CN117327782A - Heart aging biomarker and application thereof as drug target for delaying heart aging - Google Patents
Heart aging biomarker and application thereof as drug target for delaying heart aging Download PDFInfo
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- CN117327782A CN117327782A CN202311296997.5A CN202311296997A CN117327782A CN 117327782 A CN117327782 A CN 117327782A CN 202311296997 A CN202311296997 A CN 202311296997A CN 117327782 A CN117327782 A CN 117327782A
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Abstract
The application relates to a heart aging biomarker and application thereof as a drug target for delaying heart aging. The examples disclose the use of MLF1 as a biomarker in peripheral blood and/or myocardial tissue. The examples disclose the use of substances which reduce the activity and/or the expression level of MLF1 in peripheral blood and/or myocardial tissue. The examples disclose the use of a substance with MLF1 as a drug target in the preparation of a product. The examples disclose the use of mice with reduced MLF1 activity and/or expression in peripheral blood and/or myocardial tissue in the preparation and/or screening of a medicament. The examples disclose products for slowing cardiomyocyte aging. The examples disclose kits of substances for detecting MLF1 activity and/or expression level.
Description
Technical Field
The application relates to the technical field of myocardial cell aging, in particular to a heart aging biomarker and application thereof as a drug target for delaying heart aging.
Background
Age is a major risk factor for cardiovascular disease and is accompanied by myocardial remodeling and heart dysfunction during aging, including cardiomyocyte hypertrophy, myocardial fibrosis, diastolic or systolic dysfunction. The heart aging increases the sensitivity of the heart to external stimuli, increasing the incidence and mortality of heart failure in the elderly. Therefore, the research of heart aging molecular mechanism is important to clinically delay the aging-related cardiovascular diseases.
Epigenetic factor changes are one of the characteristics of aging, including changes in DNA methylation patterns, changes in histone post-translational modifications, heterochromosomal remodeling, and dysfunction of non-coding RNAs, among others. Specifically, during aging, the overall DNA methylation level gradually decreases with age, with concomitant increases in DNA methylation at specific gene sites; alterations in histone post-translational modifications are prone to transcriptional activation; heterochromatin gradually decreases during the aging process. However, how epigenetic factors regulate aging-related chromatin remodeling is still poorly understood, and the development of more epigenetic factors as biomarkers is particularly necessary.
Disclosure of Invention
The embodiment of the application discloses a heart aging biomarker and application thereof as a drug target for delaying heart aging. The biomarker is myeloid leukemia factor 1 (myeloid leukemia factor, MLF 1), which was originally identified as an MLF1-NPM fusion protein, resulting from t (3; 5) (q 25.1; q 34) chromosomal translocation. Therefore, the embodiment of the application at least discloses the following technical scheme:
in a first aspect, the examples disclose the use of MLF1 as a biomarker in peripheral blood and/or myocardial tissue. The application is A1) or A2): a1 Preparing a product for detecting myocardial cell aging; a2 Preparing a product for slowing down the aging of myocardial cells. The use is the diagnosis or treatment of non-diseases.
In a second aspect, the embodiments disclose the use of a substance that reduces the activity and/or expression of MLF1 in peripheral blood and/or myocardial tissue. The application is B1) or B2): b1 Preparing a product for detecting myocardial cell aging; b2 Preparing a product for slowing down the aging of myocardial cells. The use is the diagnosis or treatment of non-diseases.
In a third aspect, the examples disclose the use of a substance with MLF1 as a drug target in the manufacture of a product. The product functions as C1) or C2): c1 Detecting cardiomyocyte aging; c2 Detecting and/or slowing cardiomyocyte aging. The use is the diagnosis or treatment of non-diseases.
In a fourth aspect, the examples disclose the use of mice with reduced MLF1 activity and/or expression in peripheral blood and/or myocardial tissue for the preparation and/or screening of a medicament. The function of the drug is D1) or D2): d1 Detecting cardiomyocyte aging; d2 Detecting and/or slowing cardiomyocyte aging. The use is the diagnosis or treatment of non-diseases.
In a fifth aspect, embodiments disclose a product for slowing cardiomyocyte senescence, which is a substance that down regulates MLF1 gene expression, knocks down MLF1 gene, or silences MLF1 gene expression, and/or reduces MLF1 protein content and/or activity.
In a sixth aspect, the embodiments disclose a kit comprising a substance for detecting MLF1 activity and/or expression level. The detection object of the kit is peripheral blood and/or myocardial tissue. The kit is used for detecting the degree of myocardial cell aging or slowing down myocardial cell aging.
