Application of PAPPA as senescence marker and SIRTs-PAPPA in senescence regulation

Abstract

The invention discloses application of PAPPA as an aging marker and application of SIRTs-PAPPA in aging regulation and control. The invention belongs to the field of biomedicine, and particularly relates to application of PAPPA as an aging marker and application of SIRTs-PAPPA in aging regulation and control. The invention provides application of a substance for inhibiting PAPPA gene expression in preparation of a cell aging inhibitor or an anti-aging drug. Wherein the substance for inhibiting PAPPA gene expression or the substance for inhibiting PAPPA protein activity is siRNA (small interfering ribonucleic acid), and the siRNA is an RNA (ribonucleic acid) molecule of which the nucleotide sequence is SEQ ID No.1. The invention also provides application of the substance for regulating and controlling SIRTs-PAPPA pathway expression in preparation of a product for diagnosis or auxiliary diagnosis of senescence or identification of senescence cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of PAPPA as an aging marker and SIRTs-PAPPA in aging regulation. Background Art

[0002] The Sirtuin family (SIRT1-7) is a class of NAD + The sirtuin family is a family of enzymes that regulates the production of sirtuins, a type of enzyme that is involved in the production of sirtuins. These enzymes are highly conserved from archaea to mammals. The sirtuin family plays an important role in diverse cellular processes, such as apoptosis, mitochondrial biogenesis, lipid metabolism, and aging.

[0003] The gene encoding pregnancy-associated plasma protein A, also known as Pappalysin 1 (PAPPA), is located on chromosome 9, 9q33.1. The genomic sequence is approximately 248 kb in length, encoding 22 exons. The encoded protein, PAPPA, has a molecular mass of approximately 180.9 kDa. PAPPA is a secreted glycoproteinase and a metalloproteinase associated with insulin-like growth factor. During pregnancy, the placenta and decidua secrete large amounts of PAPPA into the bloodstream. Currently, PAPPA protein levels are used clinically as a screening and diagnostic tool for conditions such as Down syndrome. Summary of the Invention

[0004] The main problem to be solved by the present invention is how to delay aging.

[0005] In order to solve the above problems, the present invention provides any of the following applications:

[0006] 1) Use of substances that inhibit PAPPA gene expression in the preparation of cell aging inhibitors or anti-aging drugs;

[0007] 2) Use of substances that inhibit PAPPA gene expression in the preparation of drugs for treating cell senescence and / or preventing senescence;

[0008] 3) Use of substances that inhibit PAPPA protein activity in the preparation of cell aging inhibitors;

[0009] 4) Use of substances that inhibit PAPPA protein activity in the preparation of drugs for treating cell senescence and / or preventing senescence;

[0010] 5) Use of substances that reduce PAPPA protein content in the preparation of cell aging inhibitors;

[0011] 6) Use of a substance that reduces PAPPA protein content in the preparation of a drug for treating cell senescence and / or preventing senescence.

[0012] Herein, the amino acid sequence of the PAPPA protein is SEQ ID No. 3, and the nucleotide sequence of the gene encoding the protein is SEQ ID No. 4.

[0013] In the above application, the substance that inhibits PAPPA gene expression or the substance that inhibits PAPPA protein activity can be any of the following biological materials:

[0014] 1) a double-stranded RNA molecule, a modified substance thereof, or a pharmaceutically acceptable salt thereof;

[0015] B1) producing a DNA molecule of the double-stranded RNA molecule described in 1);

[0016] B2) an expression cassette containing the DNA molecule described in B1);

[0017] B3) a recombinant vector containing the DNA molecule described in B1);

[0018] B4) a recombinant vector containing the expression cassette described in B2);

[0019] B5) a recombinant microorganism containing the DNA molecule described in B1);

[0020] B6) a recombinant microorganism containing the expression cassette described in B2);

[0021] B7) a recombinant microorganism containing the recombinant vector described in B3);

[0022] B8) A recombinant microorganism containing the recombinant vector described in B4).

[0023] In the above application, the substance that inhibits PAPPA gene expression or the substance that inhibits PAPPA protein activity is siRNA.

[0024] Furthermore, the siRNA is an RNA molecule whose nucleotide sequence is SEQ ID No. 1 in the sequence list.

[0025] The present invention also provides a pharmaceutical composition, which contains any one of the following substances:

[0026] M1) a substance that inhibits the expression of the PAPPA gene as described above, or a substance that inhibits the activity of the PAPPA protein as described above;

[0027] M2) Cell senescence inhibitors.

[0028] In the above-mentioned pharmaceutical composition, the substance may be any of the following biological materials:

[0029] 1) a double-stranded RNA molecule, a modified substance thereof, or a pharmaceutically acceptable salt thereof;

[0030] B1) producing a DNA molecule of the double-stranded RNA molecule described in 1);

[0031] B2) an expression cassette containing the DNA molecule described in B1);

[0032] B3) a recombinant vector containing the DNA molecule described in B1);

[0033] B4) a recombinant vector containing the expression cassette described in B2);

[0034] B5) a recombinant microorganism containing the DNA molecule described in B1);

[0035] B6) a recombinant microorganism containing the expression cassette described in B2);

[0036] B7) a recombinant microorganism containing the recombinant vector described in B3);

[0037] B8) A recombinant microorganism containing the recombinant vector described in B4).

[0038] In the above pharmaceutical composition, the substance that inhibits PAPPA gene expression or the substance that inhibits PAPPA protein activity is siRNA.

[0039] Furthermore, the siRNA is an RNA molecule whose nucleotide sequence is SEQ ID No. 1 in the sequence list.

[0040] The present invention also provides the use of a substance for detecting PAPPA gene expression or a substance for detecting PAPPA protein content in any of the following:

[0041] A1) Preparation of products for diagnosing or assisting in the diagnosis of aging;

[0042] A2) preparing products for identifying or assisting in the identification of senescent cells;

[0043] A3) preparing products for screening or assisting in screening young cells;

[0044] A4) Preparation of products that assess or assist in the assessment of individual aging, organ / tissue aging, or cellular aging;

[0045] A5) Preparation of products for identifying or assisting in the identification of individual aging, organ / tissue aging, or cellular aging.

[0046] The present invention also provides the use of a substance for regulating the expression of the SIRTs-PAPPA pathway in any of the following:

[0047] A1) Preparation of products for diagnosing or assisting in the diagnosis of aging;

[0048] A2) preparing products for identifying or assisting in the identification of senescent cells;

[0049] A3) preparing products for screening or assisting in screening young cells;

[0050] A4) Preparation of products that assess or assist in the assessment of individual aging, organ / tissue aging, or cellular aging;

[0051] A5) Preparation of products for identifying or assisting in the identification of individual aging, organ / tissue aging, or cellular aging;

[0052] The SIRTs-PAPPA pathway is composed of SIRTs protein and PAPPA protein, and the SIRTs protein is at least one of the seven proteins SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 and SIRT7.

[0053] The amino acid sequence of the SIRT1 protein is SEQ ID No. 5, and the nucleotide sequence of the gene encoding the protein is SEQ ID No. 6.

[0054] The amino acid sequence of the SIRT2 protein is SEQ ID No. 7, and the nucleotide sequence of the gene encoding the protein is SEQ ID No. 8.

[0055] The amino acid sequence of the SIRT3 protein is SEQ ID No. 9, and the nucleotide sequence of the gene encoding the protein is SEQ ID No. 10.

[0056] The amino acid sequence of the SIRT4 protein is SEQ ID No. 11, and the nucleotide sequence of the gene encoding the protein is SEQ ID No. 12.

[0057] The amino acid sequence of the SIRT5 protein is SEQ ID No. 13, and the nucleotide sequence of the gene encoding the protein is SEQ ID No. 14.

[0058] The amino acid sequence of the SIRT6 protein is SEQ ID No. 15, and the nucleotide sequence of the gene encoding the protein is SEQ ID No. 16.

[0059] The amino acid sequence of the SIRT7 protein is SEQ ID No. 17, and the nucleotide sequence of the gene encoding the protein is SEQ ID No. 18.

