Screening and application of a small cell lung cancer nuclease

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to the screening of a small cell lung cancer nuclease and its application. The present invention creates an siRNA library, uses a high-content cell analyzer to calculate the fluorescence intensity, and screens out the nuclease ZC3H12A for diagnosing DNA damage during radiotherapy of small cell lung cancer, and proves that knocking down ZC3H12A increases the radiotherapy sensitivity of small cell lung cancer cells.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to the screening of a small cell lung cancer nuclease and its application. Background Art

[0002] Lung cancer is currently the malignant tumor with the highest incidence and the second highest mortality rate in the world. Small cell lung cancer accounts for about 15% of all lung cancer cases, and is characterized by rapid growth, early metastasis, and poor prognosis. Radiotherapy, as the first-line treatment for small cell lung cancer, can cause extensive exogenous DNA double-strand breaks (DSBs) and single-strand breaks (SSBs) in small cell lung cancer cells, which can lead to genomic instability and abnormal mitosis, resulting in cell death. However, small cell lung cancer often develops radiotherapy tolerance, which means that there is a mechanism in small cell lung cancer that can repair the DNA damage caused by radiotherapy to avoid death. Normal cells, after being exposed to ionizing radiation, activate the G1 checkpoint by activating the tumor suppressor gene p53. In small cell lung cancer cells, the tumor suppressor genes TP53 and RB1 are functionally inactivated. Therefore, the G1 checkpoint is missing, which means that small cell lung cancer cells rely on the activation of the G2 checkpoint to repair damaged DNA. It has been reported in the literature that after cancer cells are exposed to ionizing radiation, in addition to the exogenous breaks, they will also produce endogenous secondary breaks after a period of time. Through these secondary breaks, the G2 checkpoint in cancer cells is activated. It is inferred that this endogenous secondary break should also exist in small cell lung cancer.

[0003] The ZC3H12A gene encodes monocyte chemoattractant protein-induced protein 1, which was first discovered in 2006 as the first member of a new family of anti-inflammatory proteins and is also known as Regnase-1. It contains a CCCH-type zinc finger domain and a PilTN terminal (PIN) domain. Zinc finger proteins are generally considered to be transcription factors that bind to DNA, while the CCCH zinc finger motif binds to RNA by recognizing specific DNA binding motifs and acts as RNA binding proteins. The PIN domain has RNase activity and cuts and degrades mRNA by binding to specific stem-loop structures in transcripts. ZC3H12A was originally considered to be a transcription factor that induces apoptosis, and then more and more evidence showed that ZC3H12A plays a key role in regulating inflammation. ZC3H12A negatively regulates inflammation by degrading the mRNA of pro-inflammatory factors. In vivo studies have shown that ZC3H12A knockout mice will develop severe anemia and immune disorders. In addition, ZC3H12A has deubiquitinase activity, can target TRAF6, and negatively regulate the activation of c-Jun N-terminal kinase (JNK). At the same time, it can also affect the transduction of DNA damage signals by recruiting ubiquitin-specific protease USP10 and inhibiting the activation of genotoxic NF-κB. The results showed that ZC3H12A can regulate endogenous secondary breaks, so it is speculated that it may play a regulatory role by affecting DNA damage repair. However, there is currently almost no literature reporting on the role of ZC3H12A in DNA damage repair. Therefore, it is necessary to conduct a series of studies on the role of ZC3H12A in DNA damage repair and its application in radiotherapy of small cell lung cancer. Summary of the invention

[0004] In a first aspect, the present invention provides a marker for diagnosing DNA damage during radiotherapy or chemotherapy of small cell lung cancer, wherein the marker is a nuclease, and the nuclease is selected from one or more of ZC3H12A, MRE11, EEPD1, DIS3L2, TSEN34, PLD4 or EXO5.

[0005] Furthermore, the nuclease is preferably ZC3H12A.