Drawings
FIG. 1 shows the results related to MLF1 as a molecular marker reflecting the state of healthy aging of the heart.
(A) The data set information of heart aging such as GSE11291, GSE12480, GSE43556 of GEO database.
(B) Venn diagram of DEGs in 4 heart aging datasets.
(C) Heat map of the expression level of 21 genes significantly changed in aged hearts in 4 data sets (left), qRT-PCR detected mRNA levels of 21 heart senescence-associated genes in heart tissue of young mice of 2 months old and heart tissue of aged mice of 22 months old. * P <0.05, < P <0.01, < P <0.001, n=6.
(D) GSE11291 dataset key gene heatmaps for calorie restriction or resveratrol treatment.
(E) Western blot detection of MLF1 protein level in heart tissue of young mice of 2 months old and heart tissue of aged mice of 22 months old, 200 mu M H detection 2 O 2 Induces changes in MLF1 protein levels in AC16 cardiomyocyte senescence. * P<0.01，***P<0.001，n＝3。
FIG. 2 shows the results associated with MLF1 gene silencing to delay cardiomyocyte senescence.
(A) Western blot and quantification showed knock-down efficiency of MLF 1-specific siRNA in AC 16. * P <0.05, n=3.
(B) MLF1 knock-down pair 200 μ M H 2 O 2 Representative images (left) and quantification (right) of the proportion of induced β -gal positive cells. Scale bar, 200 μm. * P:<0.001vs siNeg.##P<0.01，###P<0.001vs Control，n＝3。
(C) Knock down of MLF1 pair 200. Mu. M H 2 O 2 Effects of treated AC16 cell senescence markers p21 and IL1B mRNA levels. * P<0.01,***P<0.001vs siNeg.##P<0.01，###P<0.001vs Control，n＝3。
(D) Western blot and quantification showed successful adenovirus-mediated MLF1 overexpression in AC16 cells. * P <0.001, n=3.
(E) MLF1 overexpression vs H 2 O 2 Representative images (left) and quantification (right) of the effect of the proportion of induced β -gal positive cells. Scale bar, 200 μm. * P<0.01，***P<0.001vs siNeg.##P<0.01，###P<0.001vs Control，n＝3。
(F) MLF1 overexpression 200. Mu. M H 2 O 2 Effect of IL1B mRNA levels of the treated AC16 cell senescence markers. * P<0.01vs siNeg.#P<0.05，##P<0.01，###P<0.001vs Control，n＝3。
FIG. 3 shows the results related to the inhibition of cardiomyocyte senescence by MLF1 via chromatin remodeling.
(A) The differential gene screening condition is a volcanic pattern with |Fold change| >1.5 and P-Value < 0.05.
(B) DEGs enrichment analysis after MLF1 knockdown.
(C) The chromatin distribution of DEGs was down-regulated after MLF1 knockdown.
(D) Subcellular distribution of MLF1 in AC16 cells.
(E) Pie charts of gene proportions up-or down-regulated after silencing MLF1 in ATAT-seq.
(F) Heat map of chromatin accessibility reduced peaks after silencing MLF1 in ATAT-seq.
(G) Venn diagram of significant gene changes in RNA-seq and ATAC-seq after silencing MLF 1.
(H) Enrichment analysis of 119 genes with reduced chromatin accessibility and concomitant reduced transcript levels following MLF1 knockdown.
(I) Wien plot of 119 genes with reduced transcript levels and heart senescence-associated genes following MLF1 knockdown with reduced chromatin accessibility.
(J) Thermal map of differential genes of 22 heart senescence versus gene set down-regulated after MLF1 knockdown.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
Myeloid leukemia factor 1 (myeloid leukemia factor, MLF 1) was originally identified as an MLF1-NPM fusion protein, resulting from a t (3; 5) (q 25.1; q 34) chromosomal translocation. The MLF1 levels in patients with myelodysplastic syndrome (myelodysplastic syndromes, MDS) and acute myelogenous leukemia (acute myeloid leukemia, AML) are significantly increased, and are closely related to the occurrence and development processes of MDS and AML diseases. In addition, the research shows that the gene deletion of the MLF1 can obviously inhibit the growth of neuroblastoma, which suggests that the MLF1 can play an important role in the occurrence process of the neuroblastoma; MLF1 gene silencing significantly inhibited proliferation of lung adenocarcinoma cells A549 cells and cell colony formation. MLF1 is highly expressed in heart tissue as a functional transcriptional regulator, but has been studied in the pathophysiological processes of the heart.
