CircRNA (Ribonucleic Acid) as cell senescence marker and regulation and control target and application of circRNA

Abstract

The invention discloses an application of a circRNA molecule as a diagnostic marker in a cell senescence diagnostic kit and an application of the circRNA molecule as a cell senescence treatment target. According to the application disclosed by the invention, the circHERC1 is found to be low in expression in natural aging and aging-inducing cell models, and the circHERC1 is over-expressed in endothelial cells, so that the multiplication capacity of the endothelial cells can be improved, the telomere length of the cells can be effectively prolonged, telomerase can be activated, the positive rate of beta-galactosidase of the cells can be reduced, and the expression quantity of aging-related proteins can be reduced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a circRNA related to telomerase activity and cell aging and an application thereof. Background Art

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

[0003] circRNA is a type of non-coding RNA molecule that exists in organisms and does not have a 5' end cap and a 3' end poly (A) tail, and forms a circular structure with covalent bonds. With the development of high-throughput sequencing and bioinformatics analysis technology, research on circRNA has been relatively rich. In recent years, circRNA has received extensive attention due to its special structure and function. circRNA can act as a circRNA "sponge", a transcriptional regulator, and plays an important role in a variety of biological functions by binding to RNA-binding proteins. Compared with other non-coding RNAs, circRNA has more stability due to its circular structure and has stronger tissue and disease specificity, making it of great research value in various diseases and physiological processes.

[0004] The purpose of the present invention is to provide a circRNA that extends the length of cell telomeres by activating telomerase, thereby inhibiting the aging of cells and organisms. Summary of the invention

[0005] Based on the above purpose, and based on a circular RNA related to the occurrence and development of non-small cell lung cancer disclosed in CN115948548A: circ_0035796, the present invention first provides the use of the circRNA molecule as a diagnostic marker in a cell aging diagnostic kit, and the sequence of the circRNA molecule is shown in the 21st-1163rd bases of SEQ ID NO.1. In the present invention, the circRNA molecule is named "circHERC1" or "circ_0035796". The kit contains a reagent for detecting the content or expression of the circRNA molecule in the sample to be detected. According to the disclosure of the present invention, the degree of cell aging can be determined by detecting the content or expression of the circRNA molecule in the cell by the kit, that is, the lower the content or expression of the circRNA molecule, the higher the degree of cell aging; vice versa. Therefore, as long as the amount of circRNA in the cell can be detected, or the amount of its precursor, metabolic derivative, modified conversion product, and the reporter gene product regulated by it, it belongs to the reagent for detecting the content or expression of the circRNA molecule in the sample to be detected as defined in the present invention. The test sample described in the present invention can be blood, body fluid, lymph fluid, secretion fluid, excrement, biopsy specimen, etc.

[0006] In a preferred embodiment, the cell aging diagnostic kit is a gene detection kit. The gene detection kit refers to a detection kit for detecting whether the circRNA molecule exists in a specimen at the nucleic acid level. The nucleic acid refers to DNA or RNA.

[0007] In a more preferred embodiment, the gene detection kit is a detection kit selected from the group consisting of a gene amplification kit, a gene hybridization kit, and a gene sequencing kit, or a detection kit that is a combination of any two or all three. The gene amplification may include polymerase chain amplification technology, isothermal amplification technology, etc., the gene hybridization may be a complementary pairing detection between DNA molecules, between RNA molecules, or between DNA and RNA molecules, and the gene sequencing refers to the sequence determination of either 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, and a combination of gene amplification and gene sequencing to determine the circRNA molecule.

[0008] In a 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 as SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0009] Secondly, the present invention provides an application of the circRNA molecule as a target for the treatment of cell aging, wherein the application is to prepare a substance that promotes or reduces the expression of the circRNA molecule into a drug for regulating cell aging.

[0010] In a preferred embodiment, the application is to prepare a vector that promotes overexpression of the circRNA molecule as a drug for delaying cell aging. The delaying aging can be evaluated using conventional evaluation methods in the art, for example, extending the length of cell telomeres, activating the activity of cell telomerase, improving the proliferation ability of endothelial cells, reducing the positive rate of β-galactosidase in cells, and reducing the gene level, transcription level and / or expression level of marker molecules of aging in cells such as P53, P21, DJ-1 and other molecules. The delaying of cell aging can be used to delay or prevent the aging of target cells for therapeutic purposes, restore, maintain and / or enhance the physiological functions of target cells, etc., and can also be used to establish a cell model or animal model for delaying cell aging for the purpose of drug evaluation and screening.