[0006] In a second aspect, the present invention provides a method for screening markers for diagnosing DNA damage during radiotherapy or chemotherapy of small cell lung cancer, the method comprising the following steps:

[0007] S1. Construction of nuclease siRNA library;

[0008] S2. small cell lung cancer cells are transfected with the siRNA library, and then irradiated to block cell division;

[0009] S3. Screening markers of cell division arrest, i.e., the markers described in the first aspect of the present invention, using a high-content cell analyzer.

[0010] Furthermore, in step S1, a nuclease library including DNA exonuclease and RNA exonuclease is constructed.

[0011] Furthermore, in step S2, the radiation treatment is performed at 4 Gy-8 Gy.

[0012] In a third aspect, the present invention provides a diagnostic kit for diagnosing DNA damage during radiotherapy of small cell lung cancer using the marker described in the first aspect, wherein the diagnostic kit contains diagnostic reagents and instructions.

[0013] Furthermore, when one or more genes among the markers ZC3H12A, MRE11, EEPD1, DIS3L2, TSEN34, PLD4 or EXO5 are expressed, it indicates that DNA damage occurs during radiotherapy or chemotherapy of small cell lung cancer.

[0014] In a fourth aspect, the present invention provides a use of the marker as described in the first aspect in the preparation of a detection reagent for diagnosing DNA damage during radiotherapy of small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Comet electrophoresis experiment of small cell lung cancer cells (NCI-H446, DMS-114) before and after ionizing radiation at different time points under microscope

[0016] Figure 2 Comet electrophoresis experiment of normal lung epithelial cells (BEAS-2B) before and after ionizing radiation at different time points under microscope

[0017] Figure 3 Microscopic observation of DNA damage after SYBRgold staining

[0018] Figure 4 Small cell lung cancer cells (NCI-H446) were transfected with siRNA library in 96-well plates

[0019] Figure 5 Immunofluorescence experiments of small cell lung cancer cells (NCI-H446, DMS-114) collected at different time points before and after ionizing radiation

[0020] Figure 6 Immunofluorescence experiments on U2os and Hela cells under 8Gy ionizing radiation

[0021] Figure 7Immunofluorescence experiments were performed on Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 4Gy ionizing radiation. After staining with DAPI (blue), ZC3H12A (green), and γH2AX (red), the cells were observed under a confocal microscope.

[0022] Figure 8 Immunofluorescence experiments were performed on Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 4Gy ionizing radiation. After staining with DAPI (blue), ZC3H12A (green), and BRCA1 (red), the cells were observed under a confocal microscope.

[0023] Fig. 9 Immunofluorescence experiments were performed on Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 4Gy ionizing radiation. After staining with DAPI (blue), dsDNA (green), and 53BP1 (red), the cells were observed under a confocal microscope.

[0024] Fig.10 Immunofluorescence experiments were performed on Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 4Gy ionizing radiation. After staining with DAPI (blue), dsRNA (green), and 53BP1 (red), the cells were observed under a confocal microscope.

[0025] Fig.11 Immunofluorescence experiments were performed on Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 4Gy ionizing radiation. After staining with DAPI (blue), DNA-RNA (green), and 53BP1 (red), the cells were observed under a confocal microscope.

[0026] Fig.12 Immunofluorescence experiments were performed on Hela cells and U2OS cells after 8 Gy ionizing radiation.

[0027] Fig.13 Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours were subjected to plate cloning experiments under 1Gy, 2Gy, and 4Gy ionizing radiation.

[0028] Fig.14 Plate cloning assay was performed on 446 cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 1Gy, 2Gy, and 4Gy ionizing radiation.

[0029] Fig.15 Western blotting experiments were performed on total protein, cytoplasm and chromatin of Hela cells under 8Gy ionizing radiation. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0032] Example 1 Comet electrophoresis

[0033] Purpose: To demonstrate that small cell lung cancer has endogenous secondary breaks after ionizing radiation, which do not exist in normal lung epithelium.

[0034] Experimental methods:

[0035] 1. Place the lysis solution in the refrigerator for >20 min in advance, heat the agarose at 100°C for 10 min and then keep it at 37°C for >30 min.

[0036] 2. Centrifuge one well of the six-well cell plate, discard the waste liquid, add 2 ml PBS and centrifuge again.