Considering the characteristics of heart aging in transcriptome, and early studies found that molecular markers of pathological cardiac hypertrophy are not entirely suitable for heart aging, it is highly necessary to screen specific molecular markers that reflect heart health, aging. And, the present inventors found that: MLF1 overexpression aggravates H 2 O 2 Induced myocardial senescence phenotype cells, whereas MLF1 gene silencing significantly improves H 2 O 2 An induced myocardial aging phenotype cell. Furthermore, the combined analysis of ATAC-seq and RNA-seq suggests that MLF1 gene silencing may regulate downstream transcription by decreasing chromatin accessibility of senescence-associated genes. Therefore, the MLF1 can be used as a reliable biomarker for reflecting the healthy aging state of the heart and can be used as an effective therapeutic target for slowing down the aging of myocardial cells.
Based on this, the examples disclose the use of MLF1 as biomarker in peripheral blood and/or myocardial tissue. The application comprises A1) or A2): a1 Preparing a product for detecting myocardial cell aging; a2 Preparing a product for slowing down the aging of myocardial cells. The use is for diagnosis or treatment of non-disease. In some embodiments, the product is a diagnostic product for determining the aging state of myocardial cells or a diagnostic-aid product for assisting diagnosis by detecting the content or the expression level of MLF1 in peripheral blood and/or myocardial tissue samples in vitro based on the content or the expression level of MLF1 in the peripheral blood and/or myocardial tissue samples, such as a kit for detecting the content or the expression level of MLF1 in the peripheral blood and/or myocardial tissue samples.
In addition, the examples also disclose the use of substances which reduce the activity and/or the expression level of MLF1 in peripheral blood and/or myocardial tissue. The application comprises B1) or B2): b1 Preparing a product for detecting myocardial cell aging; b2 Preparing a product for slowing down cardiomyocyte aging; the use is the diagnosis or treatment of non-diseases. In some embodiments, the product achieves the effect of slowing cardiomyocytes by reducing MLF1 activity in peripheral blood and/or myocardial tissue in vivo, and/or interfering with its expression, to target MLF1 specifically, e.g., interfering with the interfering RNA of MLF1 in myocardial tissue.
In addition, the embodiment also discloses application of the substance taking MLF1 as a drug target in preparing products. The product functions as C1) or C2): c1 Detecting cardiomyocyte aging; c2 Detecting and/or slowing cardiomyocyte aging. The use is the diagnosis or treatment of non-diseases. In some embodiments, the product is used for judging the aging state of myocardial cells by detecting the content or the expression level of MLF1 in-vitro peripheral blood and/or myocardial tissue samples. In some embodiments, the product achieves the effect of slowing cardiomyocytes by reducing MLF1 activity in peripheral blood and/or myocardial tissue in vivo, and/or interfering with its expression, to target MLF1 specifically.
In addition, the embodiment also discloses application of the mice with reduced MLF1 activity and/or expression level in peripheral blood and/or myocardial tissue in preparing and/or screening medicaments. The function of the drug is D1) or D2): d1 Detecting cardiomyocyte aging; d2 Detecting and/or slowing cardiomyocyte aging. The use is the diagnosis or treatment of non-diseases. In some embodiments, the effectiveness and safety of the candidate drug is determined by constructing a mouse with reduced MLF1 activity and/or expression level in peripheral blood and/or myocardial tissue as a model mouse, administering different candidate drugs to the model mouse, and evaluating the cardiomyocyte aging state of the model mouse after administration. In some embodiments, the drug is selected from one or more of a nucleic acid molecule, a carbohydrate, a lipid, a small molecule compound, an antibody, a polypeptide, a protein, a gene editing vector, a lentivirus, an adenovirus, or an adeno-associated virus.
For example, some embodiments disclose a product that slows cardiomyocyte senescence, which is a substance that down regulates MLF1 gene expression, knocks down the MLF1 gene, or silences MLF1 gene expression, and/or reduces MLF1 protein content and/or activity. In some embodiments, the product is a virus that silences the MLF1 gene. In some embodiments, the virus is an adenovirus carrying a silencing MLF1 gene siRNA. In some embodiments, the sense strand of the silencing MLF1 gene siRNA is shown in SEQ ID NO.1 and the antisense strand is shown in SEQ ID NO. 2.
For example, some embodiments disclose a kit. The kit comprises a substance for detecting the activity and/or expression level of MLF 1. The detection object of the kit is peripheral blood and/or myocardial tissue. The kit is used for detecting the degree of myocardial cell aging or slowing down myocardial cell aging. In some embodiments, the kit consists of substances that detect MLF1 activity and/or expression levels. In some embodiments, the kit comprises a primer pair as shown in SEQ ID NO. 9-10, and the kit is a qRT-PCR-based MLF1 expression level detection kit.