[0060] In the above application, the substance that regulates the expression of the SIRTs-PAPPA pathway may be substance S and substance P;

[0061] The substance S may be a substance that inhibits the expression of the SIRTs protein gene, a substance that inhibits the activity of the SIRTs protein, and / or a substance that reduces the content of the SIRTs protein;

[0062] The substance P may be a substance that inhibits the expression of the PAPPA gene, a substance that inhibits the activity of the PAPPA protein, and / or a substance that reduces the content of the PAPPA protein.

[0063] Furthermore, the substance that inhibits PAPPA gene expression or the substance that inhibits PAPPA protein activity may be any of the following biological materials:

[0064] 1) a double-stranded RNA molecule, a modified substance thereof, or a pharmaceutically acceptable salt thereof;

[0065] B1) producing a DNA molecule of the double-stranded RNA molecule described in 1);

[0066] B2) an expression cassette containing the DNA molecule described in B1);

[0067] B3) a recombinant vector containing the DNA molecule described in B1);

[0068] B4) a recombinant vector containing the expression cassette described in B2);

[0069] B5) a recombinant microorganism containing the DNA molecule described in B1);

[0070] B6) a recombinant microorganism containing the expression cassette described in B2);

[0071] B7) a recombinant microorganism containing the recombinant vector described in B3);

[0072] B8) A recombinant microorganism containing the recombinant vector described in B4).

[0073] In the above application, the substance that inhibits PAPPA gene expression or the substance that inhibits PAPPA protein activity is siRNA.

[0074] Furthermore, the siRNA is an RNA molecule whose nucleotide sequence is SEQ ID No. 1 in the sequence list.

[0075] The present invention also provides a method, which can be any of the following:

[0076] F1) A method for delaying aging of an organ, tissue or cell, comprising the step of contacting the substance for regulating the expression of the SIRTs-PAPPA pathway described in the application above with the organ, tissue or cell;

[0077] F2) A method for improving functional disorders of aging organs, tissues or cells, comprising the step of contacting the substance for regulating the expression of the SIRTs-PAPPA pathway described in the above application with the aging body, organ, tissue or cell;

[0078] F3) A method for identifying or assisting in identifying the level of senescence of an organ, tissue or cell in vitro, comprising the step of detecting the expression level of the SIRTs protein and / or the expression level of the PAPPA protein in a sample from the subject;

[0079] F4) A method for constructing senescent cells, comprising the step of introducing the substance for regulating the expression of the SIRTs-PAPPA pathway described in the application described above into recipient cells;

[0080] F5) A method for screening or assisting in screening for drugs that delay aging, comprising the step of screening for drugs that delay aging using senescent cells constructed by the method described in F4);

[0081] F6) A method for studying or assisting in studying the aging mechanism of an organism, organ, tissue, or cell, comprising the step of using senescent cells constructed by the method described in F4) to study the aging mechanism of an organism, organ, tissue, or cell;

[0082] F7) A method for changing the senescence state of cells in vitro, comprising the step of introducing the substance for regulating the expression of the SIRTs-PAPPA pathway described in the application described above into recipient cells.

[0083] In the above, the organ, tissue or cell may be an isolated organ, tissue or cell.

[0084] Furthermore, the organ may be a placenta, parathyroid gland, adipose tissue, kidney, cervix and esophagus, etc.

[0085] The cells may be stem cells or conventional primary cells.

[0086] Any of the above applications or methods may be for the purpose of diagnosing and treating a disease, or for the purpose of diagnosing and treating a non-disease.

[0087] This invention uses multi-level omics technology to systematically analyze the multi-level epigenetic changes regulated by seven members of the SIRT family (SIRT1-7), and preliminarily reveals the important role of the SIRT-PAPPA regulatory axis in the aging process of human stem cells. SIRT proteins become molecular nodes related to regulating lifespan by linking epigenetic and metabolic pathways. This project explains and compares the functions and regulatory mechanisms of SIRT family proteins, associates aging and aging-related disease models in the human stem cell research system, establishes its epigenetic regulatory network, reveals its common characteristics, and explores key biological markers of aging and intervention targets for aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1Figure 1: Establishment and testing of human embryonic stem cells deficient in SIRT1-7. A shows SIRT1-7 protein expression in human embryonic stem cells deficient in SIRT1-7; B shows equivalent genomic copy number in human embryonic stem cells deficient in SIRT1-7; C shows equivalent Ki67-positive cell proportion in human embryonic stem cells deficient in SIRT1-7; D shows equivalent clonal proliferation capacity in human embryonic stem cells deficient in SIRT1-7; E shows equivalent expression of the pluripotency markers SOX2, OCT4, and NANOG in human embryonic stem cells deficient in SIRT1-7; F shows equivalent staining for three germ layer markers in human embryonic stem cells deficient in SIRT1-7.

[0089] Figure 2 Figure 1 shows the generation of human mesenchymal stem cells deficient in SIRT1-7. A shows flow cytometry screening of CD73, CD90, and CD105-positive cells and CD34, CD45, and CD19-negative cells. B shows the expression of SIRT1-7 protein in human mesenchymal stem cells deficient in SIRT1-7. C shows the doubling curve of human mesenchymal stem cells deficient in SIRT1-7, demonstrating growth arrest after SIRT1-7 deficiency. D shows that the genome copy number of human mesenchymal stem cells deficient in SIRT1-7 is comparable.

[0090] Figure 3 Figure 1 shows the aging-related phenotypes of human mesenchymal stem cells (hMSCs) deficient in SIRT1-7. A shows an increase in the proportion of SA-β-Gal-positive hMSCs after SIRT1-7 deficiency; B shows an increase in the proliferation of hMSCs after SIRT1-7 deficiency; C shows a decrease in the proportion of Ki67-positive hMSCs after SIRT1-7 deficiency; D shows a significant shortening of the S phase of the hMSC cell cycle after SIRT1-7 deficiency; E shows an increase in IL6 secretion after SIRT1-7 deficiency; F shows a decrease in the expression of Lamin B1 and LAP2 proteins and an increase in the expression of P16 and P21 proteins after SIRT1-7 deficiency; G shows a decrease in the in vivo retention of hMSCs after SIRT1-7 deficiency.

[0091] Figure 4Identification of common downstream regulators of Sirtuin. A represents the mild upregulation of H3K9ac, H3K18ac, and H3K56ac acetylation following Sirtuin depletion, as revealed by ChIP-seq; B represents the chromatin state transition following Sirtuin depletion; C defines regions with upregulated acetylation and enhancer state transition as Sirtuin Depletion Sensitive Genomic Regions (SDSRs); D represents SDSR-mediated upregulated chromatin loops; and E represents upregulated genes associated with SDSR-mediated upregulated chromatin loops in Sirtuin-depleted human mesenchymal stem cells. The common regulatory factor shared by all seven members is PAPPA.

[0092] Figure 5 Figure 1 shows the epigenomic modifications around PAPPA. A shows the Hi-C interactions of the four chromatin loops regulating the PAPPA gene and the H3K9ac, H3K56ac, H3K18ac, and H3K27ac modifications near the four corresponding enhancers; B shows the CTCF modifications near the four PAPPA enhancers.

[0093] Figure 6 Figure 5 represents the expression level of PAPPA in human mesenchymal stem cells deficient in SIRT1-7. A shows increased RNA expression in human mesenchymal stem cells deficient in SIRT1-7; B shows increased protein expression of PAPPA in human mesenchymal stem cells deficient in SIRT1-7; C shows increased PAPPA protein secretion in human mesenchymal stem cells deficient in SIRT1-7 as detected by Western blotting; and D shows increased PAPPA protein secretion in human mesenchymal stem cells deficient in SIRT1-7 as detected by ELISA.

[0094] Figure 7 Validation of the SIRT1-7 / PAPPA regulatory axis. A shows that knocking down PAPPA in SIRT1-7-deficient human mesenchymal stem cells reduces the proportion of SA-β-Gal-positive cells; B shows that knocking down PAPPA in SIRT1-7-deficient human mesenchymal stem cells increases the proportion of Ki67-positive cells.

