CN114034870A - Application of senescence related differential expression gene in preparation of cell senescence diagnostic kit - Google Patents
Application of senescence related differential expression gene in preparation of cell senescence diagnostic kit Download PDFInfo
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Abstract
The invention discloses an application of differential expression genes related to senescence in the preparation of a cell senescence diagnostic kit, belonging to the technical field of biomedicine. Through screening and verification, the protein expression levels of three genes of differential expression genes SIRT3, E2F3 and IRS2 related to senescence and the methylation modification level of m6A RNA are found for the first time, so that the method can be used for identifying and diagnosing the senescence state of cells, solves the problem of lack of diagnostic markers in the field of cell senescence at present, and provides a reference basis for early prevention and targeted treatment of cell senilism and age-related diseases.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to application of a senescence-associated differentially expressed gene in preparation of a cell senescence diagnostic kit.
Background
Cell aging is a permanent state of cell cycle arrest. Aging cells accumulate along with the aging of the body, release a series of factors such as proinflammatory factors and proapoptotic factors, and cause the occurrence of age-related diseases and chronic diseases, such as atherosclerosis, type 2 diabetes, arthritis, idiopathic pulmonary fibrosis, liver diseases and the like. Thus, the identification, characterization and pharmacological elimination of senescent cells is of interest in the field of senescence research.
Cell aging is the basis of aging of organs and individuals, and the process of the cell aging is influenced by various complex factors such as heredity, environment and the like. For a long time, the scientific community has not been clear about the specific molecular mechanism for regulating aging, and the targets for identifying, diagnosing and intervening cellular aging also lack systematic markers.
Cellular senescence is the central developmental program of normal senescence and senescence pathology, and is strongly regulated at the post-transcriptional level. In recent years, methylation of m6A RNA has become very closely related to the occurrence and development of age-related diseases and the treatment and prognosis thereof, including obesity, immunomodulation, tumors, and the like, and can regulate the expression of target genes associated with cell proliferation, migration invasion, cell cycle progression, apoptosis, and autophagy processes. In senescent cells, we found that genes differentially expressed in young cells and senescent cells have different characteristics at the level of RNA methylation, and this differential characteristic provides evidence and methods for the identification of senescence and targets for treatment for the intervention of age-related disorders.
SIRT3 is located in mitochondria, and gene knockout enables mice to develop a variety of aging diseases, and can regulate almost every major function in mitochondrial biology, including ROS detoxification, ATP generation, etc., and various studies show that attenuation or ablation of SIRT3 is associated with accelerated development of a variety of aging diseases. No regulation and control effect of E2F3 in the aging process is reported at present, but m6A modification is involved in miRNA/E2F 3-mediated gastric cancer cell proliferation inhibition. The factors that IRS2 plays an important role in the development and treatment of tumors have also been reported to play a role in neurodegenerative diseases, but no association between their m6A modification and aging has been reported.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention aims to provide the application of the senescence-associated differentially expressed gene in preparing a cell senescence diagnostic kit.
The purpose of the invention is realized by the following technical scheme:
the application of differential expression gene related to senescence in the preparation of cell senescence diagnostic kit.
Further, the senescence-associated differentially expressed genes are one or more combinations of the following genes: SIRT3(NCBI accession No.: NM-012239.6), E2F3(NCBI accession No.: NM-001949.5), IRS2(NCBI accession No.: NM-003749.3).
Further, the kit for diagnosing cellular senescence is used for diagnosing cellular senescence by analyzing the protein expression level of senescence-associated differentially expressed genes, and/or the methylation modification level of m6A RNA in cells, that is,
(1) determining that the cell enters a senescent state when the assay results in one or more of the following combinations:
elevated sirt3 protein expression;
increased expression of the E2F3 protein;
increased or decreased expression of IRS2 protein;
elevated levels of sirt3m6A RNA methylation modification;
e.e2f3 m6A increased levels of RNA methylation modification;
increased level of methylation modification of irs2 m6A RNA;
(2) judging the cell as a replicative senescence cell when the assay results in one or more combinations of the following:
reduced expression of IRS2 protein;
increased level of methylation modification of IRS2 m6A RNA;
(3) judging the cell as a premature cell when the analysis result is one or more of the following cases:
elevated levels of sirt3m6a RNA methylation modification;
increased levels of e2f3m6A RNA methylation modification;
wherein, the protein expression condition of the senescence-associated differentially expressed gene and the m6A RNA methylation modification level in the cell are determined by a relative quantitative analysis method by taking the young cell as a control.