The present application will be further illustrated below with reference to non-limiting examples. It will be appreciated by those skilled in the art that the following examples, while indicating some embodiments of the application, are given by way of illustration only and not limitation.
1. Materials and methods
1. MLF1 gene silencing delay myocardial cell aging
(1) Cell culture conditions
Human cardiomyocyte line AC16 cells (celcook) were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS, gibco, USA), 100U/mL penicillin, 100. Mu.g/mL streptomycin, under 5% CO 2 And 37 ℃.
(2) SiRNA for silencing human MLF1 gene
Starting from the AUG initiation codon of the transcript of the human MLF1 gene, searching for an AA two-chain sequence, and recording 19 base sequences at the 3' end of the AA two-chain sequence as potential siRNA target sites, wherein the GC content of the target sequence is about 30-60%, and the target sequence is prevented from being selected near the initiation codon or a nonsense region. BLAST comparison is carried out on the siRNA sequences of the potential human MLF1 genes and the corresponding genome database, sequences homologous to other coding sequences/ESTs are removed, and proper target sequences are selected for synthesis. siRNA for silencing a human MLF1 gene: the sense strand 5'-3' is GCUCAUAAUCGUAGAGGACT, SEQ ID NO. 1; the antisense strand 5'-3' is shown in GUCCUCUACGAUUAUGAGCTT, SEQ ID No. 2.
(3) Construction of Ad-MLF1 adenoviruses
According to the following primers Adeasy-h-MLF1-K-X-F: gctgtgaccggcgcctactctggtaccGCCACCATGCGACAGATGAT, SEQ ID No. 3; adeasy-h-MLF1-K-X-R: atatcttatctagaagcttaggctcgagTTAAGCGTAATCTGGTACGT, SEQ ID No. 4; carrying out PCR amplification on the mouse MLF1 gene to obtain a target fragment; then the amplified sequence is recovered by electrophoresis and then is recombined, connected and transformed with adenovirus vector pAdEasy-EF1-MCS-CMV-EGFP, and the first generation sequencing is carried out to confirm that the recombined gene sequence is correct.
Then, the adenovirus system is adopted to carry out cell transfection on the carrier plasmid carrying the MLF1 gene, the complete culture medium is replaced for 6 hours after transfection, the culture is carried out for more than ten days, the fresh culture medium is replaced once about four and five days in the middle, then the collected cells and 1ml of culture solution are placed in a 15ml centrifuge tube, liquid nitrogen is frozen and thawed three times at 37 ℃ (thorough freezing and thawing), centrifugation is carried out at 2000rpm for 5 minutes, and the supernatant is taken as the primary stock solution of the virus liquid. After the virus was collected by repeating the amplification for three consecutive generations, a large amount of amplification of the virus was performed, and then the virus was purified by CsCI density gradient centrifugation-dialysis combined method, followed by quality detection and titer detection.
(4) MLF1 Gene-silenced AC16 cell treatment
When the confluence of the AC16 cells reaches 60 to 70 percent, the old culture medium is sucked and removed, and then the DMEM/F12 basic culture medium is added for usesiRNA transfection is carried out on the transfection reagent according to the instruction, the transfection reagent is sucked and removed after 12 hours of transfection, and fresh complete culture medium is replaced for continuous culture for 48 to 72 hours. The siRNA transfection procedure was as follows: mu.L siRNA (50 pmol) or 6. Mu.L jetPRIME transfection reagent, respectively, was added to the jetPRIME buffer, respectivelyStanding at room temperature for 5min, mixing the two materials, standing at room temperature for 15min, and adding into corresponding six-hole plate; after 12 hours of transfection, the transfection reagent is sucked and discarded, and the fresh complete culture medium is replaced for continuous culture for 48 to 72 hours.
(5) MLF1 over-expression treatment of AC16 cells
After the confluence of the AC16 cells reaches 60-70%, the old culture medium is sucked and abandoned and replaced with a DMEM/F12 basic culture medium, the AC16 cells are infected by Ad-MLF1 adenovirus or control virus with MOI of 10, and after 12 hours, the fresh culture medium is replaced for continuous culture for 48-72 hours.
(6) Construction of AC16 cell aging model
At about 60% confluence of AC16 cells, 200. Mu. M H was used 2 O 2 After 1h of treatment, the old medium was removed and replaced with fresh complete medium for further cultivation for 72h to induce the generation of AC16 cell senescence model.