[0095] Figure 8Verification of PAPPA Loop 1 functional regulation. A indicates the enhancer element that inhibits PAPPA Loop 1 in SIRT6-deficient cells, and the H3K56ac level is reduced; B indicates the enhancer element that inhibits PAPPA Loop 1 in SIRT6-deficient cells, and the frequency of interaction with the enhancer is reduced; C indicates the enhancer element that inhibits PAPPA Loop 1 in SIRT6-deficient cells, and the expression of PAPPA is reduced; D indicates the enhancer element that inhibits PAPPA Loop 1 in SIRT6-deficient cells, and the proportion of SA-b-Gal-positive cells is reduced; E indicates the enhancer element that inhibits PAPPA Loop 1 in SIRT6-deficient cells, and the proliferation ability is increased; F indicates the enhancer element that inhibits PAPPA Loop 2 in SIRT7-deficient cells, and the H3K56ac level is reduced; G indicates the enhancer element that inhibits PAPPA Loop 2 in SIRT7-deficient cells, and the frequency of interaction with the enhancer is reduced; H indicates the enhancer element that inhibits PAPPA Loop 2 in SIRT7-deficient cells, and the expression of PAPPA is reduced; I indicates the enhancer element that inhibits PAPPA Loop 2 in SIRT7-deficient cells 2 enhancer element, the proportion of SA-β-Gal-positive cells decreased; J shows that in SIRT7-deficient cells, inhibition of the enhancer element of PAPPA Loop 2 increased proliferation capacity; K shows that in SIRT6-deficient cells, SIRT6 was targeted at PAPPA Loop 1 to replenish SIRT6, and the SIRT6 protein binding level increased; L shows that in SIRT6-deficient cells, H3K56ac level decreased after targeted replenishment of SIRT6 at PAPPA Loop 1; M shows that the frequency of interaction with the enhancer was reduced after targeted replenishment of SIRT6 at PAPPA Loop 1; N shows that PAPPA expression was reduced after targeted replenishment of SIRT6 at PAPPA Loop 1; O shows that the proportion of SA-β-Gal-positive cells was reduced after targeted replenishment of SIRT6 at PAPPA Loop 1; P shows that proliferation capacity was increased after targeted replenishment of SIRT6 at PAPPA Loop 1.

[0096] Figure 9Demonstrating that PAPPA is a potential driving force of aging. A indicates increased expression of PAPPA in replicatively aged human mesenchymal stem cells; B indicates increased expression of PAPPA in UV-stimulated aged human mesenchymal stem cells; C indicates increased expression of PAPPA in hydrogen peroxide-stimulated aged human mesenchymal stem cells; D indicates increased expression of PAPPA in progeria-induced aged human mesenchymal stem cells; E indicates increased expression of PAPPA in physiologically aged human mesenchymal stem cells; F indicates increased secretion of PAPPA in replicatively aged human mesenchymal stem cells; G indicates increased secretion of PAPPA in UV-stimulated aged human mesenchymal stem cells; H indicates increased secretion of PAPPA in hydrogen peroxide-stimulated aged human mesenchymal stem cells; I indicates increased secretion of PAPPA in progeria-induced aged human mesenchymal stem cells; and J indicates increased secretion of PAPPA in physiologically aged human mesenchymal stem cells.

[0097] Figure 10 The results show that knocking down PAPPA in senescent cells can delay the aging phenotype. A shows that knocking down PAPPA in progeria cells can reduce the expression of PAPPA protein and increase the expression of Lamin B1; B shows that knocking down PAPPA in progeria cells can reduce the proportion of SA-β-Gal positive cells; C shows that knocking down PAPPA in progeria cells can increase the proliferation capacity; D shows that knocking down PAPPA in adult progeria mesenchymal stem cells can reduce the expression of PAPPA protein and increase the expression of Lamin B1. B1 expression increased; E shows that PAPPA was knocked down in adult progeria cells, and the proportion of SA-β-Gal-positive cells decreased; F shows that PAPPA was knocked down in adult progeria cells, and the proliferation capacity increased; G shows that PAPPA protein expression decreased in physiologically senescent cells due to PAPPA knockdown; H shows that the proportion of SA-β-Gal-positive cells decreased in physiologically senescent cells due to PAPPA knockdown; I shows that the proliferation capacity increased due to PAPPA knockdown in physiologically senescent cells; J shows that the proportion of Ki67-positive cells increased due to PAPPA knockdown in physiologically senescent cells; K shows a schematic diagram of PAPPA activation in wild-type mesenchymal stem cells; L shows the detection of PAPPA protein expression activation in wild-type mesenchymal stem cells; M shows that the proportion of SA-β-Gal-positive cells increased due to PAPPA activation in wild-type mesenchymal stem cells; N shows that the proliferation capacity decreased due to PAPPA activation in wild-type mesenchymal stem cells; O shows that the proportion of Ki67-positive cells decreased due to PAPPA activation in wild-type mesenchymal stem cells.

[0098] Figure 11 PAPPA is a potential biomarker of aging. A represents the upregulation of PAPPA in the skin tissue of elderly people; B represents the upregulation of PAPPA in the plasma of elderly people. DETAILED DESCRIPTION

[0099] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0100] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0101] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0102] The human embryonic stem cells with SIRT2 deficiency in the following examples (abbreviated as SIRT2 - / - hESCs) is described in Ye, Y., Yang, K., Liu, H., Yu, Y., Song, M., Huang, D., Lei, J., Zhang, Y., Liu, Z., Chu, Q., et al. (2023). SIRT2 counteracts primate cardiac aging via deacetylation of STAT3 that silences CDKN2B. Nat Aging 3, 1269–1287.10.1038 / s43587-023-00486-y. The public can obtain this biological material from the applicant for use only in repeating the experiments of the present invention and cannot be used for other purposes.

[0103] The human embryonic stem cells with SIRT3 deficiency in the following examples (abbreviated as SIRT3) - / - hESCs) is described in: Diao, Z., Ji, Q., Wu, Z., Zhang, W., Cai, Y., Wang, Z., Hu, J., Liu, Z., Wang, Q., Bi, S., et al. (2021). SIRT3 consolidates heterochromatin and counteracts senescence. Nucleic Acids Research 49, 4203–4219.10.1093 / nar / gkab161. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0104] The human embryonic stem cells lacking SIRT6 in the following examples (abbreviated as SIRT6 - / - hESCs) are described in: Pan, H., Guan, D., Liu, X., Li, J., Wang, L., Wu, J., Zhou, J., Zhang, W., Ren, R., Zhang, W., et al. (2016). SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2. Cell Res 26, 190–205.10.1038 / cr.2016.4. The public can obtain this biological material from the applicant for use only in repeating the experiments of the present invention and cannot be used for other purposes.

[0105] The SIRT7-deficient human embryonic stem cells (SIRT7 - / - hESCs) is described in Bi, S., Liu, Z., Wu, Z., Wang, Z., Liu, X., Wang, S., Ren, J., Yao, Y., Zhang, W., Song, M., et al. (2020). SIRT7 antagonizes human stem cell aging as a heterochromatinstabilizer. Protein Cell 11, 483–504.10.1007 / s13238-020-00728-4. The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0106] The human progeria mesenchymal stem cells (WRN - / -) (abbreviated as WS hMPCs) is described in: Zhang W, Li J, Suzuki K, Qu J, Wang P, Zhou J, Liu X, Ren R, Xu X, Ocampo A, Yuan T, Yang J, Li Y, Shi L, Guan D, Pan H, Duan S, Ding Z, Li M, Yi F, Bai R, Wang Y, Chen C, Yang F, Li X, Wang Z, Aizawa E, Goebl A, Soligalla RD, Reddy P, Esteban CR, Tang F, Liu GH, Belmonte JC. A Werner Syndrome Stem Cell Model Unveils Heterochromatin Alterations as a Driver of Human Aging. Science. 2015; 348(6239): 1160-1163. The public can obtain this biological material from the applicant for use only in repeating the experiments of the present invention and cannot be used for other purposes. The above-mentioned human progeria mesenchymal stem cells (WRN- / -) are human mesenchymal stem cells derived from human embryonic pluripotent stem cells in which the WRN gene is knocked out. Progeria is characterized by the knockout of the WRN gene. Here, progeria is manifested as short stature, a tendency to diabetes, premature graying of hair and baldness, increased incidence of tumors, and scleroderma of the extremities; wild-type human mesenchymal stem cells are human mesenchymal stem cells derived from human embryonic pluripotent stem cells.

