AU2021102139A4 - Application of m6a rna methylation content, and methylation-related enzymes and binding proteins thereof in preparation of senescence detection kit - Google Patents

Abstract

The present invention discloses the application of mA RNA methylation content, and methylation-related enzymes and binding proteins thereof in the preparation of a senescence detection kit. The senescence detection kit of the present invention determines cell senescence by detecting protein expressions of m6A RNA methylation content, RNA methylation enzymes METTL3, METTL14, 6 WTAP and KIAA1429, m A RNA demethylation enzymes FTO and ALKBH5, and m A RNA methylation binding proteins YTHDC1, YTHDF1 and YTHDF2; the high expression or low expression of RNA methylation enzyme METTL3 is used for determining whether the cell senescence is premature or normally replicative. The present invention can be applied to the assessment of premature senescence, replicative senescence and senescence-related diseases, and thus can be widely used in the future. Drawings A 22PDL 35PDL 49PDL PSi PSp B 120.0 ns 100.0 80.0 so4. 60.0 40.0 20.0 'Is 0.0 22PDL 35PDL 49PDL PSi PSp Fig. 1 1/3

Description

Drawings

A

22PDL 35PDL 49PDL

PSi PSp

B 120.0 ns 100.0

80.0 so4.

60.0

40.0

20.0 'Is

0.0 22PDL 35PDL 49PDL PSi PSp

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SPECIFICATION APPLICATION OF M6A RNA METHYLATION CONTENT, AND METHYLATION-RELATED ENZYMES AND BINDING PROTEINS THEREOF IN PREPARATION OF SENESCENCE DETECTION KIT

Technical Field

The present invention relates to the technical field of biomedicine, particularly relates to the application of m6A RNA methylation content, and the methylation related enzymes and binding proteins thereof in the preparation of a senescence detection kit.

Background Art

Cell senescence, i.e. replicative senescence, is a complex biological process, wherein the cells have progressively disordered physiological functions, recessing proliferation, and permanent retardation of cell cycle. Cell senescence is closely related to occurrence and development of many age-related diseases, such as atherosclerosis, osteoarthritis, Alzheimer disease, age-related macular degeneration, type-2 diabetes, and cancers or the like, and the cells are in a state of chronic and low-grade nonbacterial inflammation. Ultraviolet radiation, chemotherapy and other extraneous environmental factors may accelerate the process of cell senescence, and bring about acute senescence, i.e. premature senescence. The occurrence of premature senescence makes the functions of body organs decrease much earlier, increases risks of age-related diseases, and thus lowers life quality.

At present, detection indexes of cell senescence researches relate to senescence-related p-galactosidase, telomere and telomerase, senescence-related heterochromatin focus, senescence-related secretion phenotype, reactive oxygen species, and tumor suppression genes such as p53 and p16. These detection indexes are widely applied to senescence researches, and each has its own characteristics. However, there is yet no related patent about the detection and determination of cell premature senescence.

Cell premature senescence and replicative senescence have a lot of similar phenotypic characteristics, but they are different in inherent regulatory mechanisms. In a cell of premature senescence, there are DNA methylation of core genomes and regulatory variations of overall histone modification, unstable microenvironments of nuclear and mitochondria epigenomies, and epigenetics modification changes of senescence-related special genes. RNA methylation modification is a brand new research area of epigenetics, and m A RNA methylation is a new regulatory mechanism of RNA apparent transcriptomics, which plays an important role in maintaining normal physiological functions of a body, and in regulating age-related diseases including cancers, diabetes, cardiac functions, and immune function changes and so on.

It is searched that there are four related patents in the field of senescence diagnosis, which are respectively "Serum protein marker for indicating healthy senescence key pathway and application thereof (Publication No.: CN111366736A)", "Healthy senescence serum protein marker and application (Publication No.: CN111337688A)", "Health senescence diagnosis circRNA markers and application thereof (Publication No.: CN111424079A)", and "CircRNA small molecular marker indicating key pathways of healthy senescence and application (Publication No.: CN111363802A)". The afore-mentioned patents for invention relate to the field of health senescence diagnosis, and are mainly about circRNA and serum markers.

Summary of the Invention

The primary object of the present invention is to overcome the defects and deficiencies of prior art by providing the application of m6 A RNA methylation content, and methylation-related enzymes and binding proteins thereof in the preparation of a senescence detection kit.

The other object of the present invention is to provide a senescence detection kit.

The objects of the present invention are realized by means of the technical solution below: the application of m 6 A RNA methylation content, and methylation related enzymes and binding proteins thereof in the preparation of a senescence detection kit.

