AVV... circHERC1 Subsequently, the expression of aging-related marker proteins p21 and telomerase reverse transcriptase TERT in various organs of males was observed.

[0046] Figure 29. Western blot experiment showing that mice injected with AVV... circHERC1 Subsequently, the expression of aging-related marker proteins p21 and telomerase reverse transcriptase TERT in various organs of females was observed.

[0047] Figure 30. Aged mice injected with AVV for two days. circHERC1 Afterwards, the metabolic level of aged male mice;

[0048] Figure 31. Aged mice injected with AVV for two days circHERC1 Afterwards, the metabolic level of female aged mice;

[0049] Figure 32. Ageed mice injected with AVV circHERC1 Subsequently, the levels of IL-11, an aging-related inflammatory factor, in the blood of mice were measured.

[0050] Figure 33. Ageed mice injected with AVV circHERC1 Subsequently, the levels of IL-6, an aging-related inflammatory factor, in the blood of mice were measured.

[0051] Figure 34. Transmission electron microscopy (TEM) images of exosomes from circHERC1-overexpressing 293FT cells and vector controls;

[0052] Figure 35. Western blot analysis of exogenous body-labeled proteins in 293FT cells, cell supernatant, and exosomes;

[0053] Figure 36. Automated nanoparticle tracking analysis (NTA) of exosomes from circHERC1-overexpressing 293FT cells and vector controls;

[0054] Figure 37. qRT-PCR analysis of circHERC1 expression in exosomes from circHERC1-overexpressing 293FT cells and vector control cells;

[0055] Figure 38. Aged mice injected with EV circHERC1 Afterwards, the appearance and morphology of the mice changed;

[0056] Figure 39. Aged mice injected with EV circHERC1 Subsequently, the levels of IL-11, an aging-related inflammatory factor, in the blood of mice were measured.

[0057] Figure 40. Aged mice injected with EV circHERC1 Subsequently, the levels of IL-6, an aging-related inflammatory factor, in the blood of mice were measured.

[0058] Figure 41. Senescence staining experiment using β-galactosidase shows that: aging mice injected with EV circHERC1 Subsequently, the proportion of β-galactosidase senescence staining positive in various organs of male mice;

[0059] Figure 42. Senescence staining experiment using β-galactosidase shows that: aging mice injected with EV circHERC1 Subsequently, the proportion of β-galactosidase senescence staining positive in various organs of female mice;

[0060] Figure 43. Aged mice injected with EV circHERC1 Subsequently, changes in telomere length in various organs of male mice;

[0061] Figure 44. Aged mice injected with EV circHERC1 Subsequently, changes in telomere length in various organs of female mice;

[0062] Figure 45. AAV circHERC1 Telomere shortening rate measured in the tails of treated male (left) and female (right) mice and control group;

[0063] Figure 46. Western blot experiment showing that aged mice injected with EV circHERC Subsequently, the expression of aging-related marker proteins p21 and telomerase reverse transcriptase TERT in various organs of males was observed.

[0064] Figure 47. Western blot experiment showing that aged mice injected with EV circHERC Subsequently, the expression of aging-related marker proteins p21 and telomerase reverse transcriptase TERT in various female organs was observed. Embodiments of the present invention

[0065] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0067] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0068] Example 1: circHERC1 levels decrease with age in normal individuals' PBMCs.

[0069] I. Circular RNA circHERC1 is derived from the circular RNA circ_0035796 disclosed in CN115948548A. It was obtained through differential gene expression screening of plasma samples from patients with non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, and patients with benign lung nodules.

[0070] In this invention, the circRNA is named "circHERC1". The nucleic acid sequence of the circular RNA circHERC1 is bases 21-1163 of the sequence shown in SEQ ID NO.1.

[0071] II. Expression of circular RNA circHERC1 in normal human PBMCs

[0072] Tissue specimen source: Our research group collaborated with the Department of Laboratory Medicine of the General Hospital of the Chinese People's Liberation Army, selecting 300 healthy individuals without underlying diseases as research subjects. All patients were fully informed and signed informed consent forms before sampling.

[0073] 1. Sample selection criteria and grouping basis:

[0074] Based on the patients' clinical data, blood samples from patients with a history of cancer or infectious viral infections (such as infectious hepatitis, syphilis, and HIV) were excluded. Healthy blood samples were used as experimental subjects (as shown in Table 1) for the detection of circHERC1 and telomere length.

[0075] Table 1. Summary of Patient's Clinical Data

[0076]

[0077] 2. Reverse transcription of PBMC RNA

[0078] Blood samples were collected from the subjects. After plasma separation, PBS (Gibco, USA) was added to the original volume of the blood sample and mixed thoroughly. The mixed blood was then slowly added along the tube wall into a pre-prepared 15 mL centrifuge tube containing an equal volume of lymphocyte separation medium (Solepro, catalog number P8900). The tube was centrifuged at 400g for 15 minutes at room temperature. After centrifugation, the solution in the tube clearly separated into three layers: the top layer was PBS solution, the middle layer was lymphocyte separation medium, and the bottom layer was a layer of red blood cells. Between the top and middle layers was a thin, relatively dense white membrane, which is the PBMC layer. Carefully aspirate white membrane cells into a 15mL centrifuge tube, add PBS to the 15mL mark, and mix thoroughly by inverting repeatedly; centrifuge at 400g for 5 minutes at room temperature; the centrifuged PBMCs will settle at the bottom of the tube, discard the supernatant, add 1mL of erythrocyte lysis buffer to resuspend the cells and transfer to a 1.5mL centrifuge tube; centrifuge at 2000rpm for 5 minutes at room temperature; discard the supernatant, add 1mL of PBS to resuspend the cells again; centrifuge at 2000rpm for 5 minutes at room temperature; discard the supernatant, the white precipitate is peripheral blood mononuclear cells (PBMCs); divide the above PBMCs in half to extract RNA and DNA respectively. The reverse transcription steps for PBMC RNA are as follows:

[0079] 1) Prepare the reverse transcription mixture using an RNA reverse transcription kit (Thermo Scientific, USA) (Table 2), and keep it on ice until ready to use;

[0080] Table 2. Reverse transcription reaction system

[0081]

[0082] 2) After preparing the reverse transcription mixture, add it to each of the 8-tube sets, then add 10 μL of RNA sample to each tube to prepare a 20 μL reaction system. Gently pipette to mix, seal tightly, and centrifuge briefly.