[0107] The human progeria mesenchymal stem cells (LMNA) in the following examples G608G / + ) (HGPS hMPCs) have been described in: Wu Z, Zhang W, Song M, Wang W, Wei G, Li W, Lei J, Huang Y, Sang Y, Chan P, Chen C, Qu J, Suzuki K, Belmonte JC, Liu GH. Differential Stem Cell Aging Kinetics in Hutchinson–Gilford Progeria Syndrome and Werner Syndrome. Protein Cell. 2018.9(4):333-350. The public can obtain the biomaterial from the applicant. The biomaterial is only used to repeat the experiments of the present invention and cannot be used for other purposes. The above-mentioned human progeria mesenchymal stem cells (LMNA G608G / +) is a heterozygous mutation of the LMNA gene generated by gene editing of the LMNA gene (LMNA G608G / + Human mesenchymal stem cells derived from human embryonic pluripotent stem cells, progeria in children is manifested as atherosclerosis, cardiovascular disease and / or stroke.

[0108] The data in the following examples were processed using GraphPad statistical software. The experimental results are expressed as mean ± standard error and a two-tailed Student's t-test was used. The specific P values are attached to the bars, and P < 0.05 indicates a significant difference, P < 0.01 indicates a very significant difference, and P < 0.001 indicates an extremely significant difference.

[0109] Example 1: Obtaining SIRTs-deficient human mesenchymal stem cells and studying their aging-related phenotypes

[0110] 1. SIRT1 - / - 、SIRT4 - / - and SIRT5 - / - Generation of hESCs

[0111] (1) Gene knockout was performed using the CRISPR / Cas9 system. sgRNA1 targeting the second exon of the SIRT1 gene (the nucleotide sequence encoding sgRNA1 is: 5'-GAGATGGTCCCGGCCTCGAG-3') was cloned into the pCAG-mCherry-gRNA vector (Addgene, #87110) to obtain the sgRNA vector pCAG-mCherry-gRNA1.

[0112] (2) Gene knockout was performed using the CRISPR / Cas9 system. sgRNA2 targeting the second exon of the SIRT4 gene (the nucleotide sequence encoding sgRNA2 is: 5'-GTCTGGTATCCCCGATTCGG-3') was cloned into the pCAG-mCherry-gRNA vector to obtain the sgRNA vector pCAG-mCherry-gRNA2.

[0113] (3) Gene knockout was performed using the CRISPR / Cas9 system. sgRNA3 targeting the second exon of the SIRT5 gene (the nucleotide sequence encoding sgRNA3 is: 5'-GGGTGATGACCACGACTCGC-3') was cloned into the pCAG-mCherry-gRNA vector to obtain the sgRNA vector pCAG-mCherry-gRNA3.

[0114] The only difference between pCAG-mCherry-gRNA2, pCAG-mCherry-gRNA3 and pCAG-mCherry-gRNA1 is that the sgRNA1 sequence is replaced with sgRNA2 or sgRNA3.

[0115] (4) Wild-type human embryonic stem cells (WT hESCs) were cultured on Matrigel-coated plates in hESC medium supplemented with the ROCK inhibitor Y-27632 (Selleck) for 24 h.

[0116] (5) sgRNA vectors pCAG-mCherry-gRNA1, pCAG-mCherry-gRNA2, and pCAG-mCherry-gRNA3 and pCAG-1BPNLS-Cas9-1BPNLS-2AGFP vector (Addgene, #87109) were electroporated into WT hESCs cells using a 4D Nucleofector (Lonza).

[0117] The electroporated cells were then cultured on Matrigel-coated plates for 48 h, and GFP / mCherry double-positive cells were sorted using a fluorescence-activated cell sorting (FACS) system (BD FACS Aria II) and cultured on MEF feeder layers with hESC medium.

[0118] The obtained GFP / mCherry double-positive cells refer to hESCs cells that can simultaneously express both GFP and mCherry fluorescence.

[0119] (6) Extract and sequence the whole genome DNA of cells.

[0120] Genomic DNA of hESCs was extracted using the DNeasy Blood & Tissue Kit (QIAGEN, Cat. No. 1600022609) and sequenced by QIAGEN.

[0121] 2. Cell culture

[0122] Wild-type and SIRT1-7-deficient human embryonic stem cells were cultured in hESC medium.

[0123] The specific components of hESC culture medium are as follows: DMEM / F-12 (Thermo Fisher Scientific), 20% serum replacement (Thermo Fisher Scientific), 1% GlutaMAX (Thermo Fisher Scientific), 1% non-essential amino acids (NEAA, Thermo Fisher Scientific), 1% penicillin-streptomycin (P / S, Thermo Fisher Scientific), 55 μM β-mercaptoethanol, and 10 ng / mL bFGF (Joint Protein Central)

[0124] hMPCs were cultured on gelatin-coated plates using hMPC medium. The specific composition of hMPC medium is as follows: MEMα basal medium (Thermo Fisher Scientific, Cat. No. 32571101), 10% fetal bovine serum (FBS, Gibco, 42F9083K), 1% PS, 1% NEAA, and 1 ng / mL bFGF. When the cell density reached 95%, 1×10 cells were plated per well. 5 The cells were passaged in 6-well plates at a density of 100 cells / well.

[0125] HEK293T cells (ATCC, catalog number CRL-1573) were cultured in high-glucose DMEM (ThermoFisher Scientific) containing 10% FBS and 1% P / S.

[0126] 3. Generation and characterization of wild-type and SIRT1-7-deficient human mesenchymal stem cells (hMPCs)

[0127] Wild-type and SIRT1-7-deficient human mesenchymal stem cells (hMPCs) were differentiated from wild-type and SIRT1-7-deficient human embryonic stem cells (hMPCs).

[0128] (1) Embryoid bodies (hEBs) were derived from hESCs and cultured on Matrigel-coated plates in human mesenchymal stem cell differentiation medium. The specific composition of the human mesenchymal stem cell differentiation medium is as follows: MEMα basal medium, 10% FBS, 1% PS, 1% NEAA, 1 ng / mL bFGF, and 5 ng / mL TGFβ.

[0129] (2) After approximately one week, the differentiating cells were transferred to gelatin-coated plates and cultured in human mesenchymal stem cell culture medium.

[0130] (3) Cells triple-positively labeled with CD73, CD90, and CD105 were selected using FACS and considered as human mesenchymal stem cells, while CD34, CD43, and CD45 were used as negative markers.

[0131] 4. Clonal expansion test

[0132] (1) Human mesenchymal stem cells cultured on gelatin-coated plates were treated with TrypLE TM Enzymatic digestion into single cells.

[0133] (2) Human mesenchymal stem cells were seeded at a density of 3,000 per well in a gelatin-coated 12-well plate.

[0134] (3) After culturing for about 12 days, human mesenchymal stem cells were washed twice with PBS, fixed with 4% paraformaldehyde (PFA) for 30 minutes, and stained with crystal violet solution (Biohao) at room temperature for 30 minutes.

[0135] (4) After rinsing with running tap water, images were taken with an Epson Perfection V370 Photo or a microscope digital camera (Olympus), and the relative cell density was quantified using ImageJ.

[0136] 5. Senescence-associated β-galactosidase (SA-β-Gal) staining

[0137] (1) hMPCs cultured on 6-well plates were washed twice with PBS and treated in a fixation buffer (2% formaldehyde and 0.2% glutaraldehyde) for 5 minutes.

[0138] (2) Add 2 mL of SA-β-Gal staining solution (containing 150 mM NaCl, 2 mM MgCl2, 5 mM K4[Fe(CN)6], 5 mM K3[Fe(CN)6], 40 mM citric acid / sodium phosphate buffer, and 1 mg / mL X-Gal (AMRESCO) in water) to a 6-well plate and incubate at 37°C overnight.

[0139] (3) Images were captured with a digital microscope camera (Olympus), and the proportion of SA-β-Gal-positive cells was calculated using ImageJ.

[0140] 6. Immunofluorescence staining

[0141] (1) Human mesenchymal stem cells were seeded on gelatin-coated coverslips (Thermo Fisher Scientific) and cultured to a density of 80%. The cells were then washed once with PBS, fixed with 4% PFA for 10 minutes, and permeabilized with 0.4% Triton X-100 for 10 minutes.