The senescence includes replicative senescence (i.e. a normal senescence process) and oxidative stress-induced premature senescence.

The cells are those gradually senescent with age; preferably, the cells are human embryonic lung fibroblasts.

The senescence detection kit detects m6 A RNA methylation content, and protein expressions of RNA methylation-related enzymes and binding proteins, and regards them as markers for determining cell senescence.

The RNA methylation-related enzymes include RNA methylation enzymes and RNA demethylation enzymes. The RNA methylation enzymes are METTL3, METTL14, WTAP and KIAA1429.

The RNA demethylation enzymes are FTO and ALKBH5.

The RNA methylation binding proteins are YTHDC1, YTHDF1 and YTHDF2. 6 When a decrease of the m A RNA methylation content in the cell, and/or one or 6 at least two expressions of the m A RNA methylation enzymes METTL14, WTAP 6 and KIAA1429, the m A RNA demethylation enzymes FTO and ALKBH5, and the m6A RNA methylation binding proteins YTHDC1, YTHDF1 and YTHDF2, and/or a high expression or low expression of the RNA methylation enzyme METTL3 are detected, it shows that the cell enters into an senescence state.

The detection of a low expression of the RNA methylation enzyme METTL3 in the cell shows the replicative senescence of the cell; and the detection of a high expression of the RNA methylation enzyme METTL3 in the cell shows the premature senescence of the cell.

The protein expression is counted by using a relative quantitative analysis method, gray values of different cell bands are obtained by means of gray analysis of protein bands, a 22PDL young cell group, which is provided as 1, is taken as a comparison, and other cell expressions are compared with a gray value of the 22PDL young cell group; protein and internal reference expressions are obtained by means of Western blot detection.

The 22PDL young cell group is defined as cells of which the subculture cells have a final population doubling level (PDL) of 22, wherein the calculation formula of PDL is n=3.32(logN2-logNl)+X, N2 being a total number of the cells obtained in this generation, NI being the number of cells inoculated in the previous generation, and X being the PDL of cells of previous generation.

A senescence detection kit, comprising a quantitative detection reagent form 6 A RNA methylation in cell, and/or a protein expression detection reagent for detecting at least one of the methylation enzymes METTL3, METTL14, WTAP and KIAA1429, the demethylation enzymes FTO and ALKBH5, and m 6 A RNA methylation binding proteins.

The protein expression detection reagent is a Western blot semi-quantitative detection reagent.

The senescence detection kit is applied to non-therapeutic diagnosis and detection of senescence.

The application including a step of detecting m A RNA methylation content by using the senescence detection kit after taking cells to be detected and extracting overall RNA, and/or a step of performing protein semi-quantification by using the senescence detection kit after extracting overall protein of cells to be detected.

Relative to prior arts, the present invention has the following beneficial advantages and effects:

1. In the present invention, the m A RNA content, the RNA methylation enzymes METTL3, METTL14, WTAP and KIAA1429, the m6 A RNA demethylation enzymes FTO and ALKBH5, and m A RNA methylation binding proteins YTHDC1, YTHDF1 and YTHDF2 are used as new markers of cell senescence, and can be applied to the assessment of premature senescence, replicative senescence and senescence-related diseases, and thus can be widely used in the future.

2. Different from existing health senescence detection patents taking circRNA as the detection index and taking serum protein as the detection object, the present 6 invention takes the m A RNA methylation content, the RNA methylation-related enzymes and binding proteins as the detection objects, wherein the detection samples are not limited to serum, and is capable of accurately detecting both premature senescence and replicative senescence of histocytes caused by oxidative stress.

Description of drawings

Fig. 1 is a drawing showing the staining result of the senescence-relatedp galactosidase; wherein A is a picture (x20, Scale: 200 tm); B is the statistic result of the ratio of cells stained blue after staining; the average value standard deviation, n=3, *P<0.05, ns is the difference and has no statistical significance (P>0.05), compared with 22PDL or 49PDL.

Fig. 2 is a diagram showing the overall change of them A RNA methylation content of the cells in each group; A is the proportion of mRNA, to which m A modification occurs, among 200 ng of total RNA of cells in each group; B is the 6 relative m A content of cells in each group; compared with 22PDL or 49PDL, the average value standard deviation, n=4, *P<0.05, ns is the difference and has no statistical significance.

Fig. 3 is a diagram showing the protein expressions and relative quantification of m6A RNA methylation enzymes of cells in each group; A is the expression result of METTL3, METTL14, WTAP and KIAA1429 detected by means of Western Blot in cell senescence; B is the expression difference between the gray density value for each protein band of METTL3, METTL14, WTAP and KIAA1429 in replicative senescence and that in H2 0 2-induced cell premature senescence; the average value: standard deviation, n=3, *P<0.05, ns is the difference and has no statistical significance, compared with 22PDL or 49PDL.