The definition of the young cells is: cells with a population doubling level PDL less than or equal to 50% of their lifespan;
the replicative senescence cell is defined as: cells with a population doubling level PDL greater than or equal to 90% of their lifespan;
the premature senility cell is an aging cell induced by stress of a young cell, and the stress factors include but are not limited to oxidative stress;
wherein, the formula of PDL is N ═ 3.32(logN2-logN1) + X, N2 is the total number of cells harvested from the generation, N1 is the number of cells seeded from the previous generation, and X is PDL of the previous generation.
Further, the cell senescence status is diagnosed by the following steps:
(1) extracting total protein of the cell to be detected, and performing semi-quantification of the protein of the senescence-associated differential expression gene in the cell to be detected by taking the young cell as a control;
(2) extracting total RNA of cells to be detected; enriching m6A RNA methylation fragments by an RNA methylation co-immunoprecipitation method (MeRIP), eluting and purifying to obtain a sample to be detected; taking a sample to be detected for reverse transcription and real-time fluorescence Q-PCR quantification;
(3) and (3) diagnosing the cell senescence condition according to the semi-quantitative protein result of the senescence-associated differentially expressed gene obtained in the step (1) and the detection result of the methylation level of the m6A RNA of the senescence-associated differentially expressed gene obtained in the step (2).
Furthermore, the aging diagnosis kit contains one or more combinations of an aging related differential gene protein extraction reagent, a protein specific antibody, a Westernblot experiment reagent, an m6A RNA methylation fragment enrichment reagent, an amplification primer and an m6A RNA methylation sequencing and analysis reagent.
Wherein, the amplification primer comprises:
SIRT3-F：5'-GACCAACATGCTAGAAGTGCGG-3'；
SIRT3-R：5'-AAGCTCGGCATCTGTTGGTTAC-3'；
E2F3-F：5'-AAACCTTTCTCCTCTGGCCTCC-3'；
E2F3-R：5'-CAGGGGAGGCAGTAAGTTCACA-3'；
IRS2-F：5'-CTGTGAGAGCAGTGGGTGATGA-3'；
IRS2-R：5'-TGGAGTCTGAATGCCTAGCCAG-3'。
the cell is a cell that gradually senesces with increasing age; preferably human embryonic lung fibroblasts.
Based on the human embryonic lung fibroblast replicative senescence and the hydrogen peroxide induced premature senescence model, the gene for identifying and diagnosing cell senescence is obtained by screening through observing the change of general biological characters in the cell senescence process, the expression characteristics of senescence-related genes and the methylation characteristics of m6A RNA: SIRT3, E2F3, IRS 2.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention uses human embryonic lung fibroblasts as a model for cell senescence. Human embryonic lung fibroblast to exogenous H2O2Is less resistant than endometrial stem cells and dermal fibroblasts, is more susceptible to oxidative stress, and the process of development of its senescence characteristics has been well defined. Therefore, the human embryonic lung fibroblasts provide a useful experimental model for researching potential mechanisms and markers related to aging.
The invention establishes a replicative senescence model and a hydrogen peroxide-induced premature senescence model based on human embryonic lung fibroblasts. The model is a powerful tool for assessing cellular premature aging and cellular replicative senescence.
Through screening and verification, the protein expression level and the m6A RNA methylation modification level of 3 genes of SIRT3, E2F3 and IRS2 are found for the first time, so that the method can be used for identifying and diagnosing the senescence state of cells, solves the problem of lack of diagnostic markers in the cell senescence field at present, and provides a reference basis for early prevention and targeted treatment of cell senilism and age-related diseases.
Drawings
FIG. 1 is a diagram showing the results of morphological observation and β -galactosidase staining identification of each group of cells; wherein, A is the morphological change of each group of cells under an optical microscope, 200X, a scale: 100 μm; b is the SA- β -galactosidase staining under optical microscopy, 200 ×, scale: 100 μm; c is the blue staining ratio of SA- β -galactosidase staining, mean ± sd, n is 3, P <0.05, P <0.01, P <0.0001, ns is statistically different (P >0.05), compared to 22PDL or 49 PDL.
FIG. 2 is a graph showing protein expression of senescence-associated genes; wherein A is the protein expression condition of Western blot detection 4 genes; b, ImageJ software carries out quantitative analysis on the protein band of the methyltransferase, the 22PDL quantification is 1, and a relative quantification result is calculated; n is 3, P <0.05, P <0.01, P <0.0001, ns is statistically different (P >0.05) compared to 22PDL and 49 PDL.
FIG. 3 is a graph of the methylation levels of senescence-associated genes; wherein, A is a gel electrophoresis picture, qPCR products are subjected to agarose gel electrophoresis, M represents IP, N represents Negative control (Negative) "IgG", and P represents Positive control (Positive) "RNA Input"; b is the methylation level of m6A in the 3' UTR region of the senescence-associated gene detected by MeRIP-qPCR; 22PDL was 1 and the other 2 groups were methylation levels relative to 22PDL, n ═ 3, P <0.05, × P <0.01, × P <0.0001 compared to 22PDL and 49PDL, ns being the difference without statistical significance (P > 0.05).
