Screening and use of small cell lung cancer nuclease

Abstract

Screening and a use of a small cell lung cancer nuclease. An siRNA library is created, and a high-content cell analyzer is used to calculate the fluorescence intensity to screen for a nuclease ZC3H12A for diagnosing DNA damage in the radiotherapy process of small cell lung cancer. It is proved that knock-down of ZC3H12A can increase sensitivity to radiotherapy of small cell lung cancer cells.

Description

Screening and application of a small cell lung cancer nuclease Technical Field

[0001] 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. Background Art

[0002] Lung cancer is currently the most common malignant tumor worldwide and the second most common cause of death. Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancer cases and is characterized by rapid growth, early metastasis, and a poor prognosis. Radiotherapy, a first-line treatment for SCLC, can cause extensive exogenous DNA double-strand breaks (DSBs) and single-strand breaks (SSBs) in SCLC cells. This can lead to genomic instability, abnormal mitosis, and cell death. However, SCLC often develops radioresistance, suggesting that mechanisms exist within the SCLC system that repair radiotherapy-induced DNA damage and prevent cell death. Normal cells, exposed to ionizing radiation, activate the G1 checkpoint by activating the tumor suppressor gene p53. However, in SCLC cells, the tumor suppressor genes TP53 and RB1 are functionally inactivated, resulting in a loss of the G1 checkpoint. This means that SCLC cells rely on activation of the G2 checkpoint to repair damaged DNA. Literature reports 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. These secondary breaks activate the G2 checkpoint in cancer cells. It is inferred that this endogenous secondary break should also exist in small cell lung cancer.

[0003] The ZC3H12A gene encodes monocyte chemoattractant protein-inducing protein 1 (RCP-1), first discovered in 2006 as the first member of a new family of anti-inflammatory proteins and also known as Regnase-1. It contains a CCCH-type zinc finger domain and a PiTN-terminal (PIN) domain. Zinc finger proteins are generally considered to bind to DNA as transcription factors, while the CCCH zinc finger motif binds to RNA by recognizing specific DNA-binding motifs, acting as RNA-binding proteins. The PIN domain possesses RNase activity, binding to specific stem-loop structures within transcripts to cleave and degrade mRNA. ZC3H12A was initially thought to be a transcription factor that induces apoptosis. Subsequently, increasing evidence indicates that ZC3H12A plays a key role in regulating inflammation. ZC3H12A negatively regulates inflammation by degrading the mRNAs of pro-inflammatory factors. In vivo studies have shown that ZC3H12A knockout mice 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). It can also affect the transduction of DNA damage signals by recruiting the ubiquitin-specific protease USP10 and inhibiting the activation of genotoxic NF-κB. The results show 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 little 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 for small cell lung cancer.

[0004] Summary of the Invention

[0005] 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.

[0006] Furthermore, the nuclease is preferably ZC3H12A.

[0007] 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:

[0008] S1. Construction of nuclease siRNA library;

[0009] S2. Small cell lung cancer cells were transfected with the siRNA library and then irradiated to arrest cell division.

[0010] S3. Screening markers of cell division arrest using a high-content cell analyzer, i.e., the markers described in the first aspect of the present invention.

[0011] Furthermore, in step S1, a nuclease library including DNA exonuclease and RNA exonuclease is constructed.

[0012] Furthermore, in step S2, the radiation treatment is performed at 4 Gy to 8 Gy.

[0013] 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 comprises diagnostic reagents and instructions.

[0014] 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.

[0015] In a fourth aspect, the present invention provides a use of the marker 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

[0016] Figure 1 Comet electrophoresis of small cell lung cancer cells (NCI-H446, DMS-114) before and after ionizing radiation at different time points

[0017] Figure 2 Comet electrophoresis of normal lung epithelial cells (BEAS-2B) observed under a microscope before and after ionizing radiation at different time points

[0018] Figure 3 Microscopic observation of DNA damage after SYBRgold staining

[0019] Figure 4 Small cell lung cancer cells (NCI-H446) were transfected with siRNA library in 96-well plates

[0020] Figure 5 Immunofluorescence experiments of samples collected from small cell lung cancer cells (NCI-H446, DMS-114) at different time points before and after ionizing radiation

[0021] Figure 6 Immunofluorescence experiments on U2os and Hela cells under 8Gy ionizing radiation

[0022] Figure 7 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.

[0023] Figure 8 Immunofluorescence experiments were performed on Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) 48 hours after irradiation with 4Gy ionizing radiation. After staining with DAPI (blue), ZC3H12A (green), and BRCA1 (red), the cells were observed under a confocal microscope.

[0024] Figure 9: Immunofluorescence experiments of 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.

[0025] Figure 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.

[0026] Figure 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.