[0083] 3) Place the sample in a 96-well PCR instrument and set the reaction conditions as follows (Table 3):

[0084] Table 3. Reverse Transcription PCR Conditions

[0085]

[0086] 3. Primer Design: Primers were designed based on the gene sequences of circHERC1 and GAPDH for real-time quantitative PCR. Specific primer names and sequences are shown in Table 4.

[0087] Table 4. Primer sequence information used in PCR reactions

[0088]

[0089] 4. qRT-PCR reaction system and conditions

[0090] RNA was extracted from the PBMCs to be tested, and cDNA was obtained by reverse transcription. This cDNA was then used as a template for qRT-PCR using the following system and procedure:

[0091] According to the primer synthesis instructions, add an appropriate amount of RNase-free water to prepare a 10 μM primer solution. Use TOYOBO's THUNDERBIRD primer. TM The reaction system for the SYBR qPCR Mix Without ROX kit was prepared according to the instructions as follows (Table 5):

[0092] Table 5. qRT-PCR reaction system

[0093]

[0094] Add the prepared qRT-PCR reaction system shown in Table 5 to each well of a 96-well PCR plate. Add 18 μL of the qRT-PCR reaction system shown in Table 5 to each well, and then add 2 μL of cDNA to prepare a 20 μL reaction system. Place the plate in a real-time quantitative PCR instrument (Bio-Rad Laboratories, USA) for the reaction. The PCR reaction conditions are as follows (Table 6):

[0095] Table 6. qRT-PCR reaction conditions

[0096]

[0097] Each sample was configured with three replicates, using GAPDH as the internal reference gene. After the qPCR reaction, the average Ct value of the target gene in the three replicates for each sample was taken as the final result. The relative expression level of each target gene was calculated using the formula 2^-ΔCt, where ΔCt = Ct(target gene) - Ct(internal reference gene).

[0098] The expression of circHERC1 in PBMCs of 300 normal individuals of different ages was detected by qRT-PCR.

[0099] As shown in Figure 1, the expression of this circRNA gradually decreased with increasing age of the tested samples, and circHERC1 expression was correlated with age (P<0.05). Correlation curves were plotted in GraphPad Prim 6 based on the relative expression levels of circHERC1. The results showed that circHERC1 expression gradually decreased with increasing age, and there was a negative correlation between circHERC1 and age in PBMCs of different ages (P<0.05) (see Figure 2). This indicates that this circRNA can serve as a diagnostic molecular marker for aging.

[0100] 5. circHERC1 expression is reduced in replicative senescent umbilical cord mesenchymal cells.

[0101] RNA was extracted from each generation of naturally passaged HUVEC cells, and cDNA was obtained by reverse transcription. Real-time PCR was performed according to the above experimental procedure. The results showed that as HUVEC cells were passaged, the degree of cell senescence increased, while the expression level of circHERC1 gradually decreased (see Figure 3).

[0102] 6. circHERC1 expression is reduced in senescent cells.

[0103] HeLa cells were induced to age using both doxorubicin and hydrogen peroxide. The cDNA obtained by reverse transcription was used as a template, and real-time PCR was performed according to the above experimental procedure. The results showed that the expression level of circHERC1 in the senescent cells was reduced, and the expression level after induction was about 20% (doxorubicin induction, Figure 4) to 50% (hydrogen peroxide induction, Figure 5) of the control expression level.

[0104] Example 2: Application of circHERC1 in delaying aging

[0105] I. Construction of circHERC1 overexpression plasmids or cells, and knockdown plasmids or cells

[0106] 1. Construction of circHERC1 overexpression plasmid and knockdown plasmid

[0107] Overexpression vector pLC5-circHERC1: The DNA molecule between the EcoRI and BamHI restriction sites of the circRNA overexpression vector pLC5-ciR (Guangzhou Gise Biotechnology Co., Ltd.) was replaced with a DNA molecule containing the linear sequence of circHERC1 (SEQ ID NO. 1) to obtain the circHERC1 overexpression vector (see Figure 6 for plasmid map).

[0108] The nucleic acid sequence of the DNA molecule containing the linear circHERC1 sequence is shown in SEQ ID NO. 1, wherein positions 3-8 of SEQ ID NO. 1 are EcoRI restriction sites, positions 9-18 are forward circularization mediating sequences, positions 19-20 are AG receptors, positions 1164-1165 are GT donors, positions 1166-1175 are reverse circularization mediating sequences, and positions 1176-1181 are BamHI restriction sites.

[0109] The silencing expression vector pLKO.1-circHERC1 is obtained by replacing the DNA molecule between the AgeI and EcoRI restriction sites of the interference plasmid pLKO.1 (Addgene) with a DNA molecule that interferes with circHERC1 expression (SEQ ID NO. 6, sequence GCATCACTATCAGATATCA (linker of circular RNA, positions 1151-1163 and 21-26 of SEQ ID NO. 1).

[0110] 2. Construction of circHERC1 overexpressing cells and knockdown cells

[0111] The above-mentioned overexpression vector pLC5-circHERC1, empty vector pLC5-ciR, silent expression vector pLKO.1-circHERC1, and empty vector pLKO.1 were transfected into human umbilical vein endothelial HUVEC cells (Shanghai Qincheng Biotechnology Co., Ltd., QC473) to obtain cells transfected with pLC5-circHERC1, pLC5-ciR, pLKO.1-circHERC1, and pLKO.1, respectively.

[0112] RNA was extracted from pLC5-circHERC1 cells, pLC5-ciR cells, pLKO.1-circHERC1 cells, and pLKO.1 cells. The cDNA obtained by reverse transcription was used as a template for RT-PCR amplification.