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is only illustrated by the reference examples and is not limited to the scope of the present invention. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention.
Unless otherwise specified, the reagents and materials used in the present invention are commercially available products or products obtained by a known method.
The cells used in the following examples are human embryonic lung fibroblasts CCC-HPF-1 from the institute of basic medicine, institute of medical science, China.
Example 1
(1) Cell culture
The cells were incubated at 37 deg.C and 95% relative humidity with CO2And (5) performing aseptic culture in a cell culture box with the volume fraction of 5%. The culture solution is an L-DMEM low-sugar culture medium. When the cell confluence reaches 90%, cell passage (1: 2, 1:3 or 1: 4) is performed as required, and cell counting is performed. The formula of the Population Doubling Levels (PDL) is N-3.32 (log N2-log N1) + X, where N is the final PDL of the subcultured cells, N2 is the total number of cells harvested from the subcultured cells, N1 is the number of cells seeded at the previous generation, and X is the PDL of the cells at the previous generation.
(2) Model of senescent cells and model of replicative senescence cells
Premature senility cell model: h with Young cell group 22PDL2O2Contamination, identical numbers of 22PDL inoculated into cell culture flasks (1:3 passages), and a final concentration of 400. mu. mol/LH used when the cells grew to 50% confluence2O2And 4d of contamination, wherein the contamination is carried out once every day for 2 hours at a fixed time. Continuously infecting 4d to obtain a cell premature senescence initiation group (PSi); after 4 days of continuous infection, the continuous group of premature senility (PSp) was obtained after the continuous culture for 7 days by replacing the L-DMEM low-sugar medium without infection.
Replicative senescence cell model: when normal human embryonic lung fibroblasts were serially subcultured in vitro to 52PDL, the cells stopped proliferating and exhibited a deep replicative senescence state. According to the definition of in vitro cell culture age: obtaining young cells when the PDL of the cultured cells is less than or equal to 50% of their lifespan; obtaining senescent cells when the cell number is greater than or equal to 90%; when the concentration is between 50% and 90%, the middle-aged cells are obtained. In the HEFs model of replicative senescence in this experiment, cells were grouped into 22 PDLs for young cells, 35 PDLs for middle aged cells, and 49 PDLs for replicative senescent cells (reference, "Zhang Wen Juan, Jiweidong, Yangyiqing, etc.. P53 epigenetic modification [ J ] in the course of cell senescence. J. toxicology, 2009,23(1): 1-4.").
(3) Phenotypic characterization of senescent cells
The aging state of the cells is observed and verified by using a beta-galactosidase staining experiment. The operation steps are as follows:
1) removing the cell culture solution of the cultured cells in the 6-well plate by suction, washing the cells for 1 time by PBS, adding 1mL of beta-galactosidase staining fixing solution, and fixing the cells for 15min at room temperature;
2) cell fixative was aspirated off, cells were washed 3 times with PBS for 3min each;
3) PBS was aspirated off, and 1mL of staining working solution (β -galactosidase staining solution A10 μ L + β -galactosidase staining solution B10 μ L + β -galactosidase staining solution C930 μ L + X-Gal solution 50 μ L) was added to each well;
4) sealing the 6-hole plate with a preservative film to prevent evaporation, and placing the plate in a 37 ℃ carbon dioxide-free incubator for overnight incubation;
5) the cell senescence was observed under a normal light microscope. And selecting three different visual fields under the microscope for observing each group of cells, calculating the total number of the cells and the number of blue-stained cells in each visual field, and finally calculating the ratio of the number of the blue-stained cells to the total number of the cells.
(4) Western blot protein expression level determination
5.1 extraction and protein quantification of Total cellular proteins
And (3) extracting total cell protein: collecting cells, adding 60 mu L of RIPA lysate containing 1% PMSF into every 100 ten thousand cells, gently blowing, placing on ice for cracking for 10min, shaking, repeating for three times, carrying out centrifugation for 5min at 4 ℃, 15000rpm, and transferring the supernatant to a new 1.5mL centrifuge tube to obtain the corresponding protein sample.
The protein samples prepared above were quantified according to the BCA protein concentration assay kit protocol:
preparation of protein standards: 0.8mL of the protein standard preparation solution was added to a tube of protein standard (20mg BSA), and was dissolved sufficiently to prepare a 25mg/mL protein standard solution. Taking a proper amount of 25mg/mL protein standard, and diluting to a final concentration of 1 mg/mL;
preparing a BCA working solution: adding the reagent A and the reagent B according to the volume ratio of 50:1, and fully and uniformly mixing;
and (3) detecting the protein concentration: adding the standard substance into standard substance wells of a 96-well plate according to 0, 1, 2, 4, 8, 12, 16 and 20 μ L, and adding standard substance diluent to make up to 20 μ L; adding 20 mu L of samples, and arranging 3 compound holes in each group in parallel; adding 200 mu L of BCA working solution into each hole, and standing at 37 ℃ for 30 minutes; measuring the absorbance at 562nm by using an enzyme-labeling instrument; the protein concentration of the sample was calculated from the standard curve and the sample volume used.