[0011] In a more preferred embodiment, the vector promoting overexpression of the circRNA molecule contains a DNA molecule with a sequence as shown in bases 9 to 1175 of SEQ ID NO. 1. In a specific embodiment of the present invention, the vector is pLC5-ciR, and other gene expression vectors in the art can also be applied to the present invention.

[0012] In another preferred embodiment, the application is to prepare the vector that silences the expression of the circRNA molecule as a drug that promotes cell aging. The promotion of aging can be evaluated using conventional evaluation methods in the art, for example, shortening the length of cell telomeres, weakening the activity of cell telomerase, reducing the proliferation ability of endothelial cells, increasing the β-galactosidase positive rate of cells, and increasing the gene level, transcription level and / or expression level of marker molecules of aging in cells such as P53, P21, DJ-1, etc. The promotion of cell aging can promote the aging and even death of target cells, apoptosis, etc. for therapeutic purposes, and can also establish a cell model or animal model that promotes cell aging for the purpose of drug evaluation and screening.

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

[0014] Finally, the present invention provides an in vitro cell containing a DNA molecule having a sequence as shown in bases 9 to 1175 of SEQ ID NO. 1. The in vitro cell of the present invention refers to a cell that is separated from a parent patient and cultured independently in vivo to survive.

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

[0016] The present invention provides peripheral blood mononuclear cells transformed with a DNA molecule represented by bases 9 to 1175 of SEQ ID NO.1, and the senescence degree of the cells can be significantly improved. In a specific application, the ex vivo cells with significantly improved senescence degree can be transfused back to the parent patient, so that the cells survive in the parent patient's body and exert their inherent physiological functions. Therefore, the present invention provides the use of the above-mentioned ex vivo cells in the preparation of a drug for delaying cell senescence.

[0017] The present invention found that circHERC1 (circHERC1) was lowly expressed in both natural aging and induced aging cell models, and the expression of circHERC1 in normal peripheral blood mononuclear cells also gradually decreased with age. CircHERC1 was negatively correlated with the age and telomere length of the population in PBMCs of normal people of different ages. During the evaluation process, the shortening of telomere length in human PBMCs was also accompanied by a significant decrease in circHERC1 levels. The overexpression or knockout experiment of circHERC1 proved that it can effectively extend the length of cell telomeres and activate telomerase, indicating that circHERC1 is closely related to the aging of cells and organisms, and circHERC1 is expected to become a new biomarker of aging and an effective molecular target for delaying cell aging. The aging degree of target cells can be evaluated by detecting the content of circHERC1 in target cells, and target cells with improved aging degree can be obtained by overexpressing circHERC1 in target cells. Moreover, when the target cells are ex vivo cells, the target cells can be reinfused to achieve the therapeutic purpose of delaying the aging of the cell nucleus and organism. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 . Expression of circHERC1 in PBMCs of normal subjects of different ages; Figure 2 . Analysis of the correlation between the expression of circHERC1 in PBMCs of normal people of different age groups and age; Figure 3 . Expression of circHERC1 in naturally aged human umbilical vein endothelial cells; Figure 4 . Expression of circHERC1 in doxorubicin-induced senescent Hela cells; Figure 5 . Expression of circHERC1 in hydrogen peroxide-induced senescent Hela cells; Figure 6 . Map of plasmids used to overexpress circHERC1; Figure 7 . Expression of shRNA in HUVEC cells to verify its knockdown efficiency; Figure 8 . Expression of circHERC1 overexpression vector in HUVEC cells to verify its overexpression efficiency; Fig. 9 . The growth curve determined by CCK8 showed that high expression of circ0035796 significantly enhanced the proliferation of HUVEC cells. Fig.10 . The growth curve determined by CCK8 showed that low expression of circ0035796 significantly inhibited the proliferation of HUVEC cells; Fig.11 . The proliferation experiment 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; Fig.12 . The results of high expression of circHERC1 significantly prolonged the telomere length of HUVEC cells, and low expression of circ0035796 significantly shortened the telomere length of HUVEC cells; Fig.13 . CircHERC1 high expression significantly activated the telomerase activity of HUVEC cells; circHERC1 low expression significantly inhibited the telomerase activity of HUVEC cells; Fig.14 . The β-galactosidase senescence staining experiment showed that high expression of circHERC1 significantly inhibited the senescence of HUVEC cells, while low expression of circHERC1 promoted the senescence of HUVEC cells; Fig.15 . Circ_0035696 high expression inhibits the expression of aging-related marker proteins P53, P21, and DJ-1, while circ_0035696 low expression promotes the expression of aging-related marker proteins P53, P21, and DJ-1; Fig.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. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. 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.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] Example 1: The expression of circHERC1 in normal human PBMC decreases with age 1. Circular RNA circHERC1 is derived from the circular RNA disclosed in CN115948548A: circ_0035796, and is obtained through differentially expressed gene screening of plasma specimens from patients with non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, and benign lung nodules.