[0113] The expression levels of pLKO.1-circHERC1 transgenic cells (denoted as knockdown in the figure) and pLKO.1 transgenic cells (denoted as control in the figure) are shown in Figure 7. It can be seen that compared with pLKO.1 transgenic cells, the expression level of circHERC1 in pLKO.1-circHERC1 transgenic cells was significantly downregulated by 17.4-fold (P<0.01), indicating that cells with silenced circHERC1 expression were obtained.

[0114] Figure 8 shows the expression levels of pLC5-circHERC1 transgenic cells (represented as overexpressing cells) and pLC5-ciR transgenic cells (represented as controls). It can be seen that compared to transgenic cells with the empty vector, the expression level of circHERC1 in pLC5-circHERC1 transgenic cells was upregulated by 4.55-fold (P<0.01), indicating that cells overexpressing circHERC1 were obtained.

[0115] II. circHERC1 is related to the proliferative capacity of endothelial cells.

[0116] The proliferative capacity of endothelial cells was detected using the CCK-8 assay and the EdU assay.

[0117] 1. CCK-8 assay for cell proliferation

[0118] The CCK-8 kit is used for rapid and sensitive detection of cell proliferation. In the presence of an electron coupling agent, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt) can be reduced by mitochondrial dehydrogenases to produce a highly water-soluble orange-yellow formazan product. The intensity of the color is directly proportional to cell proliferation, and the intensity of the color is linearly related to the number of cells. Measuring the OD value at 450 nm using a microplate reader can indirectly reflect the number of viable cells.

[0119] The pLC5-circHERC1 transgenic HUVEC, pLC5-ciR transgenic HUVEC, pLKO.1-circHERC1 transgenic cells, and pLKO.1 transgenic cells obtained above were counted using a Bio-Rad cell counting chamber, and cell suspensions were prepared (density approximately 2 × 10⁻⁶). 4Cells were seeded at 100 μL per well in 96-well plates, with 3 replicates per group. After cell attachment, 10 μL of CCK-8 solution (Cell Counting Kit-8, APExBIO, catalog number K1018) was added to each well. The cells were incubated at 37°C for 120 min. The optical density (OD) at 450 nm was then measured using a microplate reader (MD Microsystems, USA). The optical density of cells in each group after 24, 48, 72, and 96 h of culture was measured using the same method. The values ​​were recorded and growth curves were plotted.

[0120] The results are shown in Figures 9 and 10, with Figure 9 representing the overexpression group and Figure 10 representing the silence group. It can be seen that in the CCK-8 experiment, after 96 h of culture, in the overexpression group, compared with pLC5-ciR transgenic cells (referred to as the control in the figure), the optical density value of pLC5-circHERC1 transgenic cells (referred to as overexpression) was significantly increased at 450 nm, while the control group had an OD value of 1.24; the OD value of circHERC1 overexpressing cells was 1.71 (P < 0.01, Figure 9). In the silence group, compared with pLKO.1 transgenic cells (referred to as the control in the figure), the optical density value of pLKO.1-circHERC1 transgenic cells (referred to as knockdown) was significantly decreased at 450 nm, while the control group had an OD value of 1.19; the OD value of circHERC1 knockdown cells was 0.88 (P < 0.01, Figure 10).

[0121] 2. EdU Experiment

[0122] Edu is a novel thymidine analog that replaces thymidine in the newly synthesized DNA during DNA synthesis. The ethynyl group on Edu can covalently react with biotin-labeled azide through the catalysis of monovalent copper ions to form a stable triazine ring. The reaction is rapid, and the newly synthesized DNA will be labeled with the corresponding biotin probe, thus displaying a different color.

[0123] EdU labeling was performed on pLC5-circHERC1 cells, pLC5-ciR cells, pLKO.1-circHERC1 cells, and pLKO.1 cells transfected with the BeyoClick Edu-594 cell proliferation assay kit (BeyoClick, catalog number C0078L). Cells were fixed with 4% paraformaldehyde for 15 min, washed twice with PBS for 5 min each time, permeabilized with 1% Triton-X100 at room temperature for 10 min, removed, and washed twice with PBS for 5 min each time. 1 mL of staining solution was prepared with the following composition: 860 μL Click Reaction Buffer, 40 μL CuSO4, 2 μL Azide 594, and 100 μL Click Additive Solution. Cells were stained with the freshly prepared staining solution under light-protected conditions for 30 min, followed by washing twice with PBS for 5 min each time. Cells were stained for DNA using a mounting medium with DAPI (Abcam, catalog number ab104139) under light-protected conditions, and then photographed under a fluorescence microscope.

[0124] The results are shown in Figure 11. From left to right, they represent the overexpression control group, the overexpression group, the knockout control group, and the knockout group. Five fields of view were collected for each group. In the overexpression group, the positive cell rate of pLC5-circHERC1 transgenic cells (referred to as overexpression in the figure) was 52.46%, and the positive cell rate of pLC5-ciR transgenic cells (referred to as control 1 in the figure) was 31.15%, with significantly increased cell proliferation activity (P<0.05). In the knockout group, the positive cell rate of pLKO.1-circHERC1 transgenic cells (referred to as knockdown in the figure) was 21.57%, and the positive cell rate of pLKO.1 transgenic cells (referred to as control 2 in the figure) was 32.39%. Silencing circHERC1 significantly reduced the number of positive endothelial cells and significantly reduced cell proliferation activity (P<0.01, Figure 11).

[0125] The above results indicate that overexpression of circHERC1 can promote endothelial cell proliferation, while knockdown of circHERC1 can inhibit endothelial cell proliferation.

[0126] III. circHERC1 is related to telomere length.

[0127] The effect of circHERC1 on cell telomeres was verified by cell telomere length detection and TRAP telomerase activity detection experiments.

[0128] 1. Telomere length detection

[0129] One day before transfection, HUVEC endothelial cells were seeded on streaked 6-well cell culture plates, with the cell density reaching 70% to 80% the next day being ideal. Transfection experiments were performed, and pLC5-ciR and the overexpression vector pLC5-circHERC1; pLKO.1 and the silencing expression vector pLKO.1-circHERC1 were used to transfect HUVEC (Beina Chuanglian Biotechnology Co., Ltd., catalog number BNCC342436) / HeLa cells (ATCC, catalog number CBP60232).