5.2SDS-PAGE electrophoretic separation of proteins
1) Preparation of SDS-PAGE electrophoretic separation gel
2) Preparation of SDS-PAGE electrophoresis concentrate
3) Slowly pouring the separation glue into a pre-assembled glue-making plate, adding absolute ethyl alcohol when the separation glue is added to the wide part of the small glass plate 2/3, sealing the glue, standing at room temperature for 30min, removing the absolute ethyl alcohol, pouring the concentrated glue, quickly inserting into a tooth comb, standing for 30min-45min, and waiting for the solidification;
4) after the electrophoresis device is assembled, adding 1 mu L of marker on the left side, sequentially adding 20 mu g of protein sample into each sample hole, and finally adding 5 mu L of marker on the right side as indicator protein;
5) and (3) setting the constant voltage to be 80V, performing electrophoresis for 20min, adjusting the voltage to be 120V when the bromophenol blue indicator protein migrates to the junction of the concentrated gel and the separation gel, and performing electrophoresis for 60min until the bromophenol blue indicator protein reaches the bottom of the gel.
5.3 transfer film (Wet transfer)
1) Taking down the rubber plate, and accurately cutting the adhesive tape of the target protein according to the indicator protein;
2) preparing a PVDF membrane with the area equivalent to that of the adhesive tape, activating the PVDF membrane in 100% methanol for 10s, then putting the PVDF membrane into distilled water for rinsing for 3min, and transferring the PVDF membrane into a membrane transferring liquid for balancing for 5 min. Simultaneously putting 2 pieces of filter paper into the membrane transferring liquid for soaking for 5 min;
3) making a traditional sandwich structure, aligning layer by layer without air bubbles, placing a rotary membrane system in an electrophoresis apparatus containing a rotary membrane buffer solution, setting a constant voltage of 200mA, and rotating the membrane for 60min by ice water bath.
5.4 immune response
1) After the wet rotation is finished, the PVDF membrane is subjected to positive and negative angle shearing to be shown, TBST is rinsed for 2 times, and a confining liquid shaking table is slowly and slightly shaken to be confined for 4 hours or is confined in a refrigerator at 4 ℃ overnight;
2) taking out the PVDF membrane, placing the PVDF membrane in a primary antibody diluted according to a proper proportion, and slowly shaking the PVDF membrane in a shaking table at room temperature to incubate for 2 hours or overnight at 4 ℃;
3) washing PVDF membrane with TBST for 10min 3 times;
4) selecting a secondary antibody type according to a primary antibody source, diluting the secondary antibody according to a ratio of 1:5000, wherein the secondary antibodies corresponding to the three proteins of SIRT3, E2F3 and IRS2 are anti-rabbitt IgG (ab 6721 of Abcam company, UK), the secondary antibody corresponding to beta-actin is anti-mouse IgG (ab 6789 of Abcam company, UK), and slowly shaking the secondary antibody for 1h by a shaking table at room temperature;
5) after the secondary antibody incubation was complete, the PVDF membrane was rinsed 3 times for 10min each with TBST.
6) Placing the PVDF film on a developing plate, mixing solution A and solution B of ECL developing solution in equal amount, uniformly dripping the mixed solution on the surface of the PVDF film, carrying out automatic exposure, and photographing and storing;
7) protein bands were semi-quantitatively analyzed using Image-Pro Plus 6.0 software.
(5) m6A RNA methylation co-immunoprecipitation
6.1 fragmentation of RNA
1) RNA concentration was adjusted to 1. mu.g/. mu.L with DEPC water. Each 200. mu.L PCR tube was filled with 18. mu.L (i.e., 18. mu.g) of total RNA. Add 2. mu.L of Fragmentation Buffer 10 × (cat. No. CS 220011). Blowing, fully mixing, and turning upside down and uniformly mixing; (for 300 u g total RNA, required 17 tube.)