[0037] 3. 10 microliters of cells, 50 microliters of agarose. Aspirate 50 microliters and apply 30-40 microliters on the glass slide. (This step is very important for subsequent observation)

[0038] 4. Refrigerate at 4℃ for 30-60 minutes. 60 minutes is best.

[0039] 5. Add lysis buffer at 4℃ for more than 2h. (Avoid light) (This step can be done overnight the next day)

[0040] 6. Add DNA precipitation solution at room temperature for 30 minutes, 4℃ for 1 hour. (Avoid light)

[0041] 7. Run electrophoresis, 25V, 30min. Black pole against slide label.

[0042] 8. Soak in water twice, 5 minutes each time. Soak in 70% alcohol once, 5 minutes.

[0043] 9. Dry for 10 min at 37°C.

[0044] 10. Stain 100 μl for 30 min at room temperature. (Avoid light) Gold staining solution (the diluted stock solution can be stored stably for several weeks at 4°C in the dark)

[0045] 11. Wash once with water and dry at 37℃ for 10 min.

[0046] 12. Observe under the microscope immediately.

[0047] Experimental results:

[0048] Samples of small cell lung cancer cells (NCI-H446, DMS-114) were collected before ionizing radiation (Control) and at different time points (10min, 6h, 12h, 24h) after 4Gy ionizing radiation for comet electrophoresis experiment. After SYBRgold staining, the DNA damage was observed under a microscope. Data are mean ± SD, n ≥ 100, **** P<0.0001, ** P = 0.0063 (Kruskal-Wallis test). Figure 1 As shown, small cell lung cancer cells NCI-H446 and DMS-114 both reached the second peak 24 hours after IR, and there was a secondary fragmentation wave.

[0049] Samples of normal lung epithelial cells (BEAS-2B) were collected before ionizing radiation (Control) and at different time points (10min, 6h, 12h, 24h) after 4Gy ionizing radiation for comet electrophoresis experiment. After SYBRgold staining, the DNA damage was observed under a microscope. Data are mean ± SD, n ≥ 100, **** P<0.000 (Kruskal-Wallis test). Figure 2 As shown, normal lung epithelial cells BEAS-2B only produced one peak after IR, and unlike tumor cells, there was no endogenous secondary break.

[0050] Because damaged DNA breaks into small molecular fragments, they will migrate out of the cell nucleus under the action of the electric field force, forming a phenomenon similar to the comet tail. Undamaged DNA will not migrate out of the cell nucleus due to the action of the electric field force. Therefore, the degree of DNA damage is judged by the length of the comet tail observed under the microscope. The longer the tail, the more serious the DNA damage.

[0051] Comet electrophoresis was performed on NCI-H446 and Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 4Gy ionizing radiation. DNA damage was observed under a microscope after SYBRgold staining. Data are mean ± SD, n ≥ 100, ****P<0.0001 (Kruskal-Wallis test). Figure 3 As shown, cells in which ZC3H12A was knocked down had reduced secondary breaks.

[0052] Example 2 High Content Cell Analyzer siRNA Library Screening

[0053] Objective: To screen for nucleases that, when knocked down, inhibit G2 checkpoint activation in small cell lung cancer cells.

[0054] Experimental methods:

[0055] 1. Construct a nuclease siRNA library including DNA exonuclease and RNA exonuclease.

[0056] 2. Small cell lung cancer cells were transfected with the siRNA library in 96-well plates.

[0057] 3. 48 hours after transfection, the transfected small cell lung cancer cells were irradiated with 4 Gy. 2 hours after irradiation, the cells were treated with Nocodazole mitotic inhibitor for 8 hours to block the cells in the G2 / M phase.

[0058] 4. Fix the cells with 4% paraformaldehyde for 10 min on ice and wash three times with PBS. Permeabilize with 0.3% Triton for 15 min and wash three times with PBS. Block with 10% FBS blocking solution at room temperature for 1 h. Add anti-histone H3 (phosphorylated S10) antibody with 488 green fluorescence at a dilution of 1:100 and incubate at 4°C overnight.