[0130] Cells stably expressing circHERC1 were selected under pressure after transfection, and their expression efficiency was verified by real-time PCR. Genomic DNA was then extracted for telomere length detection. Using a human absolute telomere length assay kit (Sciencell, Inc., catalog number 8918), after extracting the cellular genome, two qRT-PCR reactions were prepared for each DNA sample: one using telomere telogen primer stock solution and the other using internal control gene HBG primer stock solution, according to the reaction system prepared in the table below.

[0131] Table 7. Reaction system for telomere length detection

[0132]

[0133] Place the prepared reaction system into a real-time PCR instrument and perform the reaction according to the procedure in the table below.

[0134] Table 8. Telomere length detection reaction procedure

[0135]

[0136] The results showed that overexpression of circHERC1 significantly prolonged telomere length. The telomere length in the overexpression control group was 6373 bp (control group 1); the telomere length in the posterior telomere group after overexpression of circHERC1 was 8594 bp. Knockdown of circHERC1 shortened telomere length. The telomere length in the knockdown control group was 6091 bp (control group 2); the telomere length in the posterior telomere group after knockdown of circHERC1 was 4921 bp (as shown in Figure 12).

[0137] 2. TRAP telomerase activity assay

[0138] The effect of circHERC1 on cellular telomerase activity was detected using the TRAP telomerase activity assay kit (Sigma-Aldrich, catalog number S7700FR). The TRAP assay utilizes the characteristic of telomerase to add a 6-base repeat sequence to the end of a suitable oligonucleotide chain using its own RNA template region as a template. This repeat sequence was amplified by PCR, and then analyzed by polyacrylamide gel electrophoresis (PAGE) to identify the 6-base difference in the repeat sequence. circHERC1 overexpressing and knockdown cells were seeded in 24-well plates, respectively. Cells were collected into centrifuge tubes, and 3-4 volumes of CHAPs lysis buffer were added. Lysis was performed on ice for 30 min (RNase inhibitor 1:200, cocktail 1:100). The supernatant was transferred to a new tube, and 3 μL of the supernatant was added to 100 μL of a 100 μL LCA kit to determine protein concentration. CHAPs served as a blank control. The kit was diluted 1:50, incubated at 37°C for 30 min, and the OD value was measured at 565 nm. Calculate the TRAP dilution concentration, then take the corresponding volume of stock solution and dilute with CHAPs to achieve a protein concentration of 100 mg / L. Prepare the solution according to the following system:

[0139] Table 9. Reaction system for telomerase activity detection

[0140]

[0141] Table 10. Reverse Transcription PCR Conditions

[0142]

[0143] Note*: Steps 2-4 are repeated 27 times.

[0144] After PCR, dilute with 5x loading buffer. Load 10 μL of sample into 0.5x TBE buffer, perform TRAP gel electrophoresis at 200V, and develop with UV after 50 min.

[0145] Table 11. TRAP Adhesive Formulation

[0146]

[0147] The results showed that telomerase activity was significantly enhanced in cells overexpressing circHERC1 compared to the overexpression control group (control group 1), while telomerase activity was weakened in cells knocked down by circHERC1 compared to the knockdown control group (control group 2) (as shown in Figure 13).

[0148] IV. circHERC1 inhibits cell senescence

[0149] The inhibition of circHERC1 by β-galactosidase staining and senescence-related protein level detection experiments was verified.

[0150] Senescent cells have highly active β-galactosidase. Through in situ staining, using X-Gal as a substrate, a deep blue product is generated under the catalysis of β-galactosidase, which can be observed through an optical microscope.

[0151] This experiment used a cell senescence β-galactosidase staining kit (Beyotime Biotechnology, China, catalog number C0602). The steps were as follows: HUVEC endothelial cells were evenly seeded into 6-well or 12-well plates. After the cells adhered, they were gently washed with PBS buffer and the PBS was removed. 1 mL of fixative was added and the cells were fixed at room temperature for 15 min. The fixative was removed, and the cells were gently washed three times with PBS buffer, 3 min each time. The cell senescence staining solution was prepared according to the table below.

[0152] Table 12. Preparation of Cell Senescence Staining Solution

[0153]

[0154] The pH of the cell senescence staining solution was measured using a pH meter, and the pH was adjusted to 6.0 using citric acid or sodium hydroxide. The PBS buffer was removed, and 1 mL of staining working solution was added to each well. The wells were incubated overnight at 37°C, covered with aluminum foil to prevent evaporation; incubation in a CO2 incubator was prohibited. After staining, the staining solution was aspirated, and the cells were immediately observed under a microscope. Senescent cells would appear deep blue. Five fields of view were photographed and recorded. The number of blue cells and the total number of cells were counted, and the percentage of positive staining was calculated.

[0155] The results showed that circHERC1 overexpression reduced the β-galactosidase positivity rate of cells by 55.3% compared with the overexpression control group (control group 1), and reduced the degree of cell senescence. circHERC1 knockdown increased the β-galactosidase positivity rate of cells by 3.27 times compared with the knockdown control group (control group 2), and promoted cell senescence (as shown in Figure 14).

[0156] V. Detection of aging-related molecules

[0157] The expression levels of senescence markers such as P21 (which plays an important role in cell cycle control and DNA replication and repair), P53 (located upstream of P21 and closely related to cellular senescence), and DJ-1 (involved in cellular oxidative stress) were detected by Western blotting assay.

[0158] Remove the culture medium, wash with pre-chilled PBS, and discard the washing buffer. Add 300 μL of pre-chilled cell lysis buffer to HUVEC endothelial cells and lyse them thoroughly on ice. After lysis, transfer to EP tubes and centrifuge at 12,000 rpm for 10 min at 4°C.