2) The PCR instrument was preheated to 94 ℃.5 tubes were incubated in the PCR apparatus for 5min at a time with the heating lid closed. The tubes were removed from the instrument and 2 μ L of 0.5M EDTA (cat # CS203175) was added immediately to each tube. Vortex and rotate the tube, place it on ice. This step was repeated until all the RNA was fragmented;
3) the contents of all tubes were collected in a new 1.5mL EP tube and 1/10 volumes of 3M sodium acetate (pH 5.2), glycogen (final 100. mu.g/mL) and 2.5 volumes of 100% ethanol were added. The contents were mixed and incubated overnight at-80 ℃;
4) centrifuge at 15,000g for 25min at 4 ℃. Discarding the supernatant taking care not to break the precipitate, which is easily visible due to the presence of glycogen;
5) washing the precipitate with 1mL of 75% (vol/vol) ethanol, and centrifuging at 4 deg.C for 15,000g for 15 min;
6) carefully remove the supernatant by aspiration, air dry, add 300. mu.L DEPC water to resuspend the pellet.
6.2 preparation of magnetic beads for immunoprecipitation
1) Sample preparation 1 × IP buffer: the 5 × IP buffer was diluted 5-fold with DEPC water to prepare 5mL of 1 × IP buffer, i.e.: 4mL of DEPC water and 1mL of 5 XPIP buffer solution are placed in a new 15mL centrifuge tube and are placed on ice for later use;
2) labeling the appropriate number of EP tubes: 2 1.5mL microcentrifuge tubes were prepared for each set of samples, one tube for anti-m 6A antibody and one tube for negative control normal mouse IgG;
3) removing the magnetic Magna ChIP protein A/G beads, dispersing and resuspending well until no clumped beads are visible;
4) adding 50 mu L of Magna ChIP protein A/G magnetic beads into each EP tube prepared in the step 2;
5) adding 10 times of the volume of the magnetic beads into each tube of the 1 xIP buffer solution prepared in the first step, namely adding 500 mu L of MeRIP dilution buffer solution into each 50 mu L of original magnetic beads, and gently and uniformly blowing the mixture to completely resuspend the magnetic beads; placing the test tube on a magnetic separation rack for 1 min;
6) the supernatant was carefully aspirated to ensure that any magnetic beads were not aspirated. Removing the EP tube from the magnetic separation rack;
7) repeating the step 4) and the step 5), and washing once more;
8) adding 200. mu.L of 1 × IP buffer solution, resuspending 50. mu.L of magnetic beads in the amount of original magnetic beads, adding 10. mu.g of anti-m 6A antibody to the EP tube labeled with m6A, and adding 10. mu.g of IgG to the EP tube labeled with IgG as a subsequent negative control;
9) rotating and incubating for 30min at room temperature;
10) after short centrifugation, the mixture is placed on a magnetic separation rack for 1min, and then supernatant is removed;
11) the EP tube was removed from the magnetic separation rack. To each EP tube, 0.5mL of 1 × IP buffer was added and gently pipetted several times to completely resuspend the magnetic beads. Placing the EP tube on a magnetic separation frame for 1min, and removing the supernatant;
12) repeat step 10), continue washing twice. Ensuring that the supernatant is removed cleanly and only the magnetic beads are left;
13) the EP tube was removed from the magnetic separation rack and placed on ice. The lid of the EP tube was closed to prevent the beads from drying out. These samples will be used in step 3 of the following immunoprecipitation section.
6.3 immunoprecipitation (MeRIP)
1) Mu.g of total RNA (10% as Input) was removed and placed in a new EP tube labeled "RNA Input". The Input samples were stored at-80 ℃. The sample will be used for generating a standard curve or for comparison in a qPCR method, or for input control in an RNA-seq;
2) prepare 1000 μ L MeRIP reaction mix of 300 μ g total RNA (Table 1):
TABLE 1 MeRIP reaction mixtures
3) To each prepared bead-antibody tube, 500. mu.L MeRIP reaction mixture was added. Gently flick several times to completely resuspend the magnetic beads, and place on ice;
4) all EP tubes were incubated at 4 ℃ for 2h with rotation;
5) the EP tube was briefly centrifuged to remove the liquid on the lid and side of the EP tube. Placing on a magnetic separation rack for 1 min;
6) the supernatant was aspirated carefully without touching the beads;
7) removing the EP tube from the magnetic separation frame, adding 500. mu.L of precooled 1 × IP buffer solution into each tube, and gently blowing and beating for several times to completely resuspend the magnetic beads;
8) placing the EP tube on a magnetic separation frame for 1min, and then removing the supernatant;
9) repeating the above washing procedure (step 7) to step 8))2 times for a total of 3 washes;
10) the prepared EP tube was placed on ice and immediately subjected to the next elution.