[0142] (2) Human mesenchymal stem cells were blocked with 10% donkey serum (Jackson ImmunoResearch) at room temperature for 1 hour, incubated with primary antibody at 4°C overnight, and incubated with secondary antibody at room temperature for 1 hour.

[0143] (3) Images were captured using a Leica SP5 or Zeiss-LSM900 confocal system.

[0144] (4) Use ImageJ to count immunofluorescence signals.

[0145] 7. Immunohistochemical staining

[0146] Soak the tissue sections in xylene to remove wax, then rehydrate them by soaking them in anhydrous ethanol twice, 95% (twice), 85%, and 75% according to a gradient. Soak in sodium citrate repair solution with pH = 6.0 at high temperature and high pressure at 115°C for 10 minutes, then cool naturally for antigen retrieval. Soak the sections in 0.4% Triton in PBS for permeabilization and block with endogenous peroxide (3% H2O2). Block at room temperature for about 1 hour with 5% donkey serum in PBS. After incubation with antibodies at 4°C overnight, add reaction enhancement solution and bind secondary antibody. DAB color development, then stain the nuclei with hematoxylin, soak in 1% hydrochloric acid ethanol solution for differentiation, and then soak in 1% ammonia solution for anti-blueing. Finally, dehydrate with different gradient concentrations of ethanol and seal with neutral gum.

[0147] 8. Western Blot Analysis

[0148] (1) 1×10 6 Human mesenchymal stem cells or human embryonic stem cells were lysed in 1× SDS lysis buffer (62.5 mM Tris-HCl (pH = 6.8), 2% (wt / vol) SDS) in a metal bath at 105°C for 10 minutes, and protein quantification was performed using a BCA (bicinchoninic acid) kit (Dingguo, BCA02).

[0149] (2) 20 μg of the prepared protein sample was separated by SDS-PAGE and electrotransferred to a PVDF membrane (Millipore). After blocking with 5% skim milk (BBI Life Sciences) at room temperature for 1 hour, the PVDF membrane was incubated with the primary antibody at 4°C overnight and incubated with the appropriate secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature for 1 hour.

[0150] (3) Images were acquired using the ChemiDoc XRS system (Bio-Rad), and the band intensities of the target proteins were measured using ImageJ.

[0151] 9. Cell cycle analysis

[0152] (1) Human mesenchymal stem cells were harvested and fixed with 70% ethanol at -20°C overnight.

[0153] (2) After washing three times with PBS, the cells were permeabilized with 0.1% Triton X-100, incubated with 0.2 mg / mL RNase A, and stained with 0.02 mg / mL propidium iodide staining solution at 37°C for 30 minutes.

[0154] (3) Cell cycle analysis was performed using BD LSR Fortessa Cell Analyzer.

[0155] 10. Animal Experimentation

[0156] All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the Chinese Academy of Sciences. BALB / cnude mice (male, 6–8 weeks old) were housed in the animal laboratory of the Institute of Zoology, Chinese Academy of Sciences, under a 12-h light / dark cycle and provided with food and water.

[0157] For hMPC persistence assay, wild-type or SIRT1-7-deficient hMPCs infected with luciferase (Luc)-expressing virus were injected into the tibialis anterior muscle of BALB / c nude mice, and the fluorescence intensity was detected using an IVIS Spectrum Imaging System at 0 and 5 days after injection.

[0158] The results are as follows: SIRTs protein deficiency accelerated hMPC aging.

[0159] (1) To examine whether the loss of SIRT1-7 can delay the aging of hMPCs.

[0160] The SIRT1, SIRT4, and SIRT5 genes in human embryonic stem cells (hESCs) were knocked out using the CRISPR / Cas9 system, and hESCs deficient in SIRT1, SIRT4, and SIRT5 were successfully obtained as confirmed by Western blot analysis. Figure 1 According to karyotyping and genome-wide copy number variation (CNV) analysis, SIRT1, SIRT4, and SIRT5 deficiency had little impact on genomic integrity ( Figure 1 Middle B).

[0161] The SIRT1-7-deficient hESCs produced were able to express embryonic stem cell pluripotency markers such as NANOG, OCT4, and SOX2, and maintain germ layer differentiation potential; and it was found that the loss of SIRT1-7 had little effect on cell proliferation ability ( Figure 1 (CF).

[0162] These results indicate that SIRT1-7 deficiency has relatively little effect on the pluripotency of hESCs.

[0163] (2) To investigate the effects of SIRT1-7 deficiency on human mesenchymal stem cells, wild-type and SIRT1-7-deficient hESCs were differentiated into hMPCs. Both wild-type and SIRT1-7-deficient hMPCs expressed typical hMPC markers, including CD73, CD90, and CD105, but did not express other cell type markers, such as CD34, CD45, and CD19 ( Figure 2 Middle A).

[0164] The loss of SIRT1-7 proteins was confirmed by Western blot analysis ( Figure 2 Middle B). Similar to wild-type hMPCs, SIRT1-7-deficient hMPCs maintain relative genomic integrity ( Figure 2 Middle D); Cell growth arrest of hMPCs deficient in SIRT1-7 was analyzed by doubling curve analysis ( Figure 2 Middle C).

[0165] (3) The percentage of SA-β-Gal in hMPCs with SIRT1-7 deficiency increased, the clone proliferation ability was weakened, the percentage of Ki67-positive cells decreased, the cell cycle S decreased, and the IL6 secretion increased ( Figure 3 AE), and down-regulation of LAP2 and Lamin B1 and up-regulation of senescence markers P16 and P21 in hMPCs deficient in SIRT1-7 ( Figure 3 Middle (F), demonstrating that SIRT1-7 deficiency accelerates hMPC senescence.

[0166] (4) The in vivo persistence of SIRT1-7-deficient hMPCs was evaluated by implanting luciferase-labeled wild-type and SIRT1-7-deficient hMPCs into the tibialis anterior muscle of immunodeficient mice. Compared with wild-type, SIRT1-7-deficient hMPCs were found to have a weaker in vivo persistence ability ( Figure 3 Middle G).

[0167] In summary, SIRT1-7 proteins are crucial for human mesenchymal stem cell homeostasis, and their absence can lead to accelerated aging.

[0168] Example 2: Effects of PAPPA protein and SIRTs-PAPPA signaling pathway on cell senescence phenotype

[0169] 1. Lentiviral packaging

[0170] HEK293T cells were transfected with the target plasmids and the viral packaging plasmids PMD2.G (Addgene plasmid #12259) and PSPAX (Addgene plasmid #12260) using Lipofectamine 3000 transfection reagent and packaged into lentiviral vectors. Cell supernatants were collected at 48 and 72 hours, and after filtering cell debris, lentiviral particles were harvested by ultracentrifugation at 19,400 g for 2.5 hours at 4°C.

[0171] 2. CNV analysis

[0172] DNeasy Blood & Tissue Kit (QIAGEN) was used to start from 1 × 10 6 Genomic DNA was extracted from wild-type and SIRT1-7-deficient human mesenchymal stem cells (hMSCs). DNA quality control, library preparation, and sequencing were performed on an Illumina HiSeq ×10 platform. Data analysis was performed using published R packages. Raw reads were trimmed using TrimGalore and then aligned to the human hg19 genome. Read counts were calculated for each 500-kb window using the readCounter function in hmmcopy_utils (https: / / github.com / shahcompbio / hmmcopy_utils). The R / Bioconductor package HMMcopy (v1.26.0) was used to correct for copy number, GC content, and mappability.

[0173] 3. Ultraviolet (UV) and H2O2-induced cell senescence

[0174] For UV-induced cell senescence, XL-1000 UV crosslinker (Spectronics Corporation) was used at 10 J / m 2 Irradiation of about 3×10 5 Wild-type human mesenchymal stem cells were used to form a UV-irradiated senescent cell model.

[0175] To investigate H2O2-induced cellular senescence, wild-type human mesenchymal stem cells were treated with 50 μM H2O2 for approximately 24 hours. When the cells reached 95% confluence, they were passaged. Phenotypic analysis was performed on cells passaged two generations after H2O2 treatment, establishing a model of oxidative stress-induced senescence.