2. AC16 cell galactosidase (. Beta. -gal) staining
After the AC16 cell line is subjected to MLF1 gene silencing or over-expression treatment, the old culture medium in the 6-well plate is sucked and removed, and the cell line is rinsed twice by PBS for 5min each time; fixing with 4% paraformaldehyde at room temperature for 15min; after absorbing and discarding the fixing solution, PBS is used for washing twice for 5min each time; adding freshly prepared staining working solution (940. Mu.L staining solution A+10. Mu.L staining solution B+50. Mu. L X-gal), sealing with sealing film to avoid volatilization of the staining solution, and placing 6-well plate in a 37 ℃ oven for overnight staining; the next day, after the staining solution was pipetted into PBS, the photographs were taken under an inverted microscope and the proportion of β -gal positive cells were counted.
3、qRT-PCR
Total RNA was extracted from cell or tissue samples and the concentration and mass of each sample RNA was determined on a Nanpdorp instrument. RNA samples were subjected to reverse transcription using the All-in-one reverse transcription kit from Norpran. The reverse transcription system included 0.5. Mu.L Enzyme Mix, 2. Mu.L 5 Xall-in-one qRT Supermix, 2. Mu.L RNA (500 ng/. Mu.L concentration) and 5.5. Mu.L DEPC water in a total volume of 10. Mu.L. The reverse transcription procedure included 50℃for 15min, 85℃for 5min, and 4℃for 5min. Real-time fluorescent quantitative PCR (qRT-PCR) was performed using SYBR Green Master MIX from Norwezan corporation, and the reaction system was formulated to include 5. Mu.L 2X SYBR Green Master MIX, 0.5. Mu.L forward primerThe primer, 0.5. Mu.L reverse primer, cDNA 2. Mu.L and 2. Mu.L DEPC water. Adding 2 mu L/hole cDNA template into 384 hole plate corresponding micro holes, preparing the reaction system except cDNA into Mix liquid, and adding into corresponding micro holes; sealing the 384-well plate by using a sealing transparent film, and centrifuging at 3000rpm for 1min at room temperature; the 384-well plate is put into a real-time fluorescence quantitative PCR instrument to carry out PCR reaction according to a set reaction program. The reaction procedure was as follows: pre-denaturation at 95℃for 5min; PCR amplification includes denaturation at 95℃for 10s, annealing at 95℃for 10s, and extension at 72℃for 10s for 35 cycles; extending for 5min at 72 ℃; dissolution profile. Data analysis: after the reaction was completed, the data was exported and 2 was used in Excel -△△Ct The method calculates the level of each gene relative to the reference gene GAPDH or ACTB, and visual mapping was performed using GraphPad prism9.0 software. The sequences of the PCR primers used are shown in Table 1.
TABLE 1 primer sequences
| Gene Symbol | Species | Forward primer | Reverse primer |
| GAPDH | Human | tgcaaccgggaaggaaatga，SEQ ID NO.5 | gcccaatacgaccaaatcaga，SEQ ID NO.6 |
| ACTB | Human | catgtacgttgctatccaggc，SEQ ID NO.7 | ctccttaatgtcacgcacgat，SEQ ID NO8 |
| MLF1 | Human | gcgcgggaattaagtgagtc，SEQ ID NO.9 | tgagctctccctctaccatca，SEQ ID NO.10 |
| P21 | Human | tgtccgtcagaacccatgc，SEQ ID NO.11 | aaagtcgaagttccatcgctc，SEQ ID NO.12 |
| IL-1β | Human | atgatggcttattacagtggcaa，SEQ ID NO.13 | gtcggagattcgtagctgga，SEQ ID NO.14 |
4、Western blot
Total proteins were extracted from cells and heart tissue with RIPA lysis buffer, respectively, with protease inhibitor Cooktial, PMSF and phosphatase inhibitor added. Cell and tissue lysates were centrifuged at 4℃for 15mins at 12000g, the supernatant was taken, BCA protein was quantitatively assayed for protein concentration, and protein extracts of the same mass were taken for SDS-PAGE electrophoresis according to experimental requirements, followed by transfer onto PVDF membranes. The PVDF membrane was blocked with 5% skim milk for 1 hour at room temperature, the antibodies were diluted in proportion with antibody dilutions (3% BSA+1% sodium azide to 1 XTBS) and incubated overnight at 4℃in a shaker. Dilution with antibody at 1: diluting secondary antibodies of different sources according to 10000 proportion, incubating a PVDF film and the secondary antibodies for 1-2 hours at room temperature by a shaking table, and finally exposing by using a chemiluminescent instrument; data analysis gray scale statistical analysis was performed on the target strips using Image J software and visual mapping was performed using GraphPad Prism9.0 software.