6.4 elution
1) Preparation of 20mM m6A salt: 10mg of N6 methyladenosine-5' monophosphate sodium salt (m6A) (cat # CS220007) was dissolved in 1.3mL of DEPC water. Subpackaging 150 μ L of each tube, and storing at-20 deg.C;
2) preparing an elution buffer: approximately 225 μ L of 45 μ L of 5 XP buffer (CS220009) +75 μ L of 20mM m6A salt prepared in step 1 +3.5 μ L of RNase Inhibitor (CS216138) +101.5 μ L of DEPC water were prepared for each tube and the mixture was mixed well for use;
3) adding 200 mu L of elution buffer solution into the prepared magnetic beads, gently blowing and beating the elution buffer solution for several times, uniformly mixing the elution buffer solution and the magnetic beads, and completely resuspending the magnetic beads;
4) all EP tubes are placed at 4 ℃ for continuous shaking elution for 1 h;
5) centrifuging the EP tube for a short time to remove the liquid on the EP tube cover and the tube wall, and placing on a magnetic separation rack for 1 min;
6) the supernatant was transferred to a new 1.5mL EP tube, taking special care not to aspirate the beads, otherwise background noise was increased;
7) repeat steps 3) -6) above, i.e., elute twice, and combine all eluates from the same sample, with a total elution volume of 200 μ L for each sample.
6.5 purification of RNA fragments
1) The 200 μ L sample was transferred to a new 15mL conical tube according to RNeasy mini kit instructions. Add 700. mu.L Buffer RLT and mix well;
2) adding 1400 μ L of anhydrous ethanol, blowing, mixing thoroughly, centrifuging, and entering next step;
3) of these 700. mu.L samples were transferred to RNeasy MinElute spin columns and placed on a 2mL collection tube, the lid gently closed, centrifuged at ≥ 8,000 Xg (. gtoreq.10,000 rpm) for 15s, the liquid in the collection tube discarded, another 700. mu.L sample was transferred to a centrifuge tube, and the liquid discarded. Repeating the process until all samples are loaded;
4) RNeasy MinElute spin columns were placed in a new 2mL collection tube and 500. mu.L Buffer RPE was added to the spin column. The cap was gently closed and centrifuged at a speed of 8,000 Xg (10,000 rpm) or more for 15 seconds to wash the spin column membrane. Discarding the liquid in the collection tube and reusing the collection tube in the next step;
5) adding 50 μ L of 80% ethanol into RNeasy MinElute centrifugal column, slightly closing the cover, and centrifuging at a speed of more than 8000 Xg (more than 10,000rpm) for 2min to wash the membrane of the centrifugal column, and discarding the collecting tube and liquid therein;
6) placing RNeasy MinElute spin column in a new 2mL collection tube (provided in RNA purification kit), opening the cap of the centrifugation column, centrifuging at 15,000rpm for 5min, and discarding the collection tube and the liquid therein;
7) RNeasy MinElute spin columns were placed in new 1.5mL collection tubes (provided by the RNA purification kit). mu.L of RNase-free water was directly added to the center of the spin column membrane, the cap was gently closed, and the column was centrifuged at 15,000rpm for 1min to elute RNA, which could be stored at-80 ℃. The prepared RNA samples were used for subsequent qPCR.
6.6 reverse transcription
1) The m6A sample and the negative control IgG were synthesized into cDNA according to the instructions, 28. mu.L per tube (Table 2)
TABLE 2 constituents of the reverse transcription System
2) After mixing, carrying out reverse transcription reaction under the following conditions:
15min at 37 ℃ (reverse transcription)
5sec at 85 ℃ (inactivation reaction of reverse transcriptase)
4℃ forever
The synthesized cDNA was stored in a freezer at-20 ℃.
6.7 qPCR and quantitative calculation
The method comprises the following specific steps:
1) according to the m6A RNA methylation modification gene locus displayed in the sequencing result, searching a corresponding DNA sequence corresponding to mRNA in a UCSC website, and designing a Primer in Primer3 by using the sequence, wherein the sequence of the Primer is shown in Table 3.
TABLE 3 primer sequences
2) PCR reaction solution was prepared according to the composition shown in Table 4
TABLE 4 PCR reaction solution
3) Perform Real Time PCR reaction
Step1：95.0℃ 30s
Step2：95.0℃ 5s
Step3：60℃ 30s
+Plate Read
Step4：GOTO②，39more times
Step5：95.0℃for 10s
Step6：Melt Curve 65.0to 95.0℃，increment 0.5℃
For 5s+Plate Read
END
4) And outputting the Ct value.