[0059] 5. Wash three times with PBS, add Hoechst diluted 1:1000 to stain the nuclei, and incubate at room temperature for 15 minutes.

[0060] 6. Immediately perform high-content cell analyzer screening, using a 10x confocal microscope to automatically take pictures, and take 16 fields of view per well. Calculate the percentage of fluorescence intensity in each well by adjusting the software parameters in the high-content cell analyzer. (It is more convenient and faster than existing technologies, takes more pictures, and analyzes and calculates results quickly and accurately.)

[0061] Experimental results:

[0062] Small cell lung cancer cells (NCI-H446) were transfected with siRNA library in 96-well plates. 48 hours after transfection, the transfected small cell lung cancer cells were irradiated with 4Gy. After 2 hours of irradiation, the cells were treated with Nocodazole mitotic inhibitor for 8 hours, and then immunofluorescence experiments were performed. They were stained with Hoechst (blue) and histone H3 (phosphorylated S10) (green), and immediately screened with a high-content cell analyzer. The screening was automatically photographed with a 10x confocal microscope, and 16 fields of view were photographed in each well. The results are shown in Figure 2. Figure 4As shown, phosphorylated histone H3 is used as a mitotic marker, representing the number of cells entering mitosis (the number of cells whose G2 checkpoint is cancelled). Therefore, it can be seen from the results that when ZC3H12A is knocked down, the number of cells whose G2 checkpoint is cancelled is the largest. The average fluorescence intensity of the screening is calculated twice and sorted, among which ZC3H12A ranks first, and the nucleotide sequence of ZC3H12A is shown in Table 1. The nucleotide sequence of ZC3H12A is shown in SEQ ID NO.1, the nucleotide sequence of siZC3H12A after knockdown is shown in SEQ ID NO.2, and the sequences of siZC3H12A-1, siZC3H12A-2, and siZC3H12A-3 that are knocked down are shown in SEQ ID NO.3-5.

[0063] Table 1 Nucleic acid sequences of ZC3H12A markers

[0064]

[0065]

[0066]

[0067]

[0068] Example 3 Immunofluorescence

[0069] Experimental purpose: To demonstrate the role of ZC3H12A in DNA damage repair.

[0070] Experimental methods:

[0071] 1.Put about 12w of cells in each well of a 12-well plate.

[0072] 2. After overnight, transfect siRNA for 48h.

[0073] The cells were irradiated with 3.4 Gy ionizing radiation, fixed with 4% paraformaldehyde in an ice bath for 15 min about 6 h later, and washed three times with PBS.

[0074] The cells were permeabilized with 0.3% Triton for 10 min and washed three times with PBS.

[0075] 4. Block with 10% FBS for 45 min.

[0076] 5. Incubate with primary antibody at room temperature for 2 hours, wash with PBS 3 times, and incubate with secondary antibody at room temperature for 1 hour.

[0077] 6. Confocal microscopy observation.

[0078] Experimental results:

[0079] Samples of small cell lung cancer cells (NCI-H446, DMS-114) were collected before ionizing radiation (Control) and at different time points (10min, 6h, 12h, 24h) after 4Gy ionizing radiation for immunofluorescence experiments. After staining with DAPI (blue) and γH2AX (green), confocal microscopy was used to observe DNA damage. Data are mean ± SD, n ≥ 100, **** P<0.0001, *** P=0.0004, * P = 0.0103 (Kruskal-Wallis test). Figure 5 As shown, γh2ax, as a DNA damage marker, was expressed again in small cell lung cancer cells NCI-H446 and DMS-114 24 hours after IR, proving that DNA damage was aggravated again 24 hours after IR. The results were the same as comet electrophoresis, proving the existence of endogenous secondary breaks.

[0080] Immunofluorescence experiments were performed on U2os and Hela cells under 8Gy ionizing radiation. After staining with DAPI (blue), ZC3H12A (green), and BRCA1 / γH2AX (red), their co-localization was observed under a confocal microscope. The results are shown in Figure 2. Figure 6 As shown, there is a certain co-localization with BRCA1 and γH2AX in U2OS cells, indicating that it may be related to DNA damage.

