CN114317775B - Application of RNA m6A modification of NCOA4 as gamma-ray radiation marker - Google Patents
Application of RNA m6A modification of NCOA4 as gamma-ray radiation marker Download PDFInfo
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Abstract
The invention discloses an application of an RNA m6A modification level or a relative modification level of NCOA4 in preparing a product for detecting or assisting in detecting whether gamma-ray radiation is suffered or not. The invention adopts the RNA m6A modification of NCOA4 as the gamma-ray radiation marker, and can detect or assist in detecting whether an object to be detected is irradiated by gamma rays more accurately, rapidly and sensitively.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an application of RNA m6A modification of NCOA4 as a gamma-ray radiation marker.
Background
The accurate and long-term diagnosis of the radiation wounded in nuclear radiation accidents and nuclear disasters is an important basis for accident rescue and medical treatment. Currently, biological methods for detecting whether gamma radiation is encountered include clinical indications, peripheral blood lymphocyte counts, and cytogenetic techniques. The expression quantity of the protein gamma-H2 AX is a radiation marker with high specificity, is a commonly used biological dosimeter, and plays a role of gold standard in detection of various radiation samples.
However, since double strand breaks can be repaired quickly, the gamma-H2 AX expression gradually disappears with time, and the method cannot be applied to dose estimation in the later irradiation stage. Thus, there is a need for a biomarker that stabilizes for long aging.
Disclosure of Invention
The invention aims to provide an application of RNA m6A modification of NCOA4 as a gamma-ray radiation marker, which can detect or assist in detecting whether an object to be detected is irradiated by gamma rays more accurately, rapidly and sensitively.
To achieve the above object, the present invention provides an application of the RNA m6A modification level or the relative modification level of NCOA4 in preparing a product for detecting or assisting in detecting whether or not the product is subjected to gamma radiation.
Use of a system for the detection or assisted detection of whether or not gamma radiation is suffered from RNA m6A modification of NCOA4 as a marker for the preparation of a product for the detection or assisted detection of whether or not gamma radiation is suffered from.
A system for detecting the RNA m6A modification level or relative modification level of NCOA4, which is a system for detecting the RNA m6A modification level or relative modification level of NCOA4 using RNA m6A chip technology or a system for detecting the RNA m6A modification level or relative modification level of NCOA4 using MeRIP-qPCR technology.
Preferably, the system for detecting the level of RNA m6A modification or the relative level of modification of said NCOA4 comprises primers and/or reagents and/or kits and/or instruments.
Preferably, the system for detecting the level of modification or relative level of modification of the RNA m6A using MeRIP-qPCR comprises primers, kits and/or other reagents and/or instrumentation required for performing the MeRIP-qPCR.
Preferably, the system for detecting RNA m6A modification level or relative modification level further comprises a data processing system for converting the RNA m6A modification level or relative modification level from a test subject into a detection result of the test subject.
The data processing system comprises a data input module, a data comparison module and a conclusion output module; the data input module is used for inputting the numerical value of the RNA m6A modification level or the relative modification level of the NCOA4 of the object to be detected; the data comparison module is used for comparing the RNA m6A modification level or the relative modification level of the NCOA4 of the object to be detected with the object not subjected to gamma ray radiation; the conclusion output module is used for outputting a conclusion: if the RNA m6A modification level or the relative modification level of NCOA4 of the object to be detected is greater than the RNA m6A modification level or the relative modification level of NCOA4 of the object not subjected to gamma radiation, the conclusion is that the object to be detected is subjected to gamma radiation; if the level of RNA m6A modification or the relative modification of NCOA4 in the subject is less than or equal to the level of RNA m6A modification or the relative modification of NCOA4 in the subject not subjected to gamma radiation, then the conclusion is that the subject not is subjected to gamma radiation.
Preferably, the level of RNA m6A modification or relative modification of the NCOA4 gene is the level of RNA m6A modification or relative modification of the NCOA4 gene in blood or cells.
Preferably, the RNA m6A modification level or relative modification level of the NCOA4 gene is that of Ncoa4/NCOA4 gene in peripheral blood mononuclear cells isolated from peripheral blood of mice or in human HeLa cells.
Preferably, the primer for amplifying Ncoa4 of the mouse peripheral blood mononuclear cells consists of 2 pairs of primers and comprises a primer pair 1 consisting of two single-stranded DNAs shown in Seq ID No.1 and Seq ID No.2, and a primer pair 2 consisting of two single-stranded DNAs shown in Seq ID No.3 and Seq ID No. 4.