6.8 quantitative calculation of enrichment factor for methylation
1) Averaging Ct values of three multiple wells, wherein each gene respectively comprises three samples of m6A, Input and IgG;
2) normalization: delta Ctm6A＝Ctm6A–(CtInput–log210)；
3) Calculating the percentage of a certain gene in RNA under IP in the Input: % Input 2-ΔCtm6A
4) Exclusion of non-specific enrichmentAfter assembly, the ratio of the target gene in IgG: Δ Δ Ctm6A＝ΔCtm6A-ΔCtIgG
5) And finally calculating methylation enrichment times: fold attribute 2-ΔΔCtm6A。
6.9 agarose gel electrophoresis of the PCR products
1) Preparing glue with the concentration of 2 percent: 0.6g agarose +30mL 1 × TBE;
2) boiling, adding 1 μ L nucleic acid dye, mixing, pouring into a horizontal plate after the liquid is changed in temperature, and inserting into a comb;
3) after the gel is solidified, putting the gel into a horizontal electrophoresis tank, and adding 1 × TBE to immerse the DNA gel;
4) loading: adding a sample buffer Loading buffer into the DNA sample according to a corresponding proportion, Loading 5 mu L of buffer into each hole, and Loading 3 mu L of buffer into a DNA marker;
5) electrophoresis: 120V, 25 min.
6) And (3) developing: the gel was placed in a gel imager, the uv light was turned on, and the photograph was taken directly and stored.
Experimental results and analysis:
(1) cytomorphological observation and beta-galactosidase staining for identifying cell senescence
As shown in FIG. 1A, it was revealed under light microscopy that as the age of cells increased, the cell morphology became progressively larger and flattened by fiber length, the cells of the early senescence starting group PSi became larger and flattened, the cells of the PSp group became similar to 49PDL, the cell nuclei became larger, and some cells shriveled to death. As shown by the results of SA- β -galactosidase staining in FIG. 1B, the number of blue-stained cells was the greatest in 49PDL and the PSp group of cells, and SA- β -galactosidase expression was increased, 42.2 and 37.9 times that of 22PDL, respectively, with no significant difference between PSp and 49 PDL. The number of blue-stained cells in the PSi group was 16.3 times that in 22PDL, which was lower than 49PDL and PSp; as shown in FIG. 1C, SA- β -galactosidase expression gradually increased with age.
(2) Protein expression of senescence-associated genes
As shown in figure 2, SIRT3 protein expression increased with age, and expression was also higher in senescent cells than in the young group (P <0.05), with statistical differences (P <0.05), 49PDL protein expression higher than PSp (P < 0.05); protein expression of the PDL group of the E2F3 gene 49 is higher than that of the PSp group (P < 0.05). In the IRS2 gene, protein expression of PSp was higher than 49PDL (P < 0.05).
(3) RNA methylation level of senescence-associated gene m6A
After RNA fragmentation, immunoprecipitation was performed to precipitate the gene fragment containing the m6A modification, followed by real-time fluorescent quantitative PCR detection. As shown in FIG. 3A, the qPCR product was subjected to agarose gel electrophoresis, the band ranged from 100bp to 200bp, and the "IP" and "Input" samples had bands, which more intuitively indicates that the designed primers were able to successfully amplify DNA products and the negative control "IgG" had no amplified band.
Fold enrichment by methylation was calculated by qPCR assay, as shown in figure 3B, the methylation level of SIRT3 in the PSp group was higher than 22PDL and 49PDL, 3.4, 9.0 fold higher than 22PDL and 49PDL, respectively, the difference was statistically significant (P < 0.05); the methylation level of IRS2 in 49PDL was increased 3.6 times that of 22PDL (P <0.05) compared to 22PDL, the PSp group difference was not statistically significant (P >0.05), and in addition, the methylation level of 49PDL was higher than that of the PSp group, the difference was statistically significant (P < 0.05); the methylation level PSp group of E2F3 was higher than 22PDL and 49PDL, 2.7 and 2.2 times higher (P <0.05), respectively.
SIRT3 is a family of NAD + -dependent histone deacetylases with homology to Sir2, Sir2 is thought to be involved in the prolongation of lifespan associated with calorie restriction in lower organisms, SIRT3 is located within mitochondria, gene knockout enables mice to develop a variety of aging diseases, and can modulate almost every major function in mitochondrial biology, including ROS detoxification, ATP production, etc., and various studies show that attenuation or ablation of SIRT3 is associated with accelerated development of a variety of aging diseases. The invention discovers for the first time that hypermethylation of SIRT3 can promote post-transcriptional translation, thereby causing premature cell senescence.
The E2F3m6A modification is involved in miRNA/E2F 3-mediated inhibition of gastric cancer cell proliferation. The invention discovers for the first time that the methylation level of E2F3m6A in the premature senility cell is higher than that of the replicative senescence group, and the increase of the methylation level promotes protein expression, which is the reason for the premature senility of the cell. IRS2 plays an important role in tumor generation and treatmentThe factor (2) has also been reported to play a role in neurodegenerative diseases. The invention discovers for the first time that the expression of IRS2 protein is inhibited by the hypermethylation of IRS2 in replicative senescence cells, H2O2After the cells are treated, the methylation degree of IRS2 is reduced, so that the protein expression is increased, and the occurrence of premature senility is regulated and controlled.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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<120> application of senescence-associated differential expression gene in preparation of cell senescence diagnostic kit
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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Claims (8)
1. The application of the senescence-associated differentially expressed gene in the preparation of a cell senescence diagnostic kit is characterized in that:
the senescence-associated differentially expressed genes are one or more combinations of the following genes: SIRT3, E2F3, IRS 2.