Fig. 4 is a diagram showing the protein expressions and relative quantification of m6A RNA demethylation enzymes of cells in each group; A is the expression result of FTO and ALKBH5 detected by means of Western Blot in cell senescence; B is the expression difference between the gray density value for each protein band of FTO and ALKBH5 in replicative senescence and that in H2 0 2-induced cell premature senescence; the average value standard deviation, n=3, *P<0.05, ns is the difference and has no statistical significance, compared with 22PDL or 49PDL.

Fig. 5 is a diagram showing the expressions and relative quantification of RNA methylation binding proteins in the cells of each group; A is the expression results of RNA methylation binding proteins (YTHDC1, YTHDF1 and YTHDF2) detected by means of Western Blot in cell senescence; B is the expression difference between the gray density value for each protein band of YTHDC1, YTHDF1 and YTHDF2 in replicative senescence and that in H 20 2-induced cell premature senescence; the average value standard deviation, n=3, *P<0.05, ns is the difference and has no statistical significance, compared with 22PDL or 49PDL.

Embodiments

With reference to embodiments and the drawings, details of the present invention will be further described, but the embodiments of the present invention are not limited thereto.

Examples

(1) Cell culture

Human embryonic lung fibroblasts (from Cell Resource Center of Institute of Basic Medical Sciences in Chinese Academy of Medical Sciences) were placed into a cell incubator, which has a temperature of 37°C, a relative humidity of 95% and a

CO2 volume fraction of 5%, for axenic culture. The culture solution was L-DMEM low-sugar culture medium. When the cell fusion degree reached 90%, cell passage (1:2, 1:3 or 1:4) was performed according to actual necessities, and the cells were counted. The calculation formula of population doubling levels (PDL) is n=3.32(logN2-logN1)+X, wherein n is the final PDL of subculture cells, N2 is a total number of the cells obtained in this generation, NI is the number of cells inoculated in the previous generation, and X is the PDL of cells of previous generation

(2) Cell model of premature senescence and cell model of replicative senescence

Cell model of replicative senescence: when normal human embryonic lung fibroblasts were cultured to 52PDL after continuous passage in vitro, the cell proliferation stopped, and the cells were in a state of deep replicative senescence. According to the definition for the age of cell culture in vitro: when PDL of cultured cells is smaller than or equal to 50% of their lifespan, young cells will be obtained; when PDL of cultured cells is greater than or equals to 90%, senescent cells will be obtained; when PDL of cultured cells is between 50%~90%, middle-aged cells will be obtained. In the replicative senescence model of human embryonic lung fibroblasts in this experiment, the cell groups are as follows: the young cell group 22PDL, the middle-aged cell group 35PDL, and the replicative senescence cell group 49PDL (please refer to the article written by the inventor published in Journal of Toxicology, 2009, 23(01):1-4).

Cell model of premature senescence: H2 0 2 exposure was performed for the young cell group 22PDL, a same number of 22PDL were inoculated into a cell culture flask (1:3 passage), and, when the cells increased to 50% of their fusion degree, H 2 0 2 having a final concentration of 400 tmol/L was used for exposure for 4d at a fixed time each day and for 2h each time. After continuous exposure for 4d, a premature senescence initiation group (PSi) of cells was obtained; after continuous exposure for 4d, culture medium was changed to normal and the cultured was continued for 7d, thereby obtaining a premature senescence persistence group (PSp) of cells.

(3) Phenotype determination of senescence cells

The P-galactosidase staining experiment was used for observing and verifying the senescence states of replicative senescence cells and premature senescence cells in step (2). The operation steps are as follows:

1) sucking away the cell culture solution used for culturing cells from a 6-well plate, washing the plate once with 1xPBS, and adding 1 mL of p-galactosidase stain fixative for fixation of 15 min at room temperature;

2) sucking away the cell fixative, and washing the cells for three times with 1xPBS, 3 min each time;

3) sucking away 1xPBS, adding to each well 1 mL of staining working solution (10 tL of p-galactosidase staining solution A + 10 tL of p-galactosidase staining solution B + 930 tL of p-galactosidase staining solution C + 50 tL of X-Gal solution);

4) sealing the 6-well plate with plastic wrapper to prevent evaporation, and placing the sealed 6-well plate into a C0 2-free incubator at 37°C for incubation over night; and

5) observing the cell senescence conditions under an ordinary optics microscope. Three different visual fields under the microscope were selected for observing each cell group, the total number of the cells and the number of cells stained blue in each visual field were calculated, and a ratio of the number of cells stained blue to the total number of the cells was finally calculated.