Preferably, the primer for amplifying NCOA4 of Hela cells consists of 2 pairs of primers and comprises the primer pair 1 consisting of two single-stranded DNAs shown in Seq ID No.5 and Seq ID No.6, and the primer pair 2 consisting of two single-stranded DNAs shown in Seq ID No.7 and Seq ID No. 8.
Preferably, the level of RNA m6A modification or relative level of modification of Ncoa4 is the percentage of m6A modification of RNA m6A modification of Ncoa4/Ncoa4 relative to the amount of RNA (Input) after enrichment of the m6A antibody with (m 6A-IP).
Preferably, the exposure to gamma radiation is to cobalt 60-gamma radiation.
The invention provides a characteristic of variation of RNA m6A modification level or relative modification level of Ncoa4 along with gamma ray irradiation dose and a characteristic of variation of RNA m6A modification level or relative modification level of Ncoa4 along with time under the same irradiation dose, wherein the characteristic is used for evaluating the dose of gamma ray irradiation or the time after the irradiation of a subject to be tested.
The RNA m6A modification level or relative modification level of Ncoa4 tended to rise and then fall within 1, 3, 7, 14, 28 days, reaching a maximum on day 14 after irradiation, compared to the normal group not subjected to gamma radiation; compared to the normal group not subjected to gamma radiation, the RNA m6A modification level or relative modification level of Ncoa4 was significantly increased within 7 days after the irradiation when the radiation dose was not less than 0.5 Gy; the RNA m6A modification level or relative modification level of Ncoa4 was significantly increased within 1-28 days when the radiation dose was ≡2Gy compared to the normal group not subjected to gamma radiation.
Therefore, the application of the RNA m6A modification of NCOA4 as the gamma-ray radiation marker can detect whether an object to be detected is subjected to gamma-ray radiation and evaluation of radiation dosage, is beneficial to timely carrying out accident rescue and wounded treatment, and has important significance for radiation treatment.
The technical scheme of the invention is further described in detail through the drawings and the embodiments.
Drawings
FIG. 1 is a graph of the results of MeRIP-qPCR experiments on peripheral blood mononuclear cells from different batches of treated mice to detect the RNA m6A modification level or relative modification level of Ncoa 4;
FIG. 2 is a graph showing the results of MeRIP-qPCR experiments on different batches of treated Hela cells to detect the RNA m6A modification level or relative modification level of NCOA 4.
Detailed Description
The technical scheme of the invention is further described below through the attached drawings and the embodiments.
Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below, examples of which are shown in the accompanying drawings, are commercially available unless otherwise specified. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
The sources of materials, reagents and test techniques used in the examples below were as follows:
the RNA m6A-Seq chip of the mouse is provided by Shanghai Kangsheng biological information technology Co.
M6A antibody [ m6A (D9W) Rabbit mAb, cat No.: 56593]IgG (Normal rabbit IgG, cat# 2729 s) is a product of CST company in the United states; the required Protein A/G PLUS-Agarose is a product of Santa Cruz, inc. of America, under the trade designation: sc-2003; fluorescent quantitative detection Kit (KAPA)
FAST qRT-PCR kit) is a product of KAPA Biosystems, usa, cat: KK4601.
Example 1
RNA m6A modification level of Ncoa4 Gene after irradiation
Male 6-8 week old C57BL/6N mice are from Beijing Vittolihua laboratory animal technology Co., ltd, after 6.5Gy cobalt 60-gamma radiation is given to C57BL/6N mice with good health status, RNA is extracted from peripheral blood mononuclear cells obtained by peripheral blood separation at different time points (0 d, 1d, 3d, 7d, 14d and 28 d), and RNA m6A chip experiments are carried out on 6 groups of total RNA samples. RNA m6A chip experiments were performed using m6A-mRNA & lncRNA chips.
And (3) carrying out quality detection on total RNA of peripheral blood mononuclear cells before sequencing an RNA m6A chip, finding that the RNA has good quality after detection, and randomly breaking the RNA into fragments of about 100 nt. Incubating the RNA m6A modified specific antibody with the RNA fragment, grabbing the fragment with methylation modification, and sequencing; at the same time, a control (Input) sample containing only the disrupted RNA fragment was sequenced in parallel, and no RNA methylation-specific antibody was added to incubate with it.