2. The use of the senescence-associated differentially expressed gene of claim 1 in the preparation of a kit for diagnosis of cellular senescence, wherein:
the cell senescence diagnostic kit diagnoses the cell senescence by analyzing the protein expression of senescence-associated differentially expressed genes in cells and/or the methylation modification level of m6A RNA:
(1) determining that the cell enters a senescent state when the assay results in one or more of the following combinations:
elevated sirt3 protein expression;
increased expression of the E2F3 protein;
increased or decreased expression of IRS2 protein;
elevated levels of sirt3m6A RNA methylation modification;
e.e2f3 m6A increased levels of RNA methylation modification;
increased level of methylation modification of irs2 m6A RNA;
(2) judging the cell as a replicative senescence cell when the assay results in one or more combinations of the following:
reduced expression of IRS2 protein;
increased level of methylation modification of IRS2 m6A RNA;
(3) judging the cell as a premature cell when the analysis result is one or more of the following cases:
elevated levels of sirt3m6A RNA methylation modification;
increased levels of e2f3m6A RNA methylation modification;
protein expression of senescence-associated differentially expressed genes and the level of m6A RNA methylation modification in the cells were determined using relative quantitation assays with young cells as controls.
3. Use of the senescence-associated differentially expressed gene according to claim 1 or 2 in the preparation of a kit for diagnosis of cellular senescence, characterized in that:
the definition of the young cells is: cells with a population doubling level PDL less than or equal to 50% of their lifespan;
the replicative senescence cell is defined as: cells with a population doubling level PDL greater than or equal to 90% of their lifespan;
the premature senility cell is an aging cell induced by stress of a young cell, and the stress factors include but are not limited to oxidative stress;
wherein, the formula of PDL is N ═ 3.32(logN2-logN1) + X, N2 is the total number of cells harvested from the generation, N1 is the number of cells seeded from the previous generation, and X is PDL of the previous generation.
4. Use of the senescence-associated differentially expressed gene according to claim 1 or 2 in the preparation of a kit for diagnosis of cellular senescence, characterized in that:
the cell senescence condition is diagnosed by the following steps:
(1) extracting total protein of the cell to be detected, and performing semi-quantification of the protein of the senescence-associated differential expression gene in the cell to be detected by taking the young cell as a control;
(2) extracting total RNA of cells to be detected; enriching m6A RNA methylation fragments by an RNA methylation co-immunoprecipitation method, eluting and purifying to obtain a sample to be detected; taking a sample to be detected for reverse transcription and real-time fluorescence Q-PCR quantification;
(3) and (3) diagnosing the cell senescence condition according to the semi-quantitative protein result of the senescence-associated differentially expressed gene obtained in the step (1) and the detection result of the methylation level of the m6A RNA of the senescence-associated differentially expressed gene obtained in the step (2).
5. Use of the senescence-associated differentially expressed gene according to claim 1 or 2 in the preparation of a kit for diagnosis of cellular senescence, characterized in that:
the aging diagnosis kit contains one or more combinations of an aging related differential gene protein extraction reagent, a protein specific antibody, a Western blot experiment reagent, an m6A RNA methylation fragment enrichment reagent, an amplification primer and an m6A RNA methylation sequencing and analysis reagent.
6. Use of the senescence-associated differentially expressed gene according to claim 5 in the preparation of a kit for diagnosis of cellular senescence, wherein:
the amplification primer comprises:
SIRT3-F：5'-GACCAACATGCTAGAAGTGCGG-3'；
SIRT3-R：5'-AAGCTCGGCATCTGTTGGTTAC-3'；
E2F3-F：5'-AAACCTTTCTCCTCTGGCCTCC-3'；
E2F3-R：5'-CAGGGGAGGCAGTAAGTTCACA-3'；
IRS2-F：5'-CTGTGAGAGCAGTGGGTGATGA-3'；
IRS2-R：5'-TGGAGTCTGAATGCCTAGCCAG-3'。
7. use of the senescence-associated differentially expressed gene according to claim 1 or 2 in the preparation of a kit for diagnosis of cellular senescence, characterized in that:
the cells are cells that gradually age with increasing age.
8. Use of the senescence-associated differentially expressed gene according to claim 1 or 2 in the preparation of a kit for diagnosis of cellular senescence, characterized in that:
the cells are human embryonic lung fibroblasts.
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