5. Subcellular component separation
AC16 cells or 293T cells were rinsed with PBS and collected with a cell scraper, and the cell pellet was collected by centrifugation at 1000rpm at 4℃for 3 min. A lysis buffer (10mM HEPES,1.5mM MgCl2, 10mM KCl,0.5mM DTT) was added to the cell pellet, and the mixture was then subjected to lysis on ice for 30min, and the supernatant was removed by centrifugation at 12000rpm for 10min at 4 ℃. And then adding a buffer B (protease inhibitor is added into a lysis buffer A) into the cell sediment, uniformly mixing, continuing to lyse the cell sediment on ice for 10min, and centrifuging the cell sediment at 12000rpm for 10min at 4 ℃, wherein the supernatant is cytoplasm. C Buffer for precipitation (10mM Tris,10mM NaCl,3mM MgCl) 2 0.3% np40, 10% glycerol) was washed three times, centrifuged at 200g for 2min at 4 ℃ and the nuclear pellet was retained. Adding D Buffer (20mM Tris,150mM KCl,3mM MgCl) to the nuclear pellet 2 0.3% of NP40 and 10% of glycerol), and centrifuging at 12000rpm for 10min at 4 ℃ after contact ultrasonic crushing, and taking the supernatant as a cell nucleus component. Adding 4 XSDS loading, shaking, mixing, heating in a metal bath at 95 ℃ for 10min to denature proteins, and detecting subcellular localization of MLF1 by Western blot.
6. Chromatin accessibility sequencing (ATAC-seq)
(1) ATAC-seq Experimental procedure
After the AC16 cells are transfected with MLF1 specific siRNA or control siRNA for 12 hours, the fresh complete culture medium is replaced to continue to culture for 48 hours, and 105 AC16 cells are collected for subsequent experiments after cell counting; after PBS washing, the cells were lysed on ice by adding ATAC-seq lysis buffer (10 mM Tris-HCl pH 7.4, 10mM NaCl,3mM MgCl2 and 0.5% NP-40), and the supernatant was centrifuged at 1500g for 5min at 4℃to retain the nuclei; reference toDNA Library Prep Kit V2 kit instructions, adding Tn5 transposomes to the nuclear fraction and incubating for 30min at 37℃in a modification buffer; DNA purification was performed using MinElute kit, the recovered DNA was subjected to 12 cycles of PCR reaction, DNA was again purified and recovered to obtain an ATAC-seq library, and the ATAC-seq library was sent to the NodeGramineae source sequencing company Illumina Novaseq6000 sequencing platform for ATAC-seq sequencing. ATAT-seq ProgenThe initial data has been uploaded to the GEO database (GEO number: GSE 207907). (2) ATAC-seq data analysis flow
The raw data were aligned with the human reference genome (hg 38) by Bowtie2 software (version 2.3.5.1), leaving a unique alignment to reads on the reference genome; peak rolling is performed using Macs2 software with default parameters, and mitochondrial Peaks does not account for statistics; differential peak Value calculation is carried out by using DiffBind (version 1.34.0) software, and Q-Value is less than 0.05 and is a threshold Value; normalizing the reads uniquely aligned to the reference genome in a per kilobase per million mapping Reads (RPKM) normalization manner using deep software to generate bigwig data with a resolution of 100bp; peaks were annotated using the ChIPseeker software (version 1.30.3) for subsequent analysis.
7. RNA-seq transcriptome sequencing and data analysis
(1) Transcriptome sequencing of cardiac samples
Taking 3 myocardial tissues of a young mouse with the age of 2 months and 3 myocardial tissues of an old mouse with the age of 22 months, taking the myocardial tissues of a pathologic myocardial hypertrophy model mouse constructed by TAC surgery with the age of 2 months, extracting RNA samples by the method, completing the transcriptome library framework and RNA-seq sequencing by Huada sequencing company, and uploading the original transcriptome data to a GEO database (GEO numbers are GSE200741 and GSE203083 respectively).
(2) GEO data set for heart aging
In order to deeply explore the transcription characteristics of heart aging, the experiment screens transcriptome data comprising aged hearts and young hearts in a GEO database (GEO, http:// www.ncbi.nlm.nih.gov/GEO), and the transcriptome data is initially screened to obtain 20 relevant data sets; the three candidate data sets were then screened for subsequent analysis by species sources, sequencing platform, age group limiting factors (GEO-compiled GSE11291, GSE12480 and GSE43556, respectively).