[0023] In the present invention, the circRNA is named "circHERC1". The nucleic acid sequence of circular RNA circHERC1 is bases 21 to 1163 of the sequence shown in SEQ ID NO.1.

[0024] 2. Expression of circular RNA circHERC1 in PBMCs of normal subjects Source of tissue specimens: 300 normal subjects without underlying diseases were selected as research subjects. All patients were fully informed and signed informed consent before sampling.

[0025] 1. Sample selection criteria and grouping basis: According to the clinical data of the patients, blood samples from patients with a history of cancer or infectious viral infection (such as infectious hepatitis, syphilis, and HIV, etc.) were excluded. Blood samples from healthy patients were used as experimental subjects (as shown in Table 1) for the detection of circHERC1 and telomere length.

[0026] Table 1. Clinical data of patients

[0027] 2. Reverse Transcription of PBMC RNA Collect blood samples from the test subjects, add PBS (Gibco, USA) to the blood samples after plasma separation to make up to the original volume, and mix well; slowly add the mixed blood along the tube wall into a 15 mL centrifuge tube containing an equal volume of lymphocyte separation solution (Solabo, Product No. P8900) prepared in advance; centrifuge at 400g for 15 minutes at room temperature; after centrifugation, the solution in the tube is clearly divided into three layers. The upper layer is PBS solution, the middle layer is lymphocyte separation solution, and the bottom of the tube is the red blood cell layer. Between the upper and middle layers is a thin and dense white membrane, which is the PBMC layer. Carefully pipette the cells in the buffy coat layer into a 15mL centrifuge tube, add PBS to the 15mL mark, and mix by inverting repeatedly; centrifuge at room temperature at 400g for 5 minutes; the PBMCs after centrifugation sink to the bottom of the tube, discard the supernatant, add 1mL of red blood cell lysis buffer to resuspend the cells and transfer to a 1.5mL centrifuge tube; centrifuge at room temperature at 2000rpm for 5 minutes; discard the supernatant, add 1mL of PBS to resuspend the cells again; centrifuge at room temperature at 2000 rpm for 5 minutes; discard the supernatant, the white precipitate is the peripheral blood mononuclear cell (PBMC); split the above PBMCs into two to extract RNA and DNA respectively. The reverse transcription steps of PBMC RNA are as follows: 1) Prepare reverse transcription mixture (Table 2) using RNA reverse transcription kit (Thermo Company, USA) and keep on ice for later use; Table 2. Reverse transcription reaction system

[0028] 2) After preparing the reverse transcription mixture, add it to 8 tubes respectively, then add 10μL RNA sample to prepare a 20μL reaction system, gently pipette to mix, cover tightly, and centrifuge briefly; 3) Place in a 96-well PCR instrument and set the reaction conditions as follows (Table 3): Table 3. Reverse transcription PCR conditions

[0029] 3. Primer design Primers were designed based on the gene sequences of circHERC1 and GAPDH for real-time fluorescence quantitative PCR reaction. The specific primer names and sequences are shown in Table 4: Table 4. Primer sequence information used in PCR reaction