(4) Measurement of Western blot protein expression

4.1 Extraction of total protein of cells and protein quantification

Extraction of total protein of cells: replicative senescence cells and premature senescence cells in step (2) were collected, 60 tL of RIPA lysate containing 1% (v/v) PMSF was added to each million cells, gently blew and beat, placed the solution on ice for lysis of 10 min, oscillated it for three times, centrifugation was performed at 4°C and at a speed of 15000 rpm for 5 min, the supernatant was transferred to a new centrifuge tube of 1.5 mL, thereby obtaining correspondingproteinsamples.

Quantification was performed for protein samples prepared in the above way according to the operating instructions of BCA Protein Assay Kit (Beyotime Biotechnology of Shanghai):

preparation of protein standard substance: 0.8 mL of protein standard preparation solution was added and fully dissolved into a tube of protein standard (20 mg of BSA), so that a protein standard solution of 25 mg/mL was prepared; an appropriate amount of protein standard solution of 25 mg/nL was taken, and was diluted to have a final concentration of 1 mg/mL;

preparation of BCA working solution: a reagent A and a reagent B were added in a volume ratio of 50:1, and were fully mixed; and

detection of protein concentration: 0, 1, 2, 4, 8, 12, 16 and 20 tL of standard substance were added to standard substance wells on a 96-well plate, a diluent of the standard substance was added to make up to 20 tL; 20 tL of sample was added, and 3 parallel wells were provided for each group; 200 tL of BCA working solution was added to each well, and was placed at 37°C for 30 min; a microplate reader was used for measuring absorbance at 562 nm; and the protein concentration was calculated according to the standard curve and volumes of the samples used.

4.2 Protein separated by SDS-PAGE electrophoresis

1) SDS-PAGE electrophoresis separating gel was prepared;

2) SDS-PAGE electrophoresis spacer gel was prepared;

3) the separating gel was slowly perfused into a gel plate assembled in advance, and absolute ethanol was added to seal the gel when the spacer gel was added to 2/3 width of the small glass plate, after a still standing at room temperature for 30 min, absolute ethanol was removed, the spacer gel was perfused and a comb was quickly inserted; a still standing was performed for 30-45 min, awaiting for solidification;

4) after the electrophoresis device was assembled, 1 tL of marker was added to the left side, and then 20 tg of protein sample was added in turn to each loading well, and 5 tL of marker was added to the right side at last as indicating protein; and

5) the constant voltage was set to be 80 V, and the electrophoresis was performed for 20 min; the voltage was regulated to 120 V when bromophenol blue indicating protein moved to the junction of the spacer gel and the separating gel; the electrophoresis was performed for 60 min, and was stopped when the bromophenol blue indicating protein reached the bottom of the gel.

4.3 Membrane transfer (wet transfer)

1) The rubber plate was taken down, and a rubber band of object protein was precisely cut according to the indicating protein;

2) a PVDF membrane having an area similar to the rubber band was prepared, and was activated for 10 s in 100% (v/v) methyl alcohol before it was rinsed for 3 min in distilled water, and then was transferred to a transfer buffer so as to be balanced for 5 min; meanwhile, two pieces of filter paper were placed into the transfer buffer and were immersed there for 5 min; and

3) a traditional "sandwich" structure was made, wherein the layers were aligned and no bubble was left; the membrane transfer system was placed in an electrophoresis apparatus filled with transfer buffer, the constant voltage was set to be 200 mA, and the membrane was transferred in an ice-water bath for 60 min.

4.4 Immune reaction

1) After the wet transfer, the corner of PVDF membrane was cut so as to indicate the front and then back, and the PVDF membrane was rinsed twice by TBST, and then was slowly and gently shaken and sealed for 4 h by a shaker with blocking solution, or was sealed in a refrigerator at 4°C over night;

2) the PVDF membrane was taken out, and was placed into primary antibodies (which are respectively METTL3 (abl95352), METTL14 (ab220030), WTAP (abl95380), KIAA1429 (25712-1-AP), FTO (abl24892), ALKBH5 (abl95377), YTHDCl (ab220159), YTHDF1 (abl7479-1-AP), YTHDF2 (ab24744-1-AP), the internal reference being p-actin (ab8226), wherein all of them are provided by Abcam Company of UK) diluted in a proportion of 1:1000, and then was slowly and gently shaken by a shaker at room temperature for 2 h or over night so as to be incubated;

3) the PVDF membrane was washed three times with TBST, 10 min for each time;

4) corresponding secondary antibody was selected according to the primary antibody, wherein the secondary antibody used was anti-rabbit IgG (ab6721, from Abcam Company of UK), or anti-mouse IgG (ab6789) from this company, and the secondary antibody was diluted in a proportion of 1:5000, and was slowly and gently shaken at room temperature for 1 h by a shaker;

5) after the incubation of the secondary antibody, the PVDF membrane was rinsed with TBST for three time, 10 min for each time;

6) the PVDF membrane was placed on a developing plate, Solution A and Solution B mixed with the same amount of ECL developing solution were evenly dripped onto the surface of the PVDF membrane for automatic exposure, and the

pictures were taken and saved; and

7) Image-Pro Plus 6.0 software was used for semi-quantitative analysis of protein bands.