[0159] The supernatant was used for protein concentration determination. BCA working solution (Thermo Fisher Scientific, catalog number 23227) was prepared using reagent A:B (50:1). 25 μL of each standard or unknown sample was added to each well of a 96-well plate; 200 µL of working solution was added to each well and mixed thoroughly. The plate was incubated at 37°C for 30 minutes. Protein concentration was calculated by measuring the absorbance at 562 nm using a microplate reader. The protein samples were diluted with protein loading buffer (Kangwei Century Co., Ltd., catalog number 01411) and boiled for 10 minutes. The samples were then stored at -80°C for later use. Prepare an appropriate concentration of electrophoresis gel (8%-15%) according to the protein molecule size; add glycine electrophoresis buffer, 30µg sample / well, and 5μL protein marker / well; run at 80V for 1 hour, then switch to 120V, stopping electrophoresis and turning off the voltage just as the sample is fully processed; activate the PVDF membrane (Merck, Inc., catalog number IPVH00010) with methanol, and prepare it together with two black sponges and 6 filter papers, avoiding air bubbles, assemble the transfer tank, add transfer buffer, and place in an ice bath for 1 hour of transfer; after transfer, place the membrane in 5% skim milk blocking buffer and block on a slow shaker at room temperature for 1 hour; apply primary antibody P53 (Wuhan Sanying Biotechnology Co., Ltd., catalog number 10024098) diluted 1:5000; P21 CST (catalog number 2947S) diluted 1:1000; DJ-1 (CST, catalog number 5933S) diluted 1:1000; TERT (abcam, catalog number ab3202) Dilute 1:1000. Incubate overnight at 4°C; wash membrane 5 times, 5 min each time; apply secondary antibody at room temperature for 1 h; wash membrane 5 times, 5 min each time; develop and record protein using a protein developer.

[0160] The results showed that, compared with the control empty vector (control group 1), overexpression of circHERC1 reduced the expression levels of senescence-related proteins P53, P21, and DJ-1, while increasing the expression of telomerase reverse transcriptase TERT, a key component of telomerase. Conversely, knockdown of circHERC1, compared with the control vector (control group 2), increased the expression levels of P53, P21, and DJ-1 (as shown in Figure 15), and decreased TERT expression (as shown in Figure 16, where Vector represents the overexpression control group, circHERC1 the overexpression group, NC the knockdown control group, and sh circRNA the knockdown group). These results indicate that circHERC1 expression enhances cellular telomerase TERT expression and inhibits cellular senescence.

[0161] Example 3. Tail vein injection of AAVcircHERC1 Intervening in the overall aging process of aged mice.

[0162] 1. Construct an adeno-associated virus (AVV) overexpressing circHERC1.

[0163] The obtained circHERC1 sequence was inserted into the multiple cloning site of the AAV vector backbone using restriction endonuclease digestion and ligation to obtain circHERC1-overexpressing adeno-associated virus. Fourteen-month-old aged mice (half male and half female) were selected and injected intravenously via the tail vein. After three months of normal feeding, telomere length and the degree of aging of various organs in the aged mice were measured.

[0164] 2. Post-injection detection of circHERC1 expression in various mouse organs.

[0165] To investigate the systemic effects of circHERC1, aged mice were administered an AAV vector carrying circHERC1. circHERC1 and AAV control The drug was administered to 14-month-old aged mice via tail vein injection. Three months later, the expression levels of circHERC1 in various organs of the mice were detected using real-time PCR.

[0166] Extraction of AAV for testing control and AAV circHERC1 RNA from various mouse organs was reverse transcribed to obtain cDNA, which was used as a template. The primer sequence information used for the PCR reaction is shown in Table 4. qRT-PCR was performed using the following system and procedure:

[0167] According to the primer synthesis instructions, add an appropriate amount of RNase-free water to prepare a 10 μM primer solution, and prepare a 2× reverse transcription mixture (Table 2). Keep it on ice until ready to use. After preparing the reverse transcription mixture, add it to each of the eight-tube strips, then add 15 μL of RNA sample to each strip, to prepare a 30 μL reaction system. Gently pipette to mix, seal tightly, and briefly centrifuge. Place in a 96-well PCR instrument and set the reaction conditions as described in Table 3.

[0168] Add the prepared qRT-PCR reaction system shown in Table 5 to a 96-well PCR plate. Add 18 μL of the qRT-PCR reaction system shown in Table 5 to each well, and then add 2 μL of cDNA to prepare a 20 μL reaction system. Place the plate in a real-time quantitative PCR instrument for the reaction. The PCR reaction conditions are shown in Table 6.

[0169] Each sample was configured with three replicates, using U6 as the internal control gene. After the qPCR reaction, the average Ct value of the target gene in the three replicates for each sample was taken as the final result. The relative expression level of each target gene was calculated using the formula 2^-ΔCt, where ΔCt = Ct(target gene) - Ct(internal control gene).

[0170] qPCR showed that circHERC1 was strongly expressed in different organs of both male and female mice (Figure 17). circHERC1 was most highly expressed in metabolically active organs such as the liver, intestine, and kidney, while intermediate levels were detected in the heart and spleen.

[0171] 3. circHERC1 affects telomere length in mice via AAV injection.

[0172] AAV was ground using a mouse absolute telomere length assay kit (Sciencell, Inc., catalog number M8918). control and AAV circHERC1 Genomes were extracted from various tissues and organs of mice, and genomes were also extracted from the tails of mice before and after injection. For each mouse tissue DNA sample, two qRT-PCR reactions were prepared: one using telomere primer stock solution and the other using internal control gene HBG primer stock solution, according to the reaction system prepared in Table 7. The prepared reaction systems were placed in a real-time PCR instrument and reacted according to the program in Table 8.