[0030] 4. qRT-PCR reaction system and conditions Extract RNA from the PBMC to be tested, reverse transcribe to obtain cDNA as a template, and perform qRT-PCR using the following system and procedure: According to the primer synthesis instructions, add an appropriate amount of RNase-free-water to prepare a primer solution with a concentration of 10 μM. TM SYBR qPCR Mix Without ROX Kit, prepare the reaction system according to the instructions as follows (Table 5): Table 5. qRT-PCR reaction system

[0031] The prepared qRT-PCR reaction system shown in Table 5 was added to a 96-well PCR plate. 18 μL of the qRT-PCR reaction system shown in Table 5 was added to each reaction well. 2 μL of cDNA was then added to make a 20 μL reaction system. The reaction was placed in a real-time fluorescence quantitative PCR instrument (Bio-Rad, USA) for reaction. The PCR reaction conditions were as follows (Table 6): Table 6. qRT-PCR reaction conditions

[0032] Three replicate wells were set for each sample, with GAPDH as the internal reference gene. After the qPCR reaction was completed, the average Ct value of the three replicate wells of the target gene in each sample was taken as the final result. The relative expression of each target gene was calculated as 2^-△Ct, where △Ct = Ct (target gene) - Ct (internal reference gene).

[0033] qRT-PCR was used to detect the expression of circHERC1 in PBMCs of 300 normal subjects of different ages.

[0034] The results are as follows Figure 1 As shown in the figure, the expression of circHERC1 gradually decreased with the increase of the age of the sample, and the expression of circHERC1 was correlated with age (P<0.05). According to the relative expression of circHERC1, a correlation curve was drawn in GraphPad Prim 6. The results showed that the expression of circHERC1 gradually decreased with the increase of human age, and there was a negative correlation between circHERC1 and age in PBMC of people of different ages (P<0.05) (see Figure 2 ). This indicates that the circRNA can be used as a diagnostic molecular marker for body aging.

[0035] 5. circHERC1 expression is reduced in replicatively aged umbilical cord mesenchymal cells RNA was extracted from each generation of naturally passaged HUVEC cells, and cDNA was obtained by reverse transcription as a template. Real-time PCR was performed according to the above experimental process. The results showed that with the passage of HUVEC cells, the degree of cell senescence increased, and the expression level of circHERC1 gradually decreased (see Figure 3 ).

[0036] 6. circHERC1 expression is reduced in senescent cells Hela cells were induced to senescence by doxorubicin and hydrogen peroxide. The cDNA obtained by reverse transcription was used as a template. Real-time PCR was performed according to the above experimental process. The results showed that the expression level of circHERC1 in induced senescent cells was reduced, and the expression level after induction was about 20% of the expression level in the control (doxorubicin-induced, Figure 4 ) to 50% (hydrogen peroxide induced, Figure 5 ).

[0037] Example 2: Application of circHERC1 in delaying aging 1. Construction of circHERC1 overexpression plasmid or cells and knockdown plasmid or cells 1. Construction of circHERC1 overexpression plasmid and knockdown plasmid Overexpression vector pLC5-circHERC1: The DNA molecule containing the circHERC1 linear sequence (SEQ ID NO.1) was used to replace the DNA molecule between the EcoRI and BamHI restriction sites of the circRNA overexpression vector pLC5-ciR (Guangzhou Gisai Biotechnology Co., Ltd.) to obtain the circHERC1 overexpression vector (see the plasmid map for details). Figure 6 ); The nucleic acid sequence of the DNA molecule containing the circHERC1 linear 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 cyclization mediating sequences, positions 19-20 are AG acceptors, positions 1164-1165 are GT donors, positions 1166-1175 are reverse cyclization mediating sequences, and positions 1176-1181 are BamHI restriction sites.

[0038] The silencing expression vector pLKO.1-circHERC1 is a vector 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 the expression of circHERC1 (SEQ ID NO. 6, sequence GCATCACTATCAGATATCA (the linker of circular RNA, SEQ ID NO. 1 positions 1151-1163 and 21-26).