(5) Detection of overall m6A RNA methylation level (the kit was purchased from the EpiGentek Company of America)

5.1 Preparation of buffer solution and solutions

(1) Preparation of 1xwashing buffer (WB): 13 mL of WB (Oxwashing buffer) was added to 117 mL of distilled water, the final pH value being 7.2-7.5;

(2) dilution of capture antibody solution (CA): the capture antibody solution was diluted with 1xwashing buffer in a volume ratio of 1:1000;

(3) dilution of detection antibody solution (DA): the detection antibody was diluted with 1xwashing buffer in a proportion of 1:2000;

(4) dilution of enhancer solution (ES): the enhancer solution was diluted with 1xwashing buffer in a proportion of 1:5000; and

(5) building of standard curve: PC was diluted with 1xTE to 0.5 ng/tL (1 tL of PC+3 tL of TE), and positive control (PC) with the following 6 different concentrations were further prepared, i.e. 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 ng/tL.

5.2 RNA extraction and determination

5.2.1 RNA extraction

1) Cells (the replicative senescence cells and the premature senescence cells in step (2)) were scraped from a cell culture flask of 25cm 2 by pre-cooled 1xPBS, and were centrifuged for 5 min at a speed of 2000 rpm at 4°C, and the supernatant was discarded;

2) 2 mL of trizol was added, and was blown and beaten till evenly mixed, and then incubation was performed for 5 min;

3) 0.2 mL of chloroform was added, and the EP tube was shaken up and down for 15 s;

4) centrifugation was performed for 15 min at 4°C, 12000xg;

5) 400 tL of supernatant was taken and moved to a new EP tube;

6) 400 tL of isopropanol was added;

7) incubation was performed for 10 min at room temperature;

8) centrifugation was performed for 10 min at 4°C, 12000xg, and the supernatant was discarded;

9) 1 mL of 75% (v/v) ethanol was added;

10) vortext was performed for 10 s at 4°C, 7500xg, centrifugation was performed for 5 min, the supernatant was discarded, and natural drying was performed for 5~10 min;

11) 20 tL of DEPC water was added and evenly mixed, and RNA concentration and purity were detected by means of a UV spectrophotometer; and

12) RNA samples of 4 tL were sub-packaged for experiments of integrity determination, and remaining samples were preserved in a refrigerator of -20°C.

5.2.2 RNA integrity determination

1) Preparation of 1.5% agarose gel: 0.6 g of agarose powder, 8 mL of 5xTBE buffer and 32 mL of pure water were added to a conical flask of 100 mL;

2) a plastic film glove was used for sealing the opening of the conical flask, and the sealed conical flask was heated in a microwave oven till the solution was clear and transparent;

3) the conical flask was taken out, and was added with 2 tL of Goldview when the temperature of the conical flask was about 37°C, and then the conical flask was gently shaken and placed in a gel-making plate, awaiting for solidification;

4) the gel was placed into a horizontal electrophoresis tank filled with 300 mL of 1xTBE electrophoresis solution, the gel comb was taken out carefully, and spotting was performed (4 tL of RNA sample+1 tL of 5xRNA Loading buffer);

5) electrophoresis: 120V, 30min; and

6) RNA bands were observed in an automatic digital gel image analysis system.

5.3 RNA binding

(1) A micro-well plate with needed number of wells was taken out according to the requirement of the experiment, and its arrangement and sampling order is as follows;

Table 4 Sampling order of micro-well plate

Well# Connecting Connecting Connecting Connecting Connecting tube 1 tube 2 tube 3 tube 4 tube 5

(ng/well) (ng/well)

A PC 0.02 PC 0.02 NC 35PDL PSi

B PC 0.04 PC 0.04 NC 35PDL PSi

C PC 0.10 PC 0.10 NC 35PDL PSi

D PC 0.20 PC 0.20 22PDL 49PDL PSp

E PC 0.40 PC 0.40 22PDL 49PDL PSp

F PC 1.00 PC 1.00 22PDL 49PDL PSp

(2) 80 tL of BS (binding solution) was added to each well;