The m6A chip is scanned by a scanner, data signals are extracted, normalization treatment and m6A signal difference peak analysis are carried out, the change of m6A modification of RNA before and after irradiation is compared, the result shows that RNA up-regulated by m6A modification at different time points after irradiation is 110 on the first day, 1487 on the third day, 74 on the seventh day, 183 on the fourteenth day and 45 on the twenty eighth day respectively, RNA down-regulated by m6A modification is 1843 on the first day, 244 on the third day, 865 on the seventh day, 3819 on the fourteenth day and 317 on the twenty eighth day respectively, RNA m6A modification (shown in table 1) of Ncoa4 gene with obvious up-regulation at 5 time points after irradiation is selected by comparison, namely, the RNA m6A of Ncoa4 is modified to be used as a marker molecule for gamma radiation detection, and the mRNA serial number of Ncoa 4: ENSMUST00000169722, can detect whether the test subject is exposed to gamma radiation by the RNA m6A modification level or relative modification level of the Ncoa4 gene.
TABLE 1 RNA m6A chip analysis results of the Ncoa4 Gene after irradiation of mouse peripheral blood mononuclear cells
Example two
Verification of RNA m6A modification level of Ncoa4 Gene before and after irradiation Using RNA m6A chip
Male 6-8 week old C57BL/6N mice are from Beijing vitamin Toril laboratory animal technology Co., ltd, C57BL/6N mice with good health status are irradiated (control group) or are irradiated with 2Gy cobalt 60-gamma rays at different time points (1 d, 14 d), peripheral blood mononuclear cells obtained by peripheral blood separation are taken out, RNA is extracted, and RNA m6A chip experiments are carried out on 3 groups of total RNA samples. RNA m6A chip experiments were performed using m6A-mRNA & lncRNA chips.
And (3) carrying out quality detection on total RNA of peripheral blood mononuclear cells before sequencing an RNA m6A chip, finding that the RNA has good quality after detection, and randomly breaking the RNA into fragments of about 100 nt. Incubating the RNA m6A modified specific antibody with the RNA fragment, grabbing the fragment with methylation modification, and sequencing; at the same time, a control (Input) sample containing only the disrupted RNA fragment was sequenced in parallel, and no RNA methylation-specific antibody was added to incubate with it. The m6A chip is scanned by a scanner, data signals are extracted, normalization processing and m6A signal difference peak analysis are carried out, the change of m6A modification of RNA before and after irradiation is compared, and the result shows that the RNA which is up-regulated in m6A modification at different time points after irradiation is 88 on the first day and 248 on the fourteenth day respectively; the m6A modified down-regulated RNA was 43 on the first day and 550 on the fourteenth day, respectively. The comparison screening of RNA m6A modification of the Ncoa4 gene, which was more pronounced at all 2 time points after irradiation (as shown in table 2), demonstrated that the RNA m6A modification level or relative modification level of the Ncoa4 gene in peripheral blood mononuclear cells was significantly increased in mice exposed to gamma radiation relative to mice not exposed to gamma radiation.
TABLE 2 RNA m6A chip analysis results of the Ncoa4 Gene after irradiation of mouse peripheral blood mononuclear cells
Example III
Verification of RNA m6A modification level of the Ncoa4 Gene in peripheral blood mononuclear cells of mice Using MeRIP-qPCR experiments
1. Mice were irradiated with cobalt 60-gamma rays and grouped
Male 6-8 week old C57BL/6N mice were derived from Peking Vitrendylar laboratory animal technologies, inc., and C57BL/6N mice with good health status were irradiated with cobalt 60-gamma rays. The irradiation condition was room temperature, the irradiation distance was 3m, and the dose rate was 69.1cGy/min. Mice were grouped according to dose and time to isolate peripheral blood mononuclear cells after irradiation, 10 per group, and specific groupings are shown in table 3.
TABLE 3 irradiation dose and mouse grouping
2. Isolation of peripheral blood mononuclear cells
The eyeorbit of the mouse is subjected to blood taking, and peripheral blood mononuclear cells are separated by adopting a mouse peripheral blood lymphocyte separation liquid KIT (brand: TBD; product number: LTS 1092-KIT). The method comprises the following specific steps:
1) 0.5mL of anticoagulation (blood is placed for a long time and is easy to separate, premixing is needed), and 0.5mL of diluent is added and mixed uniformly. 3mL of the separation liquid is taken and added into a 15mL centrifuge tube, and after the centrifuge tube is inclined at an angle of 45 degrees, diluted peripheral blood is taken and slowly added into the centrifuge tube containing the separation liquid along the tube wall.