[0039] 2. Construction of circHERC1 overexpression and knockdown cells 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), respectively, to obtain pLC5-circHERC1 transfected cells, pLC5-ciR transfected cells, pLKO.1-circHERC1 transfected cells and pLKO.1 transfected cells.

[0040] RNA was extracted from cells transfected with pLC5-circHERC1, cells transfected with pLC5-ciR, cells transfected with pLKO.1- circHERC1, and cells transfected with pLKO.1, and reverse transcribed according to the above method to obtain cDNA as a template for the above RT-PCR amplification.

[0041] The expression results of pLKO.1- circHERC1 cells (denoted as knockdown in the figure) and pLKO.1 cells (denoted as control in the figure) are shown in Figure 7 As shown in the figure, compared with the pLKO.1 transfected cells, the expression level of circHERC1 in the pLKO.1-circHERC1 transfected cells was significantly downregulated by 17.4 times (P<0.01), indicating that cells with silenced circHERC1 expression were obtained.

[0042] The expression levels of pLC5-circHERC1 transfected cells (denoted as overexpression in the figure) and pLC5-ciR transfected cells (denoted as control in the figure) are as follows Figure 8 As shown in Figure 3, compared with the empty vector, the expression level of circHERC1 in pLC5-circHERC1 cells was upregulated by 4.55 times (P<0.01), indicating that cells overexpressing circHERC1 were obtained. 2. circHERC1 is associated with the proliferation ability of endothelial cells CCK-8 assay and EdU assay were used to detect the proliferation ability of endothelial cells.

[0043] 1. CCK-8 assay to detect cell proliferation The CCK-8 kit is used to quickly and sensitively detect cell proliferation. In the presence of an electron coupling reagent, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazolyl monosodium salt) can be reduced by dehydrogenases in mitochondria to generate a highly water-soluble orange-yellow formazan product, the color depth of which is proportional to cell proliferation, and the color depth is linearly related to the number of cells. Using an ELISA reader to measure the OD value at a wavelength of 450nm can indirectly reflect the number of living cells.

[0044] The pLC5-circHERC1-transfected HUVECs, pLC5-ciR-transfected HUVECs, pLKO.1-circHERC1-transfected HUVECs, and pLKO.1-transfected HUVECs obtained above were counted using a Bio-Rad cell counting plate and prepared into a cell suspension (density of about 2×10 4 / mL), inoculated into 96-well plates at 100μL per well, set up 3 replicates / group, and add after the cells adhere to the wall. Add 10μL of CCK-8 solution (Cell Counting Kit-8, APExBIO Product No. K1018) per well, incubate in a 37℃ cell culture incubator for 120min, and then measure the optical density OD (450nm) with an ELISA reader (MD Company, USA). Then, measure the optical density of each group of cells after 24, 48, 72, and 96 hours of culture using the same method, record the values ​​and draw growth curves.

[0045] The results are as follows Figure 9-10 As shown, Fig. 9 For the overexpression group, Fig.10 As can be seen, in the CCK-8 experiment, after 96 h of culture, in the overexpression group, compared with the pLC5-ciR transfected cells (denoted as control in the figure), the optical density value at 450 nm of the pLC5-circHERC1 transfected cells (denoted as overexpressed in the figure) was significantly increased. The OD value of the control group was 1.24; the OD value of the cells overexpressing circHERC1 was 1.71 (P < 0.01, Fig. 9 In the silencing group, compared with cells transfected with pLKO.1 (denoted as control in the figure), cells transfected with pLKO.1- circHERC1 (denoted as knockdown in the figure) had significantly lower optical density at 450 nm. The OD value of the control group was 1.19; the OD value of cells with knockdown of circHERC1 was 0.88 (P < 0.01, Fig.10 ).

[0046] 2. EdU Experiment Edu is a new type of thymidine analog. It is incorporated into the newly synthesized DNA instead of thymidine during the DNA synthesis process. The acetylene group on Edu can react covalently with biotin-labeled azide through the catalysis of monovalent copper ions to form a stable tricyclic ring. The reaction is rapid, and the newly synthesized DNA will be labeled with the corresponding biotin probe, thereby displaying a different color.