(3) 2 tL of NC, 2 tL of diluted PC, and 200 ng of RNA (1-8 pL) extracted in step 5.2 of sample RNA were added to designated wells indicated in the above Table 4, the micro-well plate was slightly tilted or was shaken several times so as to mix the solution, thereby ensuring that the solution was evenly applied to the bottom of the well;

(4) the micro-well plate was sealed with a sealing film, and incubation was performed at 37°C for 90 min; and

(5) BS (binding solution) was sucked away from each well, and each well was washed by using 150 tL of diluted WB; diluted WB was sucked into each well, and then was removed by a pipette. The washing was repeated for another two times, and the wells were washed altogether for three times.

5.4 Capture of m6A RNA

(1) 50 tL of diluted CA was added to each well, and then the micro-well plate was covered with a lid and incubation was performed at room temperature for 60 min; CA solution was discarded after the incubation;

(2) each well was washed by using 150 tL of diluted WB each time, and altogether was washed for three times, and the washing solution was discarded;

(3) 50 tL of diluted DA was added to each well, and then the micro-well plate was sealed with a sealing film and incubation was performed at room temperature for min; the diluted DA solution was sucked out;

(4) each well was washed by using 150 tL of diluted WB each time, and altogether was washed for four times, and the washing solution was discarded;

(5) 50 tL of diluted ES was added to each well, and then the micro-well plate was sealed with a sealing film and incubation was performed at room temperature for min; the diluted ES solution was removed; and

(6) each well was washed by using 150 tL of diluted WB each time, and altogether was washed for five times, and the washing solution was discarded.

5.5 Signal detection

(1) 100 tL of DS was added to each well, and incubation was performed in darkness and at room temperature for 1-10 min. Close attention was paid to the color changes in the sample wells and those in the control wells. When there was enough m6A, DS solution would turn to blue;

(2) when the color of the positive control well turned to medium blue, 100 tL of SS was added to each well so as to prevent enzymatic reaction; and

(3) the color turned to yellow after SS was added, and the absorbance values of each microplate were read at 450 nm wavelength with a microplate reader within 2~15 min.

5.6 Calculation of m 6 A content of each group

The absolute amount of m6A was qualified by using precise calculations.

A standard curve was generated, and OD values were plotted. Equation of linear regression was used for calculating the slope (OD/ng) of the standard curve, and a standard curve (comprising at least four positive control points) with the best fitting degree was selected for calculating the optimal slope. The calculation formulae of the content and percentage of m 6 A in total RNA are as follows:

m6A(ng) =Sample OD-NC OD/Slope

m6A%=mA(ng)x 6 100%/S (Note: S is the amount of the input sample RNA with ng as a unit.)

5.7 Statistical analysis

All experimental data were analyzed after relative quantification, and were expressed in the form of "average value + standard deviation". Excel 2016 and GraphPad Prism 7 software were used for plotting. SPSS 20.0 statistical software was used for the statistical analysis of the experimental results, one-way analysis of variance and Dunnett T-test were used for comparing differences between groups, and two-sided test was adopted in all cases, wherein the test level a=0.05, P<0.05, and the difference has statistical significance.

Results

(1) Determination of cell senescence occurrence via cell morphological observation and p-galactosidase staining

As shown in Fig. 1A, the biological characters of cells in the replicative senescence cell 49PDL and that in 400 tmol/L H20 2-induced cell premature senescence persistence group PSp were apparently different, wherein their volume increased, the cells became flat and vacuolated, the nucleuses became bigger, particulate matters increased and the cell spaces were wider. The positive rate of cells stained blue showed a rising trend following the aging, wherein the young cell group 22PDL was 0.0%, 35PDL was 5.2%, while the replicative senescence cell group 49PDL increased to 91.9% (P<0.05), and the cells in 49PDL group showed blue-green variations after P-galactosidase staining; a ratio of cell stained blue in the H202-induced cell premature senescence initiation group PSi was 66.5%, while the cell premature senescence persistence group PSp reached as high as 91.3%, both of which were higher than 22PDL, the difference has statistical significance (P<0.05); there was no obvious difference between the ratio of cells stained blue in PSp and that of cells stained blue in 49PDL (P>0.05), and the cell replicative senescence and the cell premature senescence had similar morphological changes. Please see Fig. 1B for details.