[0173] The above results indicate that female AAVs 对照 The telomere length shortening rate was approximately 50.63%, AAV circHERC1 The telomere shortening rate in the treated mice was approximately 10.31%; male AAV mice... 对照 The telomere length shortening rate was approximately 43.28%, AAV circHERC1 The telomere shortening rate in the treated mice was approximately 24.65%, AAV circHERC1 The treated mice showed a significantly lower rate of telomere shortening (Figure 18). This indicates that circHERC1 overexpression helps maintain telomere integrity and delays telomere wear, a hallmark of aging. Further analysis using qPCR showed that, compared with the control group, AAV... circHERC1 The treated mice exhibited significantly longer telomeres in multiple tissues, including the heart, liver, and kidney (Figures 19–20). This result supports the hypothesis that circHERC1 overexpression promotes telomere maintenance and delays telomere wear.

[0174] 4. circHERC1 affects apparent aging levels in mice via AAV injection.

[0175] Compared with the control group mice, AAV circHERC1The treated mice showed significant improvements in physical condition, exhibiting healthier fur, increased weight, and a better overall appearance. Hair that initially fell out due to natural aging began to grow back in the AAV. circHERC1 He recovered after taking the medication (Figure 21).

[0176] 5. circHERC1 affects β-galactosidase staining in various organs of mice via AAV injection.

[0177] The detection of AAV injected via tail vein in mice was performed using a β-galactosidase staining assay. control and AAV circHERC1 Sections of various organs and tissues of mice.

[0178] The detection method is the same as in Example 2 (IV).

[0179] The results showed that, compared with the control AAV control Compared to the control group, AAV injection circHERC1 The proportion of β-galactosidase positive staining in various organs of mice in the group was reduced, suggesting that circHERC1 expression reduces the aging of various organ tissues in mice, especially metabolically active organs such as the liver and kidney (Figures 22-23).

[0180] 6. circHERC1 affects the expression of aging markers in various organs of mice via AAV injection.

[0181] The expression levels of aging markers such as P21 (which plays an important role in cell cycle control and DNA replication and repair), H2A (involved in cellular DNA damage), TERT telomerase reverse transcriptase (a key component of telomerase) and P16 (a key molecule involved in cell cycle regulation) in various organs of mice were detected by Western blotting and immunohistochemistry experiments. P16 is an important aging marker protein.

[0182] Remove the culture medium, wash with pre-chilled PBS, and discard the washing solution. Grind the mouse organs and tissues, add 300 μL of pre-chilled cell lysis buffer, and lyse thoroughly on ice. After lysis, transfer to EP tubes and centrifuge at 12,000 rpm for 10 min at 4°C.

[0183] The supernatant was used for protein concentration determination. BCA working solution (Thermo Fisher Scientific, catalog number 23227) was prepared using reagent A:B (50:1). 25 μL of each standard or unknown sample was added to each well of a 96-well plate; 200 µL of working solution was added to each well and mixed thoroughly. The plate was incubated at 37°C for 30 minutes. Protein concentration was calculated by measuring the absorbance at 562 nm using a microplate reader. The protein samples were diluted with protein loading buffer (Kangwei Century Co., Ltd., catalog number 01411) and boiled for 10 minutes. The samples were then stored at -80°C for later use. Prepare an appropriate concentration of electrophoresis gel (8%-15%) based on protein molecule size; add glycine electrophoresis buffer, 30µg sample / well, and 5μL protein marker / well; run at 80V for 1 hour, then switch to 120V, stopping electrophoresis and turning off the voltage just as the sample has finished running; activate the PVDF membrane (Merck, catalog number IPVH00010) with methanol, and prepare it together with two black sponges and six filter papers, avoiding air bubbles; assemble the transfer tank, add transfer buffer, and place in an ice bath for 1 hour of transfer; after transfer, place the membrane in 5% skim milk blocking buffer and block on a slow shaker at room temperature for 1 hour; apply primary antibody P21 (CST, catalog number 2947S) diluted 1:1000; apply H2A (CST, catalog number 9718S) diluted 1:1000; TERT (abcam, catalog number ab3202) 1:1000 dilution; GAPDH (CST, catalog number 5174) 1:3000 dilution. Incubate overnight at 4°C; wash membrane 5 times, 5 min each time; apply secondary antibody at room temperature for 1 h; wash membrane 5 times, 5 min each time; develop and record protein using a protein developer.

[0184] Mouse organs (heart, liver, spleen, lung, kidney, stomach, intestines, brain, testes, etc.) were fixed in 4% paraformaldehyde solution at 4°C for 2 days. Paraffin-embedded sections were sectioned to a thickness of 5 μm and baked overnight at 60°C. Sections were dewaxed and rehydrated. Antigen retrieval was performed using sodium citrate retrieval solution, and endogenous peroxidase was blocked using an endogenous peroxidase inhibitor. After washing with PBS at room temperature, primary antibody was added and incubated overnight at 4°C. p16 (abcam, catalog number ab211542) was diluted 1:100; pH2A (CST, catalog number 9718S) was diluted 1:200. The sections were washed three times with PBS at room temperature. Enhancing enzyme-labeled goat anti-mouse / rabbit IgG polymer was added and incubated at room temperature for 1 hour. DAB staining was performed at an appropriate time. Hematoxylin staining, differentiation washing, and blue staining were performed. The sections were dehydrated, cleared, and mounted. The sections were imaged under a microscope, and the proportion of positive cells was counted.

[0185] The results showed that, compared with the control group, circHERC1-treated animals had fewer senescent cells in all tissues. Immunohistochemical analysis revealed a significant reduction in senescence markers such as p16 in multi-organ animals treated with circHERC1 (Figures 24-25). Similarly, immunostaining of DNA damage markers such as H2A showed lower levels of DNA damage in circHERC1-treated tissues (Figures 26-27). These results indicate that circHERC1 combats genotoxic stress and cellular senescence by enhancing DNA repair mechanisms. We performed Western blot analysis on TERT and p21 to assess the molecular effects of circHERC1 overexpression on telomere maintenance and cellular senescence. Western blot experiments showed reduced expression of TERT and p21 in the liver, kidney, and intestine of circHERC1-treated mice, indicating reduced cellular senescence in key metabolic tissues (Figures 28-29).

[0186] 7. circHERC1 affects metabolic levels in mice via AAV injection.

[0187] The effect of circHERC1 on the metabolic level of mice was detected by a metabolic cage experiment.