[0027] FIG12 shows immunofluorescence experiments on Hela cells and U2OS cells after 8 Gy ionizing radiation.

[0028] Figure 13 Plate cloning assay of Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) 48 hours after ionizing radiation at 1Gy, 2Gy, and 4Gy.

[0029] Figure 14: Plate cloning assay of 446 cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) 48 hours after exposure to 1Gy, 2Gy, and 4Gy ionizing radiation.

[0030] Figure 15 Western blotting experiments on total protein, cytoplasm and chromatin of Hela cells under 8Gy ionizing radiation DETAILED DESCRIPTION

[0031] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below may be combined with each other as long as they do not conflict with each other.

[0032] 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.

[0033] Example 1 Comet electrophoresis

[0034] Objective: To demonstrate that small cell lung cancer cells have endogenous secondary breaks after ionizing radiation, which are not present in normal lung epithelium.

[0035] Experimental methods:

[0036] 1. Place the lysate in the refrigerator for >20 minutes in advance. Heat the agarose at 100°C for 10 minutes and then keep it at 37°C for >30 minutes.

[0037] 2. Centrifuge one well of the six-well cell plate, discard the waste liquid, add 2 ml PBS and centrifuge again.

[0038] 3. 10 μl of cells, 50 μl of agarose. Aspirate 50 μl and spread thinly on the slide, using 30-40 μl. (This step is very important for subsequent observations)

[0039] 4. Refrigerate at 4°C for 30-60 minutes. 60 minutes is best.

[0040] 5. Add lysis buffer at 4°C for more than 2 hours (protect from light) (This step can be done the next day and can be left overnight)

[0041] 6. Add DNA precipitation solution and incubate at room temperature for 30 minutes, or at 4°C for 1 hour. (Protect from light)

[0042] 7. Run electrophoresis at 25V for 30min. Use the black electrode to label the slide.

[0043] 8. Soak in water twice, 5 minutes each time. Soak in 70% alcohol once, 5 minutes each time.

[0044] 9. Dry for 10 minutes at 37°C.

[0045] 10. Stain 100 μl for 30 minutes at room temperature (protect from light). Staining solution (the diluted stock solution can be stored stably for several weeks at 4°C in the dark)

[0046] 11. Wash once with water and dry at 37℃ for 10 min.

[0047] 12. Observe under a microscope immediately.

[0048] Experimental results:

[0049] Samples of small cell lung cancer cells (NCI-H446, DMS-114) were collected before ionizing radiation (Control) and at different time points (10 min, 6 h, 12 h, 24 h) after 4 Gy ionizing radiation for comet electrophoresis. After SYBR gold staining, DNA damage was observed under a microscope. Data are mean ± SD, n ≥ 100. **** P<0.0001, ** P=0.0063 (Kruskal-Wallis test). As shown in FIG1 , the small cell lung cancer cells NCI-H446 and DMS-114 both reached the second peak 24 hours after IR, indicating the presence of a secondary fragmentation wave.

[0050] Normal lung epithelial cells (BEAS-2B) were collected before ionizing radiation (Control) and at different time points (10 min, 6 h, 12 h, 24 h) after 4 Gy ionizing radiation for comet electrophoresis. DNA damage was observed under a microscope after SYBR gold staining. Data are mean ± SD, n ≥ 100. **** P < 0.000 (Kruskal-Wallis test). As shown in Figure 2, normal lung epithelial cells BEAS-2B only produced one peak after IR, and unlike tumor cells, there was no endogenous secondary breakage.

[0051] Because damaged DNA breaks into small fragments, they migrate out of the cell nucleus under the influence of the electric field, forming a phenomenon similar to a comet tail. Undamaged DNA, on the other hand, does not migrate out of the cell nucleus due to the electric field. Therefore, the extent of DNA damage is determined by the length of the comet tail observed under a microscope: the longer the tail, the more severe the DNA damage.

[0052] Comet electrophoresis was performed on NCI-H446 and Hela cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours after exposure to 4 Gy ionizing radiation. DNA damage was observed under a microscope after SYBR gold staining. Data are mean ± SD, n ≥ 100. **** P<0.0001 (Kruskal-Wallis test). As shown in Figure 3, secondary fragmentation was reduced in cells in which ZC3H12A was knocked down.

[0053] Example 2 High-content cell analyzer siRNA library screening

[0054] Objective: To identify nucleases that, when knocked down, inhibit G2 checkpoint activation in small cell lung cancer cells.

[0055] Experimental methods:

[0056] 1. Construct a nuclease siRNA library including DNA exonuclease and RNA exonuclease.

[0057] 2. Small cell lung cancer cells were transfected with the siRNA library in 96-well plates.

[0058] 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 arrest the cells in the G2 / M phase.