(2) Overall m6A RNA methylation changes in H2 0 2-induced cell premature senescence

As for the changes of m6 A content, as shown in Fig. 2A, in the replicative senescence cells 22PDL, 35PDL, 49PDL and H202-induced premature senescence cells in PSi and PSp, the m6 A contents in RNA of each of the groups respectively takes 0.12%, 0.07%, 0.05%, 0.1% and 0.06% of the total RNA amount. As shown in Fig. 2B, in the process of replicative senescence, compared with 22PDL, the m 6 A content of 35PDL and that of 49PDL show a downward trend, which respectively decrease by 35.1% (P<0.05) and 52.3% (P<0.05); compared with 22PDL, the m 6 A content of PSp of the premature senescence constant group in H202-induced cell premature senescence decreased by 41.1%, wherein the difference has statistical significance (P<0.05); compared with 49PDL, the mA content change of PSp is not obvious, and the difference has no statistical significance (P>0.05), showing that the premature senescence persistence group reached the cell replicative senescence level of 49PDL, and the m 6 A RNA methylation level can serve as an assessment index for cell senescence.

(3) Protein expressions of m6A RNA methylation enzymes in H20 2 -induced cell premature senescence

In cell replicative senescence, compared with the young cell group 22PDL, protein expressions of RNA methylation enzymes METTL3, METTL14, WTAP and KIAA1429 in the cell replicative senescence group 49PDL respectively decreased by 36.2% (P<0.05), 84.4% (P<0.05), 28.3% (P>0.05) and 79.0% (P<0.05); in H202 induced cell premature senescence, compared with 22PDL, the protein expression of METTL3 increased by 1.6 times in the cell premature senescence persistence group PSp (P<0.05), while the protein expressions of METTL14, WTAP and KIAA1429 respectively decreased by 66.8%, 72.5% and 35.5% (P<0.05). Compared with 49PDL, in the H202-induced premature senescence cell group PSp, the protein expressions of METTL3, METTL14 and KIAA1429 respectively increased by 2.5, 2.1 and 3.1 times, while the protein expression of WTAP decreased by 61.6%, and the differences all have statistical significance (P<0.05), please refer to Fig. 3 for details. As a result, compared with young cells, for both replicative senescence and cell premature senescence, protein expressions of METTL14, WTAP and KIAA1429 in all senescent cells significantly decreased, while the decreasing extents in these two kinds of senescence are different; as for METTL3, its expression decreased in replicative senescence cells, but significantly increased in oxidative stress-induced premature senescence cells.

(4) Protein expressions of m6A RNA demethylation enzymes in H 2 0 2 induced cell premature senescence

As shown in Fig. 4, during the process of replicative senescence, compared with 22PDL, the protein expression of RNA demethylation enzyme FTO respectively decreased by 63.7% and 82.7% in 35PDL and 49PDL, and the protein expression of ALKBH5 respectively decreased by 49.5% and 66.8% in 35PDL and 49PDL, and all of them have statistical difference (P<0.05); in H2 02-induced cell premature senescence, compared with 22PDL, the protein expression of FTO respectively decreased by 67.2% and 59.2% in PSi and PSp, the differences all have statistical significance (P<0.05), and the protein expression of ALKBH5 respectively decreased by 20.4% (P>0.05) and 91.5% (P<0.05) in PSi and PSp. Compared with 49PDL, the protein expression of FTO increased by 2.4 times in PSp, while the protein expression of ALKBH5 decreased by 74.4%, and both cases have statistical differences (P<0.05). All senescent cells have RNA demethylation enzymes with comparatively low expression levels.

(5) Expression levels of m6A RNA methylation binding proteins in H 2 0 2 induced cell premature senescence

As shown in Fig. 5, during the process of replicative senescence, compared with 6 22PDL, the m A RNA methylation binding protein YTHDCl respectively decreased by 18.6% (P>0.05) and 52.0% (P<0.05) in 35PDL and 49PDL, and YTHDF1 respectively decreased by 39.9% (P<0.05) and 62.9% (P<0.05) in 35PDL and 49PDL, and YTHDF2 respectively decreased by 12.1% (P>0.05) and 58.5% (P<0.05) in 35PDL and 49PDL; during the process of H202-induced cell premature senescence, compared with 22PDL, YTHDC1 respectively decreased by 66.3% and 66.2% in PSi and PSp, YTHDF1 respectively decreased by 55.2% and 90.1% in PSi and PSp, and all of the differences have statistical significance (P<0.05), and YTHDF2 respectively decreased by 29.5% (P>0.05) and 91.3% (P<0.05) in Psi and PSp. Compared with 49PDL, the protein expression of YTHDF1 and that of YTHDF2 in PSp respectively decreased by 73.2% and 78.9%, and the differences both have statistical significance (P<0.05), while there was no obvious change (P>0.05) in the protein expression of YTHDC1 between 49PDL and PSp. Expression characters of cell senescence are shown, and protein expressions of YTHDC1, YTHDF1 and YTHDF2 all significantly decreased relative to young cells. There are one or at least two low expressions ofm6 A RNA methylation binding proteins that can be used for assessing occurrence of cell senescence.