[0081] Immunofluorescence experiments were performed on Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 4Gy ionizing radiation. After staining with DAPI (blue), ZC3H12A (green), and γH2AX (red), the cells were observed under a confocal microscope. Data are mean ± SD, n ≥ 100, ** P = 0.0062 (unpaired t test). Figure 7 As shown, under 4GyIR conditions, the expression of γH2AX as a DNA damage marker increased after ZC3H12A knockdown, indicating that ZC3H12A knockdown increased DNA damage, suggesting that ZC3H12A may affect DNA damage repair.

[0082] Immunofluorescence experiments were performed on Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 4Gy ionizing radiation. After staining with DAPI (blue), ZC3H12A (green), and BRCA1 (red), the cells were observed under a confocal microscope. Data are mean ± SD, n ≥ 100, * P = 0.0120 (unpaired t test) Figure 8As shown, under 4GyIR conditions, BRCA1, as a key protein for homologous recombination repair, was recruited to the damage site more frequently after ZC3H12A was knocked down, indicating that ZC3H12A may affect DNA damage repair.

[0083] Immunofluorescence experiments were performed on Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 4Gy ionizing radiation. After staining with DAPI (blue), dsDNA (green), and 53BP1 (red), the cells were observed under a confocal microscope. Data are mean ± SD, n ≥ 50, ** P = 0.0089 (unpaired t test). Fig. 9 As shown in the figure, under 4GyIR conditions, after ZC3H12A was knocked down, 53BP1, as a key protein for non-homologous end recombination repair, was recruited to the damage site and increased. At the same time, dsDNA increased, further proving that ZC3H12A affects DNA damage repair.

[0084] Immunofluorescence experiments were performed on Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 4Gy ionizing radiation. After staining with DAPI (blue), dsRNA (green), and 53BP1 (red), the cells were observed under a confocal microscope. Data are mean ± SD, n ≥ 50, **** P<0.0001 (unpaired t test). Fig.10 As shown, under 4GyIR conditions, after ZC3H12A knockdown, 53BP1, as a key protein for non-homologous end recombination repair, was recruited to the damage site more frequently. At the same time, dsRNA increased, indicating that ZC3H12A knockout led to increased genomic instability.

[0085] Immunofluorescence experiments were performed on Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 4Gy ionizing radiation. After staining with DAPI (blue), DNA-RNA (green), and 53BP1 (red), the cells were observed under a confocal microscope. Data are mean ± SD, n ≥ 50, **** P<0.0001 (unpaired t test). Fig.11 As shown, under 4GyIR conditions, after ZC3H12A knockdown, 53BP1, as a key protein for non-homologous end recombination repair, was recruited to the damage site and increased. At the same time, DNA / RNA increased, which also proved that ZC3H12A knockout affected DNA damage repair and led to increased genomic instability.

[0086] Immunofluorescence experiments were performed on Hela cells and U2OS cells under 8Gy ionizing radiation. After staining with DAPI (blue) and ZC3H12A (green), the cells were observed under a confocal microscope. Data are mean ± SD, n ≥ 300, **** P<0.0001 (unpaired t test). Fig.12 As shown in the figure, the expression changes of ZC3H12A in HELA cells and U2OS were detected before and after irradiation under 8GyIR conditions. The results showed that ZC3H12A expression increased after ionizing radiation. Radiotherapy, as a means of inducing DNA damage, also promoted the increase in ZC3H12A expression. Therefore, it is believed that ZC3H12A may affect DNA damage repair.

[0087] Example 4 Plate cloning

[0088] Experimental purpose: To demonstrate the effect of ZC3H12A on the sensitivity of small cell radiotherapy.

[0089] Experimental methods:

[0090] 1. Approximately 120,000 cells were plated in each well of a 12-well plate.

[0091] 2. After overnight, transfect siRNA for 48 hours.

[0092] After 3.4 Gy ionizing radiation, cells were immediately trypsinized and centrifuged at 800 RPM for 5 minutes.