[0188] The metabolic cages were thoroughly cleaned and sterilized, and the balances used for measurement were calibrated to ensure accuracy. Each mouse was then placed in an individual metabolic cage, and basic data such as weight, identification number, and placement time were recorded. A certain amount of food and water was provided, and their initial weight and levels were recorded. Remaining food and water content was periodically weighed to assess consumption. The overall health of the mice was monitored, and any abnormalities were recorded. The duration of the experiment varied depending on the objective, but lasted for 3 days. After completion, the mice were carefully removed and weighed again. This data analysis provides insights into the metabolic health and responses of the mice under experimental conditions.

[0189] The results showed that AAV circHERC1 Male mice treated with circHERC1 exhibited better overall metabolic performance (Fig. 30), indicating that circHERC1 treatment enhanced physical activity and metabolic health. Data from female mice (Fig. 31) were consistent, with female mice treated with circHERC1 also showing improved activity and metabolic function, including higher energy expenditure during both light and dark cycles.

[0190] 8. circHERC1 affects the expression of inflammatory factors in mice via AAV injection.

[0191] IL-11 and IL-6 are currently considered to be inflammatory factors closely related to aging. In order to detect the effect of circHERC1 on inflammation, an ELISA assay was performed to detect IL-11 and IL-6.

[0192] Blood was collected from the eye sockets, and serum samples were separated from each group of mice (AAV-circ_HERC1 treatment group and control group). The expression levels of IL-6 and IL-11 were measured using a mouse IL-11 (QS42911, Beijing Qisong Biotechnology Co., Ltd.) and IL-6 ELISA kit (QS42899, Beijing Qisong Biotechnology Co., Ltd.). The test samples were added to the wells of a microplate to allow specific binding to the immobilized antigen or antibody. The microplate was washed with an appropriate washing buffer to remove unbound material. Enzyme-labeled antibodies or antigens were added: according to the experimental design, enzyme-labeled antibodies or antigens that specifically bind to the test substances were added. The microplate was washed again to remove unbound enzyme-labeled molecules. The corresponding substrate was added, which underwent a chemical reaction under the action of the enzyme to produce a colored product. A stop solution was added after a certain time to stop the enzymatic reaction. This is typically done to facilitate reading results at different time points or to save results for subsequent analysis, measuring the optical density (OD) value of each well at 450 nm. A standard curve is constructed. The concentration of each sample is calculated based on the equation of the standard curve.

[0193] We measured AAV circHERC1 Cytokine levels in the serum of treated mice. The cytokine IL-11, associated with tissue repair and fibrosis, and another pro-inflammatory cytokine, IL-6. Male AAV. 对照 The serum IL-11 level in the group of mice was approximately 43.57 pg / μL, and AAV... circHERC1 The serum IL-11 level in treated mice was approximately 39.87 pg / μL, and in female AAV mice... 对照 The serum IL-11 level in the group of mice was approximately 43.01 pg / μL, and AAV... circHERC1 The serum IL-11 level in the treated mice was approximately 39.31 pg / μL, indicating that IL-11 plays a role in AAV. circHERC1 The levels of AAV in the serum of treated mice were reduced (Figure 32); male AAV 对照 The serum IL-6 level in the group of mice was approximately 47.32 pg / μL, and the AAV level was... circHERC1 The serum IL-6 level in treated mice was approximately 37.36 pg / μL, and in female AAV mice... 对照 The serum IL-6 level in the group of mice was approximately 47.21 pg / μL, and the AAV level was... circHERC1 The serum IL-6 level in the treated mice was approximately 37.21 pg / μL, indicating that IL-6 plays a role in AAV. circHERC1 The levels of circHERC1 in the serum of treated mice were reduced (Figure 33). These changes suggest that circHERC1 may regulate the inflammatory response, potentially promoting tissue repair and reducing age-related tissue damage.

[0194] Example 4. Tail vein injection of EV circHERC1 Intervening in aging in aged mice.

[0195] 1. Exosome preparation

[0196] Exosomes were used to load circHERC1 and control vectors. Culture supernatants of 293FT cells and 293FT cells overexpressing circHERC1 were collected. First, the cells were removed by centrifugation at 300×g for 10 minutes, and the supernatant was transferred to a new centrifuge tube. Then, the cells were centrifuged at 2000×g for 10 minutes to remove cell debris. The clarified supernatant was transferred to a clean sample vial, and EVs were separated using a high-performance exosome separation system (EXODUS H-600, Huixin Life Science & Technology). EVs were recovered from the chip using 1 mL of phosphate-buffered saline (PBS) to obtain exosomes loaded with circHERC1 and control vectors. Fourteen-month-old aged mice, half male and half female, were selected and injected with 1×10⁻⁶ oz.5 every other day. 11 Exosomes were continuously injected for 3 months, and the telomere length and aging degree of various organs in aged mice were then measured.

[0197] 2. Validation of extracellular oosomes from 293FT cells overexpressing circHERC1

[0198] To investigate the therapeutic potential of extracellular vesicles (EVs) overexpressing circHERC1 in alleviating age-related decline, 293FT cells overexpressing circHERC1 were used to generate circHERC1-containing EVs. TEM imaging confirmed that the isolated EVs exhibited typical exosome morphology (Fig. 34). Western blotting (Fig. 35) and NTA (Fig. 36) further confirmed that the EVs were rich in exosome markers (ALIX, TSG101, CD9) and had an appropriate size distribution. Furthermore, as shown by quantitative PCR, the relative expression level of circHERC1 in the exosome control vector was approximately 1.32, while the relative expression level of circHERC1 in the exosome-loaded circHERC1 vector was approximately 30.02, confirming the presence of circHERC1 in the EVs (Fig. 37).

[0199] 3. Injecting EVs circHERC1 It then affects the overall coat color and amount in mice.

[0200] To evaluate EV circHERC1 The in vivo effects, using overexpressed EV circHERC1Mice treated with EVs showed significant improvement in coat color, regaining shine and increasing hair volume. Compared to control mice, mice treated with EVcircHERC1 showed significant improvements in physical condition, with control mice exhibiting healthier fur and a better overall appearance, even superior to mice treated with AAVcircHERC1 (Figure 38).