6 To sum up, the m A content decreases in the cell replicative senescence and the 6 H202-induced premature senescence, while there is no obvious difference in m A content between these two kinds of senescence; expression profiles of RNA methylation enzymes are different between the cell replicative senescence and the

H20 2-induced premature senescence; compared with normal young cells, METTL3 has high expression in cells of the H 20 2-induced premature senescence, and has low expression in cells of the replicative senescence; METTL14, WTAP and KIAA1429, as well as FTO and ALKBH5 all have low expression in both cells of H2 0 2 -induced premature senescence and cells of replicative senescence; and m 6 A RNA methylation binding proteins YTHDC1, YTHDF1 and YTHDF2 have low expression in cells of H2 0 2-induced premature senescence and cells of replicative senescence. METTL3 can serve as a specific indicator for distinguishing normal replicative senescence and oxidative stress-induced premature senescence of cells.

The above embodiments are preferable ones of the present invention; however, the embodiments of the present invention are not limited thereto, and any change, modification, replacement, combination and simplification without deviating from the principle substance and principles of the present invention should all be regarded as equivalent substitute modes, and should all be contained within the scope of protection of the present invention.

Claims (10)

1. CLAIMS 1. Application of m 6 A RNA methylation content, and methylation-related enzymes and binding proteins thereof in the preparation of a senescence detection kit.
2. 2. The application according to claim 1, wherein, 6 the senescence detection kit detects an m A RNA methylation content, and protein expressions of RNA methylation-related enzymes and binding proteins, and regards them as markers for determining cell senescence;

   the senescence includes replicative senescence and oxidative stress-induced premature senescence; the cells gradually senescent with age; and

   the RNA methylation-related enzymes include RNA methylation enzymes and RNA demethylation enzymes.
3. 3. The application according to claim 2, wherein the cells are human embryonic lung fibroblasts;

   the RNA methylation enzymes are METTL3, METTL14, WTAP and KIAA1429;

   the RNA demethylation enzymes are FTO and ALKBH5; and

   the RNA methylation binding proteins are YTHDC1, YTHDF1 and YTHDF2.
4. 4. The application according to claim 3, wherein, when a decrease of the m A RNA methylation content in the cell, and/or one or at least two expressions of the 6 6 mA RNA methylation enzymes METTL14, WTAP and KIAA1429, the m A RNA demethylation enzymes FTO and ALKBH5, and the m A RNA methylation binding proteins YTHDC1, YTHDF1 and YTHDF2, and/or a high expression or low expression of the RNA methylation enzyme METTL3 are detected, it shows that the cell enters into an senescence state.
5. 5. The application according to claim 4, wherein the detection of a low expression of the RNA methylation enzyme METTL3 in the cell shows the cell replicative senescence; the detection of a high expression of the RNA methylation enzyme METTL3 in the cell shows the cell premature senescence.
6. 6. The application according to claim 5, wherein the protein expression is counted by using a relative quantitative analysis method, gray values of different cell bands are obtained by means of gray analysis of protein bands, a 22PDL young cell group, which is provided as 1, is taken as a comparison, and other cell expressions are compared with a gray value of the 22PDL young cell group; protein and internal reference expressions are obtained by means of Western blot detection; and

   the 22PDL young cell group is defined as cells of which the subculture cells have a final population doubling level (PDL) of 22, wherein the calculation formula of PDL is n=3.32(logN2-logNl)+X, N2 being a total number of the cells obtained in this generation, NI being the number of cells inoculated in the previous generation, and X being the PDL of cells of previous generation.
7. 7. A senescence detection kit, comprising: a quantitative detection reagent for m6A RNA methylation in cell, and/or a protein expression detection reagent for detecting at least one of the methylation enzymes METTL3, METTL14, WTAP and KIAA1429, the demethylation enzymes FTO and ALKBH5, and m A RNA methylation binding proteins.
8. 8. The senescence detection kit according to claim 7, wherein the protein expression detection reagent is a Western blot semi-quantitative detection reagent.
9. 9. The application of the senescence detection kit according to claim 7 or 8 to non-therapeutic diagnosis and detection of senescence.
10. 10. The application according to claim 9, wherein the application including a 6 step of detecting m A RNA methylation content by using the senescence detection kit after taking cells to be detected and extracting overall RNA, and/or a step of performing protein semi-quantification by using the senescence detection kit after extracting overall protein of cells to be detected.