[0047] According to the BeyoClick Edu-594 Cell Proliferation Assay Kit (Biyuntian, Cat. No. C0078L), pLC5-circHERC1 cells, pLC5-ciR cells, pLKO.1-circHERC1 cells, and pLKO.1 cells were EdU labeled, fixed with 4% paraformaldehyde for 15 min, washed twice with PBS for 5 min each, permeabilized with 1% Triton-X100 at room temperature for 10 min, the permeabilization solution was removed, and washed twice with PBS for 5 min each. 1 mL of staining solution was prepared, and the formula was as follows: 860 μL-ClickReaction Buffer, 40 μL-CuSO4, 2 μL-Azide 594, 100 μL-Click Additive Solution. The cells were stained with the prepared staining solution for 30 minutes in the dark, and washed twice with PBS for 5 minutes each. The cells were stained for DNA using Mounting medium with DAPI (Abcam, Catalog No. ab104139) in the dark and then photographed under a fluorescence microscope.

[0048] The results are as follows Fig.11 As shown, from left to right are the overexpression control group, overexpression group, knockout control group, and knockout group, and 5 fields of view were collected respectively. In the overexpression group, the positive cell rate of cells transfected with pLC5-circHERC1 (represented as overexpression in the figure) was 52.46%, and the positive cell rate of cells transfected with pLC5-ciR (represented as control 1 in the figure) was 31.15%, and the cell proliferation activity was significantly increased (P<0.05,); in the knockout group, the positive cell rate of cells transfected with pLKO.1-circHERC1 (represented as knockdown in the figure) was 21.57%, and the positive cell rate of cells transfected with pLKO.1 (represented as control 2 in the figure) was 32.39%. Silencing circHERC1 significantly reduced the number of positive cells in endothelial cells and significantly reduced the cell proliferation activity (P<0.01, Fig.11 ).

[0049] The above results indicate that overexpression of circHERC1 can promote endothelial cell proliferation, while knockdown of circHERC1 can inhibit endothelial cell proliferation.

[0050] 3. circHERC1 is associated with cell telomere length

[0051] Cell telomere length detection experiment and TRAP telomerase activity detection experiment were used to verify the effect of circHERC1 on cell telomeres.

[0052] 1. Cell telomere length detection The day before transfection, endothelial cells HUVEC were seeded on a 6-well cell culture plate with lines, and the cell density was preferably 70% to 80% on the next day; transfection experiments were performed, and pLC5-ciR and overexpression vector pLC5-circHERC1; pLKO.1 and silenced expression vector pLKO.1-circHERC1 were transfected into HUVEC (Beina Chuanglian Biotechnology Co., Ltd., catalog number BNCC342436) / Hela cells (ATCC, catalog number CBP60232), respectively.

[0053] After transfection, cells that stably express circHERC1 were screened by pressure, and their expression efficiency was verified by real-time PCR, and then the genome was extracted for telomere length detection. After extracting the cell genome using the Human Absolute Telomere Length Detection Kit (Sciencell, USA, Cat. No. 8918), two qRT-PCR reactions were prepared for each DNA sample, one using telomere tel primer stock solution and the other using internal reference gene HBG primer stock solution, and the reaction system was prepared according to the table below.

[0054] Table 7. Telomere length detection reaction system

[0055] Place the prepared reaction system into the real-time PCR instrument and perform the reaction according to the procedure in the table below.

[0056] Table 8. Telomere length detection reaction procedure

[0057] The results showed that overexpression of circHERC1 could significantly prolong the length of cell telomeres. The telomere length of the overexpression control group was 6373 bp (control group 1); the telomere length of circHERC1 overexpression was 8594 bp; knockdown of circHERC1 shortened the length of cell telomeres. The telomere length of the knockdown control group was 6091 bp (control group 2); the telomere length of circHERC1 knockdown was 4921 bp ( Fig.12 as shown).