(3) Transcriptome data analysis procedure
The original data is compared with a reference genome or the probe name is converted into a corresponding official gene name based on platform annotation; eliminating probe information without a gene name, eliminating genes with low expression level to prevent interference to analysis results; carrying out differential gene analysis on each data set by using an R language Limma package, wherein the differential gene screening condition is |fold change| >1.5 and P-Value is <0.05; the Metascape (http:// Metascape. Org) online enrichment analysis tool performs joint enrichment analysis on the differential genes of the multiple data sets; enrichment analysis is carried out by an Enrich (https:// maayanlab. Closed/Enrich /) online tool, and related databases comprise ChEA2022, GO gene ontology, KEGG signal path, wiki signal path and the like; the GSEA software uses default parameters for genome enrichment analysis of transcriptome or GEO chip data.
8. Statistical analysis
Data are expressed as mean ± standard deviation. For normal distribution variables with equal variance, the differences between the two groups were evaluated using independent Student's t test; multiple comparisons are analyzed by adopting one-way analysis of variance and Tukey post-hoc test; p <0.05 represents a statistical significance. Data statistical analysis using SPSS (v 22) (IBM, usa) software, graphic visualization uses GraphPad and R language.
2. Results
1. MLF1 can be used as a molecular marker for reflecting heart health aging state
In this example, a search of the GEO database for heart senescence-associated data sets (fig. 1A) identified 21 genes (18 up-regulated genes and 3 down-regulated genes) as synchronized changes in four heart senescence data sets (fig. 1B) by wien's diagram, suggesting that this gene set could be a specific marker of heart senescence. Moreover, qRT-PCR verified that most of the genes changed in aged and young mouse myocardial tissues consistent with transcriptomes (fig. 1C), demonstrating higher accuracy in the combined screening of heart aging molecular markers from multiple data sets.
Numerous studies have reported that including calorie restriction (calorie restriction, CR), exercise and medication can delay and even reverse the progression of heart aging to some extent. Thus, this example further looks for a list of core genes based on the effects of CR and resveratrol drug intervention on heart aging transcription reprogramming. The changes in four genes, BCL 2-like 11 (BCL 2 like 11, BCL2l 11), myeloid leukemia factor 1 (MLF 1), cadherin 22 (Cdh 22) and Myh7, during heart aging can be significantly reversed by CR and resveratrol suggesting that this gene combination can serve as a reliable molecular marker that accurately predicts heart health and aging status (fig. 1D).
Among them, in agreement with the conclusion of the human protein profile database, this example found that MLF1 was highly expressed in the heart and distributed mainly in cardiac myocytes. The study of this example found that MLF1 protein levels were significantly reduced in aged mouse heart tissue compared to young hearts, at H 2 O 2 The significant decrease in the induced AC16 cardiomyocyte senescence model (fig. 1E) suggests that MLF1 can not only serve as a specific biomarker reflecting healthy senescence in the heart, possibly a key target for delaying cardiac senescence.
2. MLF1 gene silencing can delay myocardial cell aging
To investigate the role of MLF1 in cardiomyocyte senescence, this example silences MLF1 expression with specific siRNA and overexpresses MLF1 with adenovirus, and in H 2 O 2 The induction of AC16 human cardiomyocyte senescence model was verified. As a result, MLF1 gene silencing significantly reduced the protein level of MLF1 (FIG. 2A), and knocking down MLF1 significantly reduced H 2 O 2 The proportion of β -gal positive cells induced (FIG. 2B), suggesting that MLF1 gene silencing significantly reduces H 2 O 2 Proportion of senescent cells induced. While MLF1 gene silencing significantly reduced the elevation of senescence marker p21 and SASP marker IL1B (fig. 2C). MLF1 adenovirus overexpression significantly increased protein levels of MLF1 (FIG. 2D), significantly increased H 2 O 2 The ratio of induced β -gal positive cells to IL1B levels (FIG. 2E, F), suggesting that MLF1 gene overexpression significantly increased H 2 O 2 The proportion of senescent cells induced and the level of senescence. Thus, it is shown that MLF1 gene silencing promotes heart aging and that inhibition of MLF1 expression may be an effective measure in delaying or reversing myocardial cell aging.
3. MLF1 regulates senescence-associated transcriptomes by chromatin remodeling
To explore the potential molecular mechanisms of MLF1 gene silencing to improve cardiomyocyte senescence, this example employed transcriptome sequencing analysis. As a result, it was found that after MLF1 gene silencing, transcriptome reprogramming tended to repress transcription, and when the differential gene screening conditions were set to |Fold Change| >1.5, P-Value <0.05, 816 genes were significantly down-regulated after MLF1 knockdown, and 288 genes were up-regulated (FIG. 3A). Enrichment analysis of the differential genes revealed that significantly down-regulated differential gene and extracellular matrix composition after MLF1 gene silencing correlated with extracellular activation (fig. 3B), and that the down-regulated differential genes were clustered on the chromosome (fig. 3C). Subcellular isolation experiments of the AC16 human cardiomyocyte line found that MLF1 was predominantly localized to the nucleus and formed stable binding to chromatin in an insoluble form, suggesting that MLF1 may function by modulating chromatin remodeling (fig. 3D).