2) The tube was placed in a centrifuge and centrifuged at 500g for 20min. Blood cells were separated into 4 layers in a centrifuge tube, white lymphocyte layers (upper layer not aspirated, lower layer not aspirated) were carefully aspirated, transferred into a fresh centrifuge tube, added with wash solution or PBS to 10mL, and mixed well with a dropper.
3) Centrifugation at 1100rpm for 15min, discarding supernatant, resuspension with 1mL of wash or PBS, cell counting was performed by taking 20. Mu.L, the remaining cells were added to a fresh 1.5mL EP tube, centrifugation at 1100rpm for 15min, discarding supernatant, resuspension with 1mL of Trizol.
3. RNA extraction of peripheral blood mononuclear cells
The phenol chloroform method for extracting RNA comprises the following specific steps:
1) 200. Mu.L of chloroform was added to Trizol resuspended peripheral blood mononuclear cells, vigorously shaken for 15s, and allowed to stand for 5min until the liquid was separated.
2) Centrifuge at 12000g for 15min at 4 ℃.
3) Sucking supernatant (avoiding sucking other layer liquid) into new EP tube, adding equal volume of isopropanol, shaking, mixing, and incubating at-20deg.C for 1 hr.
4) Centrifuge at 14000rpm for 30min at 4℃and discard the supernatant.
5) 1mL of pre-chilled 75% alcohol was added, vortexed, centrifuged at 14000rpm at 4℃for 3min, and the supernatant was discarded.
6) Repeating the above steps.
7) The EP tube was back-buckled on absorbent paper, dried at room temperature for 10min, and added with 50 mu LRNase free water to resuspend the precipitate.
8) Total RNA concentration was determined using an ultra-micro spectrophotometer Nano-300, purity was assessed at a ratio of A260nm/A280nm, and RNA was quantified.
4. Fragmentation of RNA:
1) The concentration of the above RNA was adjusted to 10 ng/. Mu.L with RNase free water and the total amount of RNA was ensured to at least 2. Mu.g.
2) 200. Mu.L of the RNA sample was pipetted into a fresh EP tube, 2. Mu.L of RNase inhibitor was added, thermo Fisher Scientific LabServ was used TM The Model 120 ultrasonic crusher is used for ultrasonic crushing, and the ultrasonic crushing is set to be 20% of power, 1s of ultrasonic waves and 2s of interval, and the ultrasonic waves are carried out for 15 times.
3) Sucking 20 mu L of the RNA sample after ultrasonic treatment into a new EP tube, marking as Input, and storing at-80 ℃ for standby;
4) The remaining post-ultrasound RNA samples were placed on ice for IP experiments.
5. Merp experiments:
1) Preparing the following reagents:
IP reaction solution: 50mmol/L Tris-HCl (pH=7.4), 150mmol/L NaCl, 0.1% NP-40;
1×ip buffer: 10mM Tris-HCl (pH=7.4), 150mM NaCl, 0.1% NP-40;
1 x wash buffer: 10mM Tris-HCl (pH=7.4), 50mM NaCl, 0.1% NP-40;
elution buffer: 10mM Tris-HCl (pH=7.4), 1mM EDTA, 0.05% SDS.
(Note: the above solutions were prepared with RNase free water)
2) beads were incubated with antibody:
the experiment is divided into an m6A antibody IP group and an IgG IP group for comparison, a required 1.5mLEP tube is marked, 20 mu L of Protein A/G PLUS-Agarose is respectively added, 500 mu L of IP reaction liquid is added, after shaking and mixing, the supernatant is washed after centrifugation at 5000rpm for 2min at 4 ℃,500 mu L of IP reaction liquid is added after washing for 2 times, 1 mu G of m6A antibody is added to the m6A antibody IP group, 1 mu G of IgG antibody is added to the IgG IP group, shaking table rotation is carried out at 4 ℃ for 4h, after incubation is completed, the supernatant is washed after centrifugation at 5000rpm for 2min, 500 mu L of IP reaction liquid is added, and the washing steps are repeated, so that antibody-coupled beads are obtained.