[0058] 2. TRAP telomerase activity detection experiment The TRAP telomerase activity detection kit (Sigma-Aldrich, catalog number S7700FR) was used to detect the effect of circHERC1 on cell telomerase activity. The TRAP detection method uses the characteristics of telomerase, which can use the template region of its own RNA as a template in vitro to add a 6-base repeat sequence at the end of a suitable oligonucleotide chain. The repeat sequence is amplified by PCR, and then the 6-base difference ladder is performed by polyacrylamide (PAGE) gel electrophoresis. CircHERC1 overexpressed and knocked down cells were plated in 24-well plates, collected in centrifuge tubes, and 3-4 times the volume of CHAPs lysis buffer was added. Lysis was performed on ice for 30 minutes (RNase inhibitor 1:200, cocktail 1:100), and the supernatant was transferred to a new tube. 3μL+100μL BCA kit was taken to detect protein concentration, and CHAPs was used as a blank control. The kit was diluted 1:50, incubated at 37℃ for 30 minutes, and the OD value was measured at 565nm. Calculate the TRAP dilution concentration, then take the corresponding volume of stock solution and dilute it with CHAPs to make the protein concentration 100 mg / 1L, and configure it according to the following system: Table 9. Telomerase activity detection reaction system

[0059] Table 10. Reverse transcription PCR conditions

[0060] Note*: Steps 2-4 were repeated 27 times.

[0061] After PCR is completed, add 5x loading buffer for dilution. Load 10 μL of sample, 0.5x TBE buffer, TRAP gel electrophoresis at 200V, UV development after 50 minutes.

[0062] Table 11. Formulation for TRAP glue

[0063] The results showed that the telomerase activity in cells with circHERC1 overexpression was significantly enhanced compared with that in the overexpression control group (control group 1), while the telomerase activity in cells with circHERC1 knockdown was weakened compared with that in the knockdown control group (control group 2). Fig.13 as shown).

[0064] CircHERC1 inhibits cell senescence β-galactosidase staining experiments and aging-related protein level detection experiments were used to verify that circHERC1 inhibited cell senescence.

[0065] Senescent cells have highly active β-galactosidase. Through in situ staining, using X-Gal as a substrate, a dark blue product will be generated under the catalysis of β-galactosidase, which can be observed under an optical microscope.

[0066] This experiment uses a cell senescence β-galactosidase staining kit (China Biotech Company, Cat. No. C0602) for reaction, and the steps are as follows: endothelial cells HUVEC are evenly inoculated in a six-well plate or a 12-well plate, wait for the cells to adhere to the wall, gently wash the cells with PBS buffer and remove the PBS, add 1 mL of fixative, and fix at room temperature for 15 min. Remove the fixative, and gently shake and wash with PBS buffer 3 times, 3 min each time. Prepare the cell senescence staining solution according to the table below.

[0067] Table 12. Preparation of cell senescence staining solution

[0068] Use a pH meter to measure the pH of the cell senescence staining solution, and use citric acid or sodium hydroxide to adjust the pH of the solution to 6.0. Remove the PBS buffer, add 1 mL of the staining working solution to each well, incubate overnight in a 37°C ordinary incubator, and wrap with tin foil to prevent the liquid from evaporating. Do not incubate in a carbon dioxide incubator. After the staining is completed, remove the staining solution and observe it under a microscope immediately. Senescent cells will appear dark blue. Select five fields of view to take pictures and record, count the blue cells and the total number of cells, and calculate the staining positive ratio.

[0069] The results showed that circHERC1 overexpression reduced the β-galactosidase positive rate of cells by 55.3% compared with the overexpression control group (control group 1), and the degree of cell senescence was reduced. circHERC1 knockdown increased the β-galactosidase positive rate of cells by 3.27 times compared with the knockdown control group (control group 2), promoting cell senescence (such as Fig.14 as shown).

[0070] 5. Detection of Aging-Related Molecules Protein immunoblotting experiments were used to detect the expression levels of marker molecules of aging in cells, such as P21 (which plays an important role in cell cycle control, DNA replication and repair), P53 (located upstream of P21 and closely related to cell aging), and DJ-1 (involved in cellular oxidative stress).

[0071] Remove the culture medium, wash with pre-cooled PBS and discard the washing solution. Add 300ul pre-cooled cell lysis solution to the endothelial cells HUVEC and fully lyse them on ice; after lysis, transfer to EP tubes and centrifuge at 12000rpm for 10min at 4℃.