[0176] Through continuous subculture, a replicative senescent cell model was obtained.

[0177] 4. Chromatin Immunoprecipitation (ChIP)

[0178] For chromatin immunoprecipitation, wild-type and SIRT1-7-deficient human mesenchymal stem cells were first cross-linked in 10% FBS-1% formaldehyde in PBS at room temperature for 10 minutes. The cross-linking reaction was then terminated by incubation with 0.125 M glycine for 5 minutes at room temperature. The cell suspension was then centrifuged at 3,000 rpm for 8 minutes at 4°C, the supernatant discarded, and the cross-linked, fixed cell sample was washed with PBS.

[0179] 130 μL of Lysis buffer was added to the cell sample, vortexed, and incubated on ice for 5 minutes. Ultrasonication was performed using a Covaris S220 focused sonicator (Covaris) under appropriate conditions. After sonication, RIPA solution containing protease inhibitors was added for neutralization. The cell lysate was then centrifuged at 12,000 g / min for 10 minutes at 4°C. The supernatant was transferred to a suspension of Protein A Dynabeads (Thermo Fisher Scientific, 10004D) pre-incubated with the target antibodies H3K9ac (Abacm, ab4441), H3K18ac (Abcam, ab1191), H3K27ac (Abacm, ab4729), or H3K56ac (Active Motif, 39281) and incubated overnight at 4°C. The next day, Complete elution buffer containing protease inhibitors was added to the beads after washing with RIPA buffer and TE buffer, and elution was performed in a metal bath at 68°C with shaking. The eluted DNA solution was extracted with phenol-chloroform-isoamyl alcohol solution and ethanol precipitation was used to isolate the DNA. Furthermore, the isolated DNA was used for library construction using the KAPA Hyper Prep kit (New England Biolabs, KK8504). The constructed sequencing library was sequenced on an Illumina NovaSeq or X Ten sequencer using a 150-bp read-length paired-end sequencing method.

[0180] 5. Construction of CTCF CUT&Tag

[0181] Initially, 1×10 6Live wild-type and SIRT1-7-deficient human mesenchymal stem cells were assayed using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903-02) according to the manufacturer's instructions. Anti-CTCF antibodies (1:25) (Cell Signaling Technology, 2899S) were incubated overnight at 4°C. All libraries were amplified for 13 cycles and sequenced by Annoroad Gene Technology Co., Ltd.

[0182] 6. HiC library construction

[0183] Collect 5×10 6 Wild-type and SIRT1-7-deficient human mesenchymal stem cells were cross-linked with 1% (vol / vol) formaldehyde for 10 minutes at room temperature. Cross-linking was terminated with glycine at a final concentration of 125 mM, and the cell pellet was collected by centrifugation at 3,000 × rpm for 10 minutes. Cross-linked hMPCs were resuspended in lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2% IGEPAL CA-630, and 1× protease inhibitor cocktail (Selleck)) and incubated on ice for 20 minutes to disrupt cell membranes. Nuclei were collected by centrifugation and digested with MboI restriction endonuclease (New England Biolabs) overnight at 37°C. The digested DNA was then biotinylated with biotin-14-dCTP (Invitrogen) and blunt-end ligated using T4 DNA ligase (New England Biolabs). After ligation, the biotinylated DNA was incubated overnight at 65°C with proteinase K (New England Biolabs) to reverse crosslinks, and the DNA was purified by phenol-chloroform extraction. The purified DNA was treated with T4 DNA polymerase (New England Biolabs) to remove fragments without biotinylated ends. Genomic DNA was further fragmented into 200-500 bp fragments using a Covaris S220 sonicator. Library construction was then completed following a workflow that included end repair, A-tailing, adapter ligation, and PCR amplification. DNA sequencing was performed using the Illumina HiSeq X-Ten platform.

[0184] 7. Chromosome conformation capture (3C)

[0185] Collect 1×10 6Human mesenchymal stem cells (hMPCs) expressing wild-type or SIRT2, SIRT6, and SIRT7 were cross-linked with 1% (vol / vol) formaldehyde for 10 minutes at room temperature. Cross-linking was terminated with glycine at a final concentration of 125 mM, and the hMPC pellet was collected by centrifugation at 3,000 × rpm for 10 minutes. hMPCs were lysed and digested with DpnII (NEB, R0543) at 37°C overnight. Religation was performed using T4 ligase, and DNA was purified. RT-qPCR was used for analysis.

[0186] Loop 1-E: 5'-TTATAGTTCAGGCGGGAGA-3';

[0187] Loop 1-P: 5'-TTGAGTTGCTGGGGAGTCTC-3';

[0188] Loop 2-E: 5'-GGTAATGGAACTTGCCCCTC-3';

[0189] Loop 2-P: 5'-ATCAAGGGTTAGGGTGGGTC-3';

[0190] GAPDH-3C-Forward: 5'-CCCGCAAGGCTCGTAGAC-3';

[0191] GAPDH-3C-Reverse: 5'-CCACATCGCTCAGACACCAT-3'.

[0192] 8. Small interfering RNA (siRNA)-mediated PAPPA knockdown

[0193] Using si-PAPPA and nonspecific control siRNA (si-NC) targeting the PAPPA gene, synthesized by RiboBio, and transfected into wild-type and SIRT1-7-deficient cells, respectively, using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) according to the manufacturer's instructions, si-PAPPA and si-NC were harvested 48-72 hours after transfection for PAPPA expression and aging-related phenotype analysis. PAPPA expression and aging-related phenotype analysis were performed as described in Steps 5, 6, and 8 of the previous example.

[0194] The sequence information of si-PAPPA is as follows: 5'-GGAGACCUCUGCAAUGAUA-3' (SEQ ID No. 1).

[0195] The sequence information of si-NC is as follows: 5′-GGCUCUAGAAAAGCCUAUGC-3′ (SEQ ID No. 2).

[0196] 9. Using the CRISPRi (dCas9-KRAB) system, we can induce local heterochromatinization on the PAPPA enhancer element, thereby disrupting the chromatin loop structure.

[0197] Six sgRNAs were designed near the enhancer elements of Loop 1 and Loop 2 of PAPPA and synthesized by Qingke Biosequencing Company.

[0198] The specific sequence information of the 6 sgRNAs in Loop 1 is as follows:

[0199] sgRNA 1:5'-TCCGGGTGAACTTCTGGAAA-3';

[0200] sgRNA 2:5'-ATCTAGATAGAAAGAACCGT-3';

[0201] sgRNA 3:5'-CAGTCAACACGACTCAACAG-3';

[0202] sgRNA 4:5'-TAGCAACATCGTGTGTAAGA-3';

[0203] sgRNA 5:5'-TATTGCCCCTGTTGGGGGCTA-3';

[0204] sgRNA 6:5'-GTGATGTGTCATCATGGTAC-3'.

[0205] The specific information of the 6 sgRNAs in Loop 2 is as follows:

[0206] sgRNA 7:5'-GTTGAACTGAATTCCCTTAT-3';

[0207] sgRNA 8:5'-TATCAACACGGACCGCACCA-3';

[0208] sgRNA 9:5'-AGAGGTCTGGATGCTACCAA-3';

[0209] sgRNA 10:5'-GGGGTTGTCATATCTTGGTG-3';

[0210] sgRNA 11:5'-GTGTGTTCTAATTGGCAGTA-3';

[0211] sgRNA 12:5'-AAAGGGACATCTTCTGATGT-3'.

[0212] The synthesized sgRNA was constructed into the plasmid pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro (Addgene plasmid #71236). The specific steps were as follows: the sgRNA primer was annealed and then 5' phosphorylated, the vector was linearized with Esp3I enzyme, and then ligated with T4 DNA ligase, and finally transformed to obtain a recombinant vector containing the corresponding loop 1 and loop 2 sgRNA.