Subsequently, this example also uses transposase accessible chromatin high throughput sequencing (ATAC-seq) for in-depth analysis. After MLF1 knockdown, chromatin accessibility exhibited overall inhibition, 8825 closed peaks (30.8%) and 140 open peaks (0.05%) were detected (fig. 3E), and combining ATAC-seq and RNA-seq results indicated that MLF1 increased chromatin accessibility and thus activated gene transcription, and MLF1 gene silencing exhibited overall transcriptional inhibition (fig. 3F). After gene annotation of the peak of ATAC-seq, the gene sets with increased or decreased chromatin opening and the gene sets with up-or down-regulated transcriptome were intersected by Veen plots, and 119 gene sets with decreased chromatin opening and decreased transcription level were found after MLF1 gene silencing, far more than those with increased chromatin opening with increased transcription level (fig. 3G). Reduced chromatin accessibility is accompanied by significant enrichment of the gene sets down-regulated by transcription in TGF-B regulated extracellular matrix and IL-1 regulated extracellular matrix, key genes including TGFBR1, IL1B, IL RA, etc. (figure 3H). By plotting the differential gene of heart aging with 119 differential genes silenced and regulated by MLF1 genes (FIG. 3I), it was found that MLF1 gene silencing can significantly reduce the elevation of heart aging process key genes including NPPB, IL15RA, etc. (FIG. 3J).
The results show that MLF1 can be used as a molecular marker for reflecting heart health aging, MLF1 gene silencing can delay myocardial cell aging, MLF1 regulates aging-related transcriptome through chromatin remodeling, and therefore myocardial cell aging is improved, and MLF1 can be used as a potential target for delaying heart aging.
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.
Claims (10)
1. Use of MLF1 as biomarker in peripheral blood and/or myocardial tissue, A1) or A2):
a1 Preparing a product for detecting myocardial cell aging;
a2 Preparing a product for slowing down cardiomyocyte aging;
the use is the diagnosis or treatment of non-diseases.
2. Use of a substance that reduces MLF1 activity and/or expression level in peripheral blood and/or myocardial tissue, as B1) or B2):
b1 Preparing a product for detecting myocardial cell aging;
b2 Preparing a product for slowing down cardiomyocyte aging;
the use is the diagnosis or treatment of non-diseases.
3. Use of a substance with MLF1 as drug target for the preparation of a product with the function C1) or C2):
c1 Detecting cardiomyocyte aging;
c2 Detecting and/or slowing cardiomyocyte aging;
the use is the diagnosis or treatment of non-diseases.
4. The use according to claim 3, wherein the subject of MLF1 is an in vitro peripheral blood and/or myocardial tissue sample.
5. Use of mice with reduced MLF1 activity and/or expression in peripheral blood and/or myocardial tissue in the preparation and/or screening of a medicament; the function of the drug is D1) or D2):
d1 Detecting cardiomyocyte aging;
d2 Detecting and/or slowing cardiomyocyte aging;
the use is the diagnosis or treatment of non-diseases.
6. The use according to claim 5, wherein the medicament is selected from one or more of a nucleic acid molecule, a carbohydrate, a lipid, a small molecule compound, an antibody, a polypeptide, a protein, a gene editing vector, a lentivirus, an adenovirus or an adeno-associated virus.
7. A product for slowing cardiomyocyte senescence, which is a substance that down regulates MLF1 gene expression, knocks down MLF1 gene, or silences MLF1 gene expression; and/or, substances that reduce the content and/or activity of MLF1 proteins.
8. The product of claim 7, which is a virus that silences the MLF1 gene;
alternatively, the virus is an adenovirus carrying a silencing MLF1 gene siRNA;
optionally, the sense strand of the silencing MLF1 gene siRNA is shown as SEQ ID NO.1, and the antisense strand is shown as SEQ ID NO. 2.
9. A kit comprising a substance for detecting the activity and/or expression level of MLF 1;
the detection object of the kit is peripheral blood and/or myocardial tissue;
the kit is used for detecting the degree of myocardial cell aging or slowing down myocardial cell aging.
10. The kit according to claim 6, which consists of a substance for detecting the activity and/or expression level of MLF 1;
optionally, the kit comprises primer pairs shown as SEQ ID NO. 9-10.
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