[0072] Take the supernatant for protein concentration determination, and prepare BCA working solution (Thermo Corporation, USA, catalog number 23227) with reagent A:B (50:1). Add 25μL of each standard or unknown sample to the well of the 96-well plate; add 200μL of working solution to each well and mix thoroughly. Incubate at 37℃ for 30 minutes. Calculate the protein concentration after measuring the absorbance at 562nm on the microplate reader, dilute the protein sample with protein loading buffer (China Kangwei Century Company, catalog number 01411), boil it in boiling water for 10 minutes, and store it at -80℃ for use. Prepare electrophoresis gel of appropriate concentration (8%-15%) according to the size of protein molecules; add glycine electrophoresis solution, add 30µg sample / well and protein marker 5μL / well; run at 80V for 1h, then switch to 120V, stop electrophoresis and turn off the voltage when the sample is just finished; PVDF membrane (Merck, USA, catalog number IPVH00010) is activated with methanol, made together with two black sponges and 6 filter papers to avoid bubbles, assemble the transfer tank, add transfer solution, and place it in an ice bath for transfer for 1h; after the transfer is completed, the membrane is placed in the blocking solution 5% skim milk powder and blocked at room temperature for 1h on a slow shaking table; apply primary antibody P53 (Wuhan Sanying Biological Company, catalog number 10024098) 1:5000 dilution; P21 CST, catalog number 2947S) 1:1000 dilution; DJ-1 (CST, catalog number 5933S) 1:1000 dilution; TERT (abcam, catalog number ab3202) 1:1000 dilution. Place in 4℃ refrigerator overnight; wash the membrane 5 times, 5 min each time; use secondary antibody at room temperature for 1 hour; wash the membrane 5 times, 5 min each time; develop and record using protein developer.

[0073] The results showed that compared with the empty vector control group (control group 1), the expression levels of aging-related proteins P53, P21, and DJ-1 were all reduced in the overexpressed circHERC1 group, while the key component of telomerase, telomerase reverse transcriptase TERT, was increased; compared with the empty vector control group (control group 2), the expression levels of P53, P21, and DJ-1 were all increased in the knockdown circHERC1 group (e.g. Fig.15 TERT expression decreased (as shown in Fig.16 As shown, Fig.16 In the figure, Vector was the overexpression control group, circHERC1 was the overexpression group, NC was the knockdown control group, and sh circRNA was the knockdown group). The above results showed that the expression of circHERC1 enhanced the expression of telomerase TERT and inhibited cell senescence.

Claims

1. The use of circRNA molecules as diagnostic markers in a cell aging diagnostic kit is characterized in that: The sequence of the circRNA molecule is shown in bases 21 to 1163 of SEQ ID NO.1, and the kit contains a reagent for detecting the content or expression amount of the circRNA molecule in the sample to be detected.

2. The use according to claim 1, characterized in that: The cell aging diagnostic kit is a gene detection kit.

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

4. The use according to claim 3, characterized in that 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 as SEQ ID NO.2 and SEQ ID NO.3 respectively.

5. Application of circRNA molecules as targets for the treatment of cellular aging, characterized in that: The sequence of the circRNA molecule is shown in bases 21 to 1163 of SEQ ID NO.1, and the application is to prepare a substance that promotes or reduces the expression of the circRNA molecule as a drug for regulating cell aging.

6. The use according to claim 5, characterized in that: The application is to prepare the vector that promotes the overexpression of the circRNA molecule into a drug for delaying cell aging.

7. The use according to claim 6, characterized in that The vector for promoting overexpression of the circRNA molecule contains a DNA molecule whose sequence is shown in bases 9 to 1175 of SEQ ID NO.

1.

8. The use according to claim 5, characterized in that: The application is to prepare the vector for silencing the expression of the circRNA molecule as a drug for promoting cell senescence.

9. The use according to claim 8, characterized in that The vector for silencing the expression of the circRNA molecule contains a DNA molecule with a sequence as shown in SEQ ID NO.

6.

10. An in vitro cell containing a DNA molecule having a sequence as shown in bases 9 to 1175 of SEQ ID NO.

1.

11. The isolated cell according to claim 10, characterized in that The ex vivo cells are peripheral blood mononuclear cells.

12. Use of the in vitro cells according to claim 10 or 11 in preparing a drug for delaying cell senescence.

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