[0213] pLV hU6-s gRNA hUbC-dC as9-KRAB-T2 a-Pur o-gRNA2, pLV hU6-s gRNA hUbC-dCas9-KRAB-T2 a-Puro-gRNA3, pLV hU6-s gRNA hUbC-dCas9-KRAB-T2 a-Puro-gRNA4, pLV hU6-s gRNA hUbC-dCas9-KRAB-T2 a-Puro-gRNA5, pLV hU6-s gRNA hUbC-dCas9-KRAB-T2 a-Puro-gRNA6, pLV hU6-s gRNA hUbC-dCas9-KRAB-T2 a-Puro-gRNA7, pLV hU6-s gRNA hUbC-dCas9-KRAB-T2 a-Puro-gRNA8, pLV hU6-s gRNA hUbC-dCas9-KRAB-T2 a-Puro-gRNA9, pLV hU6-s gRNA hUbC-dCas9-KRAB-T2 a-Puro-gRNA10, pLV hU6-s gRNA hUbC-dCas9-KRAB-T2 a-Puro-gRNA11, pLV hU6-s gRNA hUbC-dCas9-KRAB-T2 a-Puro-gRNA12 and pLV hU6-s gRNA hUbC-dCas9-KRAB-T2 a-Puro-gRNA1 differ only in that the sgRNA1 sequence is replaced with sgRNA2, sgRNA3, sgRNA4, sgRNA5, sgRNA6, sgRNA7, sgRNA8, sgRNA9, sgRNA10, sgRNA11, and sgRNA12.

[0214] The recombinant vectors targeting Loop 1 (sgRNA1-6) were mixed in equal amounts, and the recombinant vectors targeting Loop 2 (sgRNA7-12) were mixed in equal amounts, packaged into lentivirus, and then infected human mesenchymal stem cells lacking SIRT6 (for Loop 1, labeled sg-Loop 1 group, control group labeled Control) or SIRT7 (for Loop 2, labeled sg-Loop 2 group, control group labeled Control). This was done to achieve local heterochromatinization of these two enhancer elements. Forty-eight hours after infection, cells were treated with puromycin (Puro) for approximately seven days. Chromatin acetylation levels, enhancer-promoter interaction strength, changes in PAPPA expression, and aging-related phenotype analysis were then measured.

[0215] 10. Directed tethering of SIRT6 to the enhancer element of PAPPA using dCas9-SIRT6

[0216] Near the Loop 1 enhancer element of PAPPA, where there is a binding site for SIRT6 protein, six sgRNAs were designed and constructed into the pU6-sgRNA EF1Alpha-puro-T2A-BFP plasmid (Addgene plasmid #60955). Specifically, the sgRNA primers were annealed and then 5' phosphorylated. The vector was linearized with BstXl / Blpl double enzymes and then ligated with T4 DNA ligase before transformation.

[0217] The specific sequence information of the 6 sgRNAs is as follows:

[0218] sgRNA 1: 5'-TCCGGGTGAACTTCTGGAAA-3',

[0219] sgRNA 2: 5'-ATCTAGATAGAAAGAACCGT-3',

[0220] sgRNA 3: 5'-CAGTCAACACGACTCAACAG-3',

[0221] sgRNA 4: 5'-TAGCAACATCGTGTGTAAGA-3',

[0222] sgRNA 5: 5'-TATTGCCCCTGTTGGGGGCTA-3',

[0223] sgRNA 6: 5'-GTGATGTGTCATCATGGTAC-3',

[0224] The only difference between pU6-sgRNA EF1Alpha-puro-T2A-BFP-gRNA2, pU6-sgRNA EF1Alpha-puro-T2A-BFP-gRNA3, pU6-sgRNA EF1Alpha-puro-T2A-BFP-gRNA4, pU6-sgRNAEF1Alpha-puro-T2A-BFP-sgRNA5, pU6-sgRNA EF1Alpha-puro-T2A-BFP-sgRNA6 and hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro-gRNA1 is that the sgRNA1 sequence is replaced with sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6.

[0225] The dCas9-VP64-EGFP vector (addgene #61422) was double-digested with BamHI and NheI to remove the VP64 fragment, and the linearized vector was recovered. The SIRT6 cDNA fragment was then ligated to obtain the recombinant pLenti-dCas9-SIRT6 plasmid.

[0226] The dCas9-VP64-EGFP vector (addgene#61422) was double-digested with BamHI and NheI to remove the VP64 fragment, and the linearized vector was recovered. The vector fragment was repaired to a blunt end using NEBNext End repair module (E6050L), and DNA Clean& The vector was purified by enzyme digestion with 5-5 (Zymo research, D4003) and self-ligated with T4 DNA ligase to obtain the recombinant pLenti-dCas9 plasmid as a control.

[0227] The six recombinant plasmids (pU6-sgRNA EF1Alpha-puro-T2A-BFP-sgRNA (1-6)) were mixed with equal mass and packaged into lentivirus with pLenti-dCas9-SIRT6 (pLenti-dCas9 served as a control). These lentiviruses were then used to infect human mesenchymal stem cells (human mesenchymal stem cells) lacking SIRT6 (experimental group labeled dCas9-SIRT6, control group labeled Control) to achieve targeted re-expression of SIRT6 protein. Forty-eight hours after infection, cells were treated with puromycin (Puro) for approximately seven days. Acetylation levels, enhancer-promoter interaction strength, changes in PAPPA expression, and aging-related phenotypes were then measured.

[0228] Refer to the gRNA cloning in lentiCRISPRv1, lentiCRISPRv2 and lentiGuide-Purovectors protocol at https: / / www.addgene.org / pooled-library / zhang-human-gecko-v2 / .

[0229] 11. CRISPR / dCas9-mediated PAPPA transcriptional activation

[0230] Young wild-type human mesenchymal stem cells were infected with lentiviruses carrying sg-NTC or sg-PAPPA in a LentiSAM-v2 vector (Addgene plasmid #75112) expressing dCas9-VP64.

[0231] 48 hours after infection, cells were treated with blasticidin (InvivoGen) and hygromycin (InvivoGen) for approximately 7 days, followed by detection of PAPPA expression and analysis of senescence-related phenotypes.

[0232] Here are the results:

[0233] 1) H3K9ac, H3K18ac, and H3K56ac acetylation levels were moderately increased in SIRT1-7-deficient hMPCs ( Figure 4 Middle A), chromatin state changes ( Figure 4 B), defining three regions with upregulated acetylation and chromatin state transition as Sirtuin-depleted sensitive genomic regions (SDSRs) ( Figure 4 Middle C).

[0234] SDSR regions are associated with enhanced promoter-enhancer interactions (chromatin looping) ( Figure 4 Middle D), induced a number of up-regulated genes, and PAPPA was the only key downstream factor co-regulated by SIRT1-7 ( Figure 4 Middle E).

[0235] Further exploration of the epigenetic modification characteristics near PAPPA revealed that after Sirtuin loss, four chromatin loops were formed near PAPPA, and the acetylation modification near the enhancer region corresponding to the chromatin loop was upregulated ( Figure 5 Middle A).

[0236] The dynamic extrusion and formation of chromatin loop domains are mainly mediated by structural proteins including CTCF. Previous studies have also reported that enhancer elements tend to recruit CTCF. Therefore, we analyzed CTCF signals and used CUT&Tag analysis to find that in Sirtuin-deficient cells, CTCF binding signals at the nascent chromatin loop enhancer sites of PAPPA were increased ( Figure 5 Middle B).

[0237] Experiments have also confirmed that in Sirtuin-deficient human mesenchymal stem cells, PAPPA RNA levels increase, and protein expression and secretion levels increase ( Figure 6 (in AD).

[0238] 2) Knockdown of PAPPA slows down the aging of Sirtuin-deficient human mesenchymal stem cells.

[0239] Knocking down PAPPA expression in Sirtuin-deficient human mesenchymal stem cells using siRNA can reduce the proportion of SA-β-Gal positive cells and enhance cell proliferation ( Figure 7 Further evidence of the PAPPA / Sirtuin regulatory axis.

[0240] 3) CRISPRi intervention to block acetylation levels in enhancer elements

[0241] In human mesenchymal stem cells lacking SIRT6, the CRISPRi system was used to target Loop 1 of PAPPA, which reduced the acetylation level of this region, the frequency of promoter-enhancer interactions, the expression of PAPPA, the proportion of SA-β-Gal-positive cells, and enhanced cell proliferation. Figure 8 Similar phenomena were observed when intervening in PAPPA Loop 2 in SIRT7-deficient human mesenchymal stem cells ( Figure 8 Chinese FJ).