[0201] 4. Injecting EVs circHERC1 It subsequently reduced the expression of inflammatory factors in mouse serum.

[0202] To assess inflammation, we used ELISA to measure the EV10 levels. circHERC1 The levels of IL-11 and IL-6 in the serum of treated mice. The IL-11 level in the serum of mice in the male buffer group was approximately 39.76%, while the IL-11 level in the serum of mice in the exosome group was approximately 38.51%. circHERC1 The serum IL-11 level in the female buffer group was approximately 32.01%; the serum IL-11 level in the female buffer group was approximately 37.25%; and the serum IL-11 level in the exosome group was approximately 36.84%. circHERC1 The serum IL-11 level in the EV group mice was approximately 29.45%, indicating that compared with the PBS and EV controls, EV... circHERC1 Treatment resulted in a significant decrease in IL-11 levels (Figure 39); the serum IL-6 level in the male buffer group was approximately 41.56%, while the serum IL-6 level in the exosome group was approximately 39.35%, and the EV... circHERC1 The serum IL-6 level in the female buffer group was approximately 30.56%; the serum IL-6 level in the female buffer group was approximately 53.24%; and the serum IL-6 level in the exosome group was approximately 40.78%. circHERC1 The serum IL-6 level in the EV group mice was approximately 38.03%, indicating that compared with the PBS and EV controls, EV... circHERC1 Treatment resulted in a significant decrease in IL-6 levels (Figure 40); this suggests that circHERC1-overexpressing EVs may enhance tissue repair and regulate inflammation.

[0203] 5. Injecting EVs circHERC1 It inhibited the aging of various organs in mice.

[0204] The results showed that SA-β-gal staining of the tissues indicated reduced cellular senescence in EVcirc-treated mice, suggesting a reduction in age-related cellular damage (Figures 41-42).

[0205] 6. Injecting EVs circHERC1 This subsequently affects the telomere length in mice.

[0206] The experimental procedure was the same as above. Quantitative PCR analysis of telomere length in these tissues showed that telomeres in mice treated with EVcircHERC1 were significantly longer compared with the control group treated with PBS and EV. This indicates that overexpression of circHERC1 by EVs helps maintain the integrity of telomeres in various organs in vivo (Figures 43-44). EVcircHERC1 treatment slowed telomere shortening in both male and female mice. As shown by quantitative PCR of telomere length in tail tissue, the telomere shortening rate was approximately 55.78% in the buffer group, approximately 52.02% in the exosome group, and approximately 5% in the telomere length shortening rate in the EVcircHERC1-treated mice (Figure 45).

[0207] 7. circHERC1 affects aging and the expression of aging markers in various organs of mice via exosome injection.

[0208] From EV circHERC1 Western blot analysis of tissues (liver, lung, kidney, intestine) from treated mice revealed upregulation of TERT and downregulation of p21, indicating enhanced telomerase activity and reduced senescence in the tissues (Figures 46-47). Compared with AAV... circHERC1 Compared to EV, circHERC1 The treatment showed a more significant anti-aging effect in mice.

[0209] The results show that EV circHERC1和 AAV circHERC1均 It significantly enhanced telomerase activity, reduced cellular senescence, maintained telomere integrity, and improved multiple aspects of health in aging mice, with better exosome delivery. In summary, our results indicate that circHERC1 delivery represents a promising therapeutic strategy to alleviate aging and improve healthy lifespan, potentially providing a novel intervention for age-related diseases.

Claims

1. Use of circRNA molecules as diagnostic markers in a diagnostic kit for cell senescence, characterized in that, The sequence of the circRNA molecule is shown as bases 21-1163 of SEQ ID NO.1, and the kit contains reagents for detecting the content or expression level of the circRNA molecule in the sample to be tested.

2. Use according to claim 1, characterized in that, The cell senescence diagnostic kit is a gene detection kit.

3. Use according to claim 2, characterized in that, The gene detection kit is any one of the gene amplification kit, gene hybridization kit, and gene sequencing kit, or any combination of two or all three.

4. Use according to claim 3, characterized in that, The gene amplification kit is a reverse transcription PCR kit, and the sequences of the upstream and downstream primers for the PCR are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

5. Use of a circRNA molecule as a target for cell senescence therapy, characterized in that, The sequence of the circRNA molecule is shown as bases 21-1163 of SEQ ID NO.1, and the application is to prepare a drug for regulating cellular senescence from a substance that promotes or reduces the expression of the circRNA molecule.

6. Use according to claim 5, characterized in that, The application involves preparing a drug that delays cell aging from a substance that promotes the overexpression of the circRNA molecule.

7. Use according to claim 6, characterized in that, The substance that promotes the overexpression of the circRNA molecule is a vector for overexpressing the circRNA molecule.

8. Use according to claim 7, characterized in that, The vector overexpressing the circRNA molecule contains a DNA molecule with the sequence shown in bases 9-1175 of SEQ ID NO.

1.

9. Use according to claim 6, characterized in that, The substance that promotes the overexpression of the circRNA molecule is an exosome containing the circRNA molecule.

10. Use according to claim 6, characterized in that, The substance that promotes the overexpression of the circRNA molecule is a cell that secretes exosomes containing the circRNA molecule.

11. Use according to claim 5, characterized in that, The application involves preparing a drug that promotes cell senescence by using a vector that silences the expression of the circRNA molecule.

12. Use according to claim 11, characterized in that, The vector for silencing the expression of the circRNA molecule contains a DNA molecule with the sequence shown in SEQ ID NO.

6.

13. An ex vivo cell containing a DNA molecule with the sequence shown in bases 9-1175 of SEQ ID NO.

1.

14. The ex vivo cell of claim 13, wherein, The isolated cells are peripheral blood mononuclear cells.

15. The use of the ex vivo cells according to claim 13 or 14 in the preparation of a drug for delaying cell aging.

Images