[0093] 4. Count the cells, add 2 ml of culture medium to a six-well plate after dilution, and inoculate 1500 cells per well.

[0094] 5. After 14 days, discard the waste liquid, wash with PBS three times, and fix with 4% paraformaldehyde for 15 minutes.

[0095] 6. Incubate with crystal violet stain at room temperature for 30 minutes, rinse with clean water and dry.

[0096] Experimental results:

[0097] Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours were subjected to plate cloning experiments under 1Gy, 2Gy, and 4Gy ionizing radiation. The number of cell clones was counted 14 days later, and the survival score = the number of cell clones at each dose / the number of cell clones at 0Gy. Data are mean ± SD, N = 3, * P=0.01, *** P=0.0007, *** P = 0.0004 (unpaired t test). Fig.13As shown, under different ionizing radiation doses, the clone numbers of ZC3H12A knockdown and NC groups were compared in HELA cells, showing that after ZC3H12A knockdown, the decrease in clone number was more significant than that in the negative control group, indicating that ZC3H12A knockdown can increase the radiotherapy sensitivity of cells.

[0098] Plate cloning experiments were performed on 446 cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours under 1Gy, 2Gy, and 4Gy ionizing radiation. The number of cell clones was counted 14 days later, and the survival score = the number of cell clones at each dose / the number of cell clones at 0Gy. Data are mean ± SD, N = 3, ** P=0.0015, * P=0.0285, * P = 0.0232 (unpaired t test). Fig.14 As shown, under different ionizing radiation doses, the clone numbers of ZC3H12A knockdown and NC groups were compared in NCI-H446 cells, showing that after ZC3H12A knockdown, the decrease in clone number was more significant than that in the negative control group, indicating that ZC3H12A knockdown can increase the radiotherapy sensitivity of cells.

[0099] Example 5: Separation of subcellular proteins and Western blot

[0100] Experimental purpose: To detect the recruitment of ZC3H12A in various cellular components after IR.

[0101] Experimental methods:

[0102] 1. Treat cells with 8Gy ionizing radiation.

[0103] 2. Use the ThermoScientific Subcellular Protein Isolation Kit to separate the cell components.

[0104] 3. Perform protein electrophoresis detection using the separated subcellular proteins.

[0105] 4. Electrophoresis at 80v for 30min, 120v for 1h. Electrotransfer at 400mA for 40min.

[0106] Block with 5.5% skim milk for 1 h and incubate with 1 antibody at 4°C overnight.

[0107] 6. Incubate with secondary antibody at room temperature for 1 hour before exposure to light for detection.

[0108] Experimental results:

[0109] Western blotting was performed on total protein, cytoplasm and chromatin of Hela cells under 8Gy ionizing radiation. Rad51 recruitment was used as a positive control, and H3 and β-Tubulin were used as internal controls. Fig.15 As shown in the figure, the components of HELA cells were extracted under 8GyIR. The expression of ZC3H12A was detected at different time points after IR. The expression of ZC3H12A in total protein increased after IR, and the expression of ZC3H12A in chromatin also showed an increase. However, the expression level of ZC3H12A in the cytoplasm was almost unchanged. Therefore, after being exposed to ionizing radiation, ZC3H12A may be recruited to chromatin to play a role.

Claims

1. A method for screening markers for diagnosing DNA damage during radiotherapy of small cell lung cancer, the method comprising the following steps: S1. Construction of nuclease siRNA library; S2. The small cell lung cancer cells were transfected with the siRNA library, and the small cell lung cancer cells were irradiated after transfection to block cell division; wherein, The radiation treatment is performed at 4 Gy; S3. Screening of cell division arrest markers, namely ZC3H12A, by high-content cell analyzer.

2. A method for screening markers for diagnosing DNA damage during radiotherapy of small cell lung cancer according to claim 1, characterized in that: In step S1, a nuclease siRNA library including DNA exonuclease and RNA exonuclease is constructed.

3. Use of ZC3H12A in the preparation of a detection reagent for diagnosing DNA damage during radiotherapy of small cell lung cancer.

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