[0242] 4) dCas9-SIRT6 targeted complementation

[0243] In human mesenchymal stem cells lacking SIRT6, targeted replenishment of SIRT6 at the Loop 1 site of PAPPA revealed decreased acetylation levels in this region, reduced promoter-enhancer interaction frequency, decreased PAPPA expression, reduced the proportion of SA-β-Gal-positive cells, and enhanced cell proliferation ( Figure 8 Chinese KP).

[0244] 5) Knockdown of PAPPA alleviated the senescence of hMPCs and progeria cells.

[0245] To further explore the therapeutic potential of PAPPA for delaying hMPC aging, it was found that the expression and secretion levels of PAPPA were reduced in cell models of replicative aging, UV irradiation-induced aging, oxidative stress-induced aging, progeria-induced aging, and physiological aging ( Figure 9 (AJ in Chinese).

[0246] 6) By knocking down PAPPA protein in replicative senescence hMPCs (RS-hMPCs), progeria-derived hMPCs (HGPS-hMPCs and WS-hMPCs), and hMPCs from healthy elderly people through siRNA, knocking down PAPPA can delay cell senescence in these aging hMPC models, as manifested by a decrease in the percentage of SA-β-Gal-positive cells, an increase in the proportion of Ki67-positive cells, and an increase in monoclonal expansion capacity. In the progeria cell models (HGPS-hMPCs and WS-hMPCs), an increase in Lamin B1 protein levels was also observed when PAPPA was knocked down ( Figure 10 (AJ in Chinese).

[0247] 7) Activation of PAPPA accelerates the aging process of hMPCs. PAPPA is a potential driving force of aging.

[0248] Activation of endogenous PAPPA using the CRISPR / dCas9 system promoted hMPC senescence, as evidenced by an increase in the percentage of SA-β-Gal-positive cells, a decrease in monoclonal expansion, and a decrease in Ki67-positive cells. Figure 10 KO).

[0249] 8) PAPPA protein is upregulated in the skin tissue and plasma of the elderly and can serve as a potential biological marker of aging.

[0250] Under ethical requirements, skin tissues of young and old individuals (defined as young at 18-35 years old and old at 55 and above) were collected for immunohistochemical staining. The expression of PAPPA in skin tissues of old individuals was high ( Figure 11 Similarly, the PAPPA content in plasma protein increases in elderly individuals ( Figure 11 Middle B).

[0251] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Application, characterized in that, The application is any of the following: 1) Use of substances that inhibit PAPPA gene expression in the preparation of cell aging inhibitors or anti-aging drugs; 2) Use of substances that inhibit PAPPA gene expression in the preparation of drugs for treating cell senescence and / or preventing senescence; 3) Use of substances that inhibit PAPPA protein activity in the preparation of cell aging inhibitors; 4) Use of substances that inhibit PAPPA protein activity in the preparation of drugs for treating cell senescence and / or preventing senescence; 5) Use of substances that reduce PAPPA protein content in the preparation of cell aging inhibitors; 6) Use of a substance that reduces PAPPA protein content in the preparation of a drug for treating cell senescence and / or preventing senescence.

2. The use according to claim 1, characterized in that The substance that inhibits PAPPA gene expression or the substance that inhibits PAPPA protein activity is any of the following biological materials: 1) a double-stranded RNA molecule, a modified substance thereof, or a pharmaceutically acceptable salt thereof; B1) producing a DNA molecule of the double-stranded RNA molecule described in 1); B2) an expression cassette containing the DNA molecule described in B1); B3) a recombinant vector containing the DNA molecule described in B1); B4) a recombinant vector containing the expression cassette described in B2); B5) a recombinant microorganism containing the DNA molecule described in B1); B6) a recombinant microorganism containing the expression cassette described in B2); B7) a recombinant microorganism containing the recombinant vector described in B3); B8) A recombinant microorganism containing the recombinant vector described in B4).

3. The use according to claim 1 or 2, characterized in that The substance that inhibits PAPPA gene expression or the substance that inhibits PAPPA protein activity is siRNA.

4. The use according to claim 3, characterized in that The siRNA is an RNA molecule whose nucleotide sequence is SEQ ID No. 1 in the sequence table.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition contains any of the following substances: M1) a substance that inhibits the expression of the PAPPA gene described in claim 1, or a substance that inhibits the activity of the PAPPA protein described in claim 1; M2) Cell senescence inhibitors.

6. The pharmaceutical composition according to claim 5, characterized in that The substance is any of the following biological materials: 1) a double-stranded RNA molecule, a modified substance thereof, or a pharmaceutically acceptable salt thereof; B1) producing a DNA molecule of the double-stranded RNA molecule described in 1); B2) an expression cassette containing the DNA molecule described in B1); B3) a recombinant vector containing the DNA molecule described in B1); B4) a recombinant vector containing the expression cassette described in B2); B5) a recombinant microorganism containing the DNA molecule described in B1); B6) a recombinant microorganism containing the expression cassette described in B2); B7) a recombinant microorganism containing the recombinant vector described in B3); B8) A recombinant microorganism containing the recombinant vector described in B4).

7. Use of a substance for detecting PAPPA gene expression or a substance for detecting PAPPA protein content in any of the following: A1) Preparation of products for diagnosing or assisting in the diagnosis of aging; A2) preparing products for identifying or assisting in the identification of senescent cells; A3) preparing products for screening or assisting in screening young cells; A4) Preparation of products that assess or assist in the assessment of individual aging, organ / tissue aging, or cellular aging; A5) Preparation of products for identifying or assisting in the identification of individual aging, organ / tissue aging, or cellular aging.

8. Use of substances that regulate the expression of the SIRTs-PAPPA pathway in any of the following: A1) Preparation of products for diagnosing or assisting in the diagnosis of aging; A2) preparing products for identifying or assisting in the identification of senescent cells; A3) preparing products for screening or assisting in screening young cells; A4) Preparation of products that assess or assist in the assessment of individual aging, organ / tissue aging, or cellular aging; A5) Preparation of products for identifying or assisting in the identification of individual aging, organ / tissue aging, or cellular aging; The SIRTs-PAPPA pathway is composed of SIRTs protein and PAPPA protein, and the SIRTs protein is at least one of the seven proteins SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 and SIRT7.

9. The use according to claim 8, characterized in that The substances regulating the expression of the SIRTs-PAPPA pathway are substance S and substance P; The substance S is a substance that inhibits the expression of the SIRTs protein gene, a substance that inhibits the activity of the SIRTs protein, and / or a substance that reduces the content of the SIRTs protein; The substance P is a substance that inhibits the expression of the PAPPA gene, a substance that inhibits the activity of the PAPPA protein and / or a substance that reduces the content of the PAPPA protein.

10. The method, characterized in that The method is any of the following: F1) A method for delaying aging of an organ, tissue or cell, comprising the step of contacting the substance for regulating the expression of the SIRTs-PAPPA pathway as claimed in claim 8 with the organ, tissue or cell; F2) A method for improving functional disorders of aged organs, tissues or cells, comprising the step of contacting the substance for regulating the expression of the SIRTs-PAPPA pathway as claimed in claim 8 with the aged body, organ, tissue or cell; F3) A method for identifying or assisting in identifying the level of senescence of an organ, tissue or cell in vitro, comprising the step of detecting the expression level of the SIRTs protein and / or the expression level of the PAPPA protein in a sample from the subject; F4) A method for constructing senescent cells, comprising the step of introducing the substance for regulating the expression of the SIRTs-PAPPA pathway as claimed in claim 8 into recipient cells; F5) A method for screening or assisting in screening for drugs that delay aging, comprising the step of screening for drugs that delay aging using senescent cells constructed by the method described in F4); F6) A method for studying or assisting in studying the aging mechanism of an organism, organ, tissue, or cell, comprising the step of using senescent cells constructed by the method described in F4) to study the aging mechanism of an organism, organ, tissue, or cell; F7) A method for changing the senescence state of cells in vitro, comprising the step of introducing the substance for regulating the expression of the SIRTs-PAPPA pathway as claimed in claim 8 into recipient cells.

Images