3) RNA immunoprecipitation:
adding 500 mu L of IP reaction solution into antibody-coupled beads, respectively adding 90 mu L of fragmented RNA into m6A antibody IP group and IgG IP group, rotating at 4 ℃ for incubation overnight, centrifuging at 5000rpm for 2min, discarding supernatant, adding 500 mu L of IP reaction solution to wash RNA which is not combined with the beads, shaking and mixing uniformly, centrifuging at 4 ℃ at 5000rpm for 2min, discarding supernatant, washing for 3 times, then adding 500 mu L of washing buffer to wash the beads, shaking and mixing uniformly, centrifuging at 4 ℃ at 5000rpm for 2min, discarding supernatant, and washing for 3 times.
4) Elution and purification of m6A modified RNA:
100. Mu.L of elution buffer was added to the above sample, 3. Mu.L of proteinase K was added thereto, and the mixture was incubated at 50℃for 30 minutes with rotation, and the eluted RNA was purified again by phenol chloroform and finally dissolved in 20. Mu. LRNase free water.
6. Reverse transcription of RNA from peripheral blood mononuclear cells to cDNA
The kit used for reverse transcription is a MonScriptTM RTIII AII-in-One Mix reverse transcription kit, and comprises the following specific steps:
1) Taking out the components from the kit, putting the components on ice for dissolving, uniformly mixing the dissolved components, and putting the components on ice for standby after short centrifugation.
2) Reverse transcription systems were formulated in 200. Mu.L of RNase-free inlet PCR tubes as shown in Table 4.
TABLE 4 reverse transcription system (20. Mu.L)
3) Mixing, and placing into a PCR instrument for reverse transcription reaction. The PCR procedure was set as follows: the first step is at 55 ℃ for 15min; the second step is 85 ℃ for 5min; and in the third step, the temperature is 4 ℃ for 10min.
4) The cDNA product after reverse transcription was removed and diluted by adding 100. Mu. L RNase free water.
7、qPCR
1) Specific primers were designed for potential sites of RNA m6A modification of mouse Ncoa4, the primer sequences are as shown in table 5:
TABLE 5 real-time fluorescent quantitative PCR primer sequences
2) The kit for real-time quantitative PCR is KAPA
The FAST qRT-PCR kit was loaded according to the system shown in table 6 and sub-loaded into 96 well plates, and three repeated reaction systems, i.e., three sets of parallel assays, were performed for each sample.
TABLE 6qPCR sample addition System
3) Centrifuging the 96-well plate at 3000rpm for 5min, putting the mixture into a qPCR instrument for PCR reaction, and setting a qPCR program as follows: the first step is 95 ℃ for 5min; the second step is 95 ℃ for 5s; third, 60 ℃ for 30s; the second step to the third step are repeated for 40 times; fourth step: dissolution profile; fifth step: preserving at 4 ℃.
4) Data were analyzed to calculate the m6A modification level or relative modification level of RNA according to the following formula and compare the differences in% Input between groups.
The calculation formula is as follows: Δct=ct (RIP) - [ Ct (Input) -log 2 (dilution times)]M6A modification level% input=2 of RNA -△Ct 。
The average data of three times after 3 MeRIP-qPCR experiments for m6A modification levels or relative modification levels of RNA of Ncoa4 are shown in fig. 1, wherein a is the determination of the required RNA amount for the MeRIP-qPCR experiments to detect the m6A modification levels or relative modification levels of RNA of Ncoa4, and when the required RNA for m6A antibody IP is not less than 2 μg, the irradiation group (6.5 Gy irradiation dose, 1 day after irradiation) compares the m6A modification levels or relative modification levels of RNA of Ncoa4 with the normal group (not subjected to gamma radiation), with p < 0.0001; primer 1 (346-480) of Ncoa4 detects changes in RNA m6A modification level or relative modification level of Ncoa4 at different radiation doses and time points, and at the same time point, the irradiation groups (0.2 Gy, 0.5Gy, 1Gy, 2Gy, 4Gy, 6.5Gy, 10Gy irradiation doses) respectively compare with the normal groups (not subjected to gamma radiation) for RNA m6A modification level or relative modification level of Ncoa4, p < 0.01, p < 0.001, p < 0.0001; primer 2 (658-847) with C as Ncoa4 detects changes in RNA m6A modification level or relative modification level of Ncoa4 at different radiation doses and time points, and at the same time point, the irradiation groups (0.2 Gy, 0.5Gy, 1Gy, 2Gy, 4Gy, 6.5Gy, 10Gy irradiation doses) respectively compare with the normal groups (not subjected to gamma radiation), wherein p < 0.1, p < 0.01, p < 0.001, p < 0.0001.
Both pairs of primers for detection of RNA m6A modification of Ncoa4 showed: RNA m6A modification of Ncoa4 increased significantly with increasing radiation dose, gradually over 1-14 days, and gradually over 14-28 days; when the radiation dose is more than or equal to 0.5Gy, the RNA m6A modification level or the relative m6A modification level of the Ncoa4 gene is obviously increased within 7 days after the radiation; when the radiation dose is more than or equal to 2Gy, the RNA m6A modification level or the relative m6A modification level of the Ncoa4 gene is obviously higher than that of a group which is not subjected to gamma rays in 1-28 days. RNA m6A modification of the Ncoa4 gene can respond to the stimulation of ionizing radiation in a short time (1 day), and is suitable for detecting the irradiation dose of more than or equal to 0.5 Gy; meanwhile, when the irradiation dose is higher, the RNA m6A modification of the target gene can be maintained at a high level for a longer time (28 days), and the method is suitable for dose evaluation for a longer time after irradiation.
Example IV
Verification of RNA m6A modification level of NCOA4 Gene in human Hela cells Using MeRIP-qPCR experiments
1. Cell culture
Hela cells are human cervical cancer cells, which are cultured in DMEM medium containing 10% fetal calf serum, and 5% CO is placed 2 Subculture in an incubator at 37 ℃.
2. Irradiation with cobalt 60-gamma rays
HeLa cells in good growth state were irradiated with cobalt 60-gamma rays. Wherein the irradiation condition of the cobalt 60-gamma ray irradiation is room temperature, the irradiation dose is 10Gy, the irradiation distance is 3 meters, and the dose rate is 69.1cGy/min. Control (non-irradiated) cells (labeled Ctrl) were harvested, followed by 10Gy of cells (labeled IR) 1 hour after irradiation.
3. RNA extraction of Hela cells
Extracting RNA by phenol chloroform method.
4. Fragmentation of RNA
5. MeRIP experiment
6. Reverse transcription of RNA from Hela cells to cDNA
The kit used for reverse transcription was MonScriptTM RTIII AII-in-One Mix reverse transcription kit, reverse transcription system, as shown in Table 4.
7、qPCR：
1) Specific primers were designed against the potential sites of RNA m6A modification of the human NCOA4 gene, and the primer sequences are shown in Table 7.
TABLE 7 real-time fluorescent quantitative PCR primer sequences
2) The kit for real-time quantitative PCR is KAPA
The FAST qRT-PCR kit was loaded according to the system shown in table 6 and sub-loaded into 96 well plates, and three repeated reaction systems, i.e., three sets of parallel assays, were performed for each sample.
3) Centrifuging the 96-well plate at 3000rpm for 5min, putting the mixture into a qPCR instrument for PCR reaction, and setting a qPCR program as follows: the first step is 95 ℃ for 5min; the second step is 95 ℃ for 5s; third, 60 ℃ for 30s; the second step to the third step are repeated for 40 times; fourth step: dissolution profile; fifth step: preserving at 4 ℃.
4) Data were analyzed to calculate the m6A modification level or relative modification level of RNA according to the following formula and compare the differences in% Input between groups.
The calculation formula is as follows: Δct=ct (RIP) - [ Ct (Input) -log 2 (dilution times)]M6A modification level% input=2 of RNA -△Ct 。
The average data of three times after 3 MeRIP-qPCR experiments for the RNA m6A modification level or relative modification level of NCOA4 is shown in fig. 2, wherein primer 1 (219-319) for a NCOA4 detects the RNA m6A modification level or relative modification level of NCOA4, and the irradiated group (10 Gy irradiation dose, 1 hour after irradiation) compares the RNA m6A modification level or relative modification level of NCOA4 with the normal group (not subjected to gamma radiation), p < 0.001; primer 2 (796-897) of NCOA4 detects the RNA m6A modification level or relative modification level of NCOA4, and the irradiation group (10 Gy irradiation dose, 1 hour after irradiation) compares the RNA m6A modification level or relative modification level of NCOA4 with that of the normal group (not subjected to gamma-ray irradiation), p < 0.001.
The significant difference in the m6A modification level or relative modification level of NCOA4 RNA compared to the non-irradiated (0 Gy) dose of 10Gy indicates that the detection of the m6A modification level or relative modification level of NCOA4 RNA can detect whether the subject is exposed to gamma radiation.
According to the invention, RNA m6A-seq is carried out on peripheral blood mononuclear cells of mice treated by gamma-ray radiation, genes with significant differences in RNA m6A modification after radiation are analyzed through various analysis software and algorithms, the RNA m6A modification level or relative modification level of Ncoa4 is found to be significantly increased at a plurality of time points after radiation, meRIP-qPCR is further utilized in a sample queue of peripheral blood mononuclear cells of independent mice, the change rule of RNA m6A modification of Ncoa4 after gamma-ray radiation along with radiation dose and time after radiation is found, meRIP-qPCR is further utilized in human Hela cells, and compared with a group which does not suffer gamma-ray radiation, the RNA m6A modification level or relative modification level of NCOA4 after gamma-ray radiation is found to be significantly increased.
Furthermore, the change characteristics of the RNA m6A modification after gamma-ray irradiation treatment are clarified, and the RNA m6A modification level of the gene is found to be significantly increased at a plurality of time points Ncoa4 after gamma-ray irradiation treatment, and the RNA m6A modification level of the gene is used for detecting gamma-ray irradiation.
Therefore, the application of the RNA m6A modification of NCOA4 as the gamma-ray radiation marker can detect whether an object to be detected is subjected to gamma-ray radiation and evaluation of radiation dosage, is beneficial to timely carrying out accident rescue and wounded treatment, and has important significance for radiation treatment.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.
Claims (9)
1. Use of a system for detecting or aiding in the detection of whether or not gamma radiation is suffered from RNAm6A modification level or relative modification level of NCOA4 as a marker in the manufacture of a product for detecting or aiding in the detection of whether or not gamma radiation is suffered from.
2. A system for detecting RNAm6A modification level or relative modification level of NCOA4, characterized by: the system for detecting the RNAm6A modification level or the relative modification level of NCOA4 is a system for detecting the RNAm6A modification level or the relative modification level of NCOA4 by using an RNA m6A chip technology or a system for detecting the RNAm6A modification level or the relative modification level of NCOA4 by using a MeRIP-qPCR technology.
3. A system for detecting RNAm6A modification level or relative modification level of NCOA4 according to claim 2, wherein: the system for detecting the level of RNAm6A modification or the relative level of modification of the NCOA4 comprises primers and/or reagents and/or kits and/or instruments.
4. A system for detecting RNAm6A modification level or relative modification level of NCOA4 according to claim 2, wherein: the system for detecting the level of RNAm6A modification or the relative level of modification using MeRIP-qPCR comprises primers, kits and/or reagents and/or instrumentation required for performing MeRIP-qPCR.
5. A system for detecting RNAm6A modification level or relative modification level of NCOA4 according to claim 2, wherein: the system for detecting RNAm6A modification level or relative modification level further comprises a data processing system for converting the RNAm6A modification level or relative modification level from a test subject into a detection result of the test subject.
6. A system for detecting RNAm6A modification level or relative modification level of NCOA4 according to claim 2, wherein: the level of RNAm6A modification or the relative level of modification of the NCOA4 gene is that of the NCOA4 gene in blood or cells.
7. A system for detecting RNAm6A modification level or relative modification level of NCOA4 according to claim 6, wherein: the RNAm6A modification level or relative modification level of the NCOA4 gene is that of the Ncoa4 gene in peripheral blood mononuclear cells isolated from peripheral blood of mice or that of the NCOA4 gene in human HeLa cells.
8. A system for detecting RNAm6A modification level or relative modification level of NCOA4 according to claim 7, wherein: the primer for amplifying Ncoa4 of the peripheral blood mononuclear cells of the mice consists of 2 pairs of primers, and comprises a primer pair 1 consisting of two single-stranded DNAs shown by Seq ID No.1 and Seq ID No.2, and a primer pair 2 consisting of two single-stranded DNAs shown by Seq ID No.3 and Seq ID No. 4.
9. A system for detecting RNAm6A modification level or relative modification level of NCOA4 according to claim 7, wherein: the primer for amplifying NCOA4 of Hela cells consists of 2 pairs of primers and includes a primer pair 1 consisting of two single-stranded DNAs shown in Seq ID No.5 and Seq ID No.6, and a primer pair 2 consisting of two single-stranded DNAs shown in Seq ID No.7 and Seq ID No. 8.
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