[0059] 4. Fix the cells with 4% paraformaldehyde for 10 minutes on ice and wash three times with PBS. Permeabilize with 0.3% Triton for 15 minutes and wash three times with PBS. Block with 10% FBS blocking buffer for 1 hour at room temperature. Add anti-histone H3 (phospho-S10) antibody with 488 green fluorescence at a 1:100 dilution and incubate at 4°C overnight.

[0060] 5. Wash three times with PBS, add Hoechst 1:1000 dilution to stain the nuclei, and incubate at room temperature for 15 minutes.

[0061] 6. Immediately perform screening using a high-content cell analyzer. Automatically image 16 fields per well using a 10x confocal microscope. Calculate the percentage of fluorescence intensity per well by adjusting the software parameters in the high-content cell analyzer. (This is more convenient and faster than existing techniques, captures more images, and provides faster and more accurate analysis and calculation results.)

[0062] Experimental results:

[0063] Small cell lung cancer cells (NCI-H446) were transfected with the siRNA library in a 96-well plate. 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 the Nocodazole mitosis 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 using a high-content cell analyzer. Screening was performed using a 10x confocal microscope and 16 fields of view were taken per well. The results are shown in Figure 4. Phosphorylated histone H3 is used as a mitotic marker, representing the number of cells entering mitosis (the number of cells whose G2 checkpoint was canceled). Therefore, it can be seen from the results that when ZC3H12A was knocked down, the number of cells whose G2 checkpoint was canceled was the highest. The average fluorescence intensity of the screening was calculated twice and ranked. Among them, ZC3H12A ranked first. 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 knockdown siZC3H12A-1, siZC3H12A-2, and siZC3H12A-3 are shown in SEQ ID NO.3-5.

[0064] Table 1 Nucleic acid sequence of ZC3H12A marker

[0065] Example 3 Immunofluorescence

[0066] Experimental purpose: To prove the role of ZC3H12A in DNA damage repair.

[0067] Experimental methods:

[0068] 1.Plate approximately 12 w of cells per well of a 12-well plate.

[0069] 2. After overnight, transfect siRNA for 48 hours.

[0070] 3. After 4 Gy ionizing radiation, cells were fixed in 4% paraformaldehyde on ice for 15 min approximately 6 hours later and then washed three times with PBS.

[0071] The cells were permeabilized with 0.3% Triton for 10 min and washed three times with PBS.

[0072] 4. Block with 10% FBS for 45 minutes.

[0073] 5. Incubate with primary antibody at room temperature for 2 hours, wash three times with PBS, and incubate with secondary antibody at room temperature for 1 hour.

[0074] 6. Confocal microscopy observation.

[0075] Experimental results:

[0076] Small cell lung cancer cells (NCI-H446, DMS-114) were collected before ionizing radiation (Control) and at different time points (10 min, 6 h, 12 h, 24 h) after 4 Gy ionizing radiation for immunofluorescence experiments. After staining with DAPI (blue) and γH2AX (green), DNA damage was observed by confocal microscopy. Data are mean ± SD, n ≥ 100, **** P<0.0001, *** P=0.0004, * P = 0.0103 (Kruskal-Wallis test). The results are shown in Figure 5. γh2ax, a DNA damage marker, increased again in small cell lung cancer cells NCI-H446 and DMS-114 24 hours after IR, demonstrating that DNA damage was further aggravated 24 hours after IR. The results were consistent with comet electrophoresis, demonstrating the presence of endogenous secondary breaks.

[0077] Immunofluorescence experiments were performed on U2OS and HeLa cells exposed to 8 Gy of ionizing radiation. After staining with DAPI (blue), ZC3H12A (green), and BRCA1 / γH2AX (red), colocalization was observed under a confocal microscope. As shown in Figure 6, colocalization with BRCA1 and γH2AX was observed in U2OS cells, suggesting a possible association with DNA damage.

[0078] Immunofluorescence analysis was performed on HeLa cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours after exposure to 4 Gy ionizing radiation. Cells were stained with DAPI (blue), ZC3H12A (green), and γH2AX (red) and observed under a confocal microscope. Data are mean ± SD, n ≥ 100. ** P = 0.0062 (unpaired t test). The results are shown in Figure 7. Under 4GyIR conditions, the expression of γH2AX, a DNA damage marker, increased after ZC3H12A knockdown, indicating that ZC3H12A knockdown increased DNA damage, suggesting that ZC3H12A may affect DNA damage repair.

[0079] Immunofluorescence analysis was performed on HeLa cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours after exposure to 4 Gy ionizing radiation. Cells were stained with DAPI (blue), ZC3H12A (green), and BRCA1 (red) and observed under a confocal microscope. Data are mean ± SD, n ≥ 100. *P = 0.0120 (unpaired t test) The results are shown in Figure 8. Under 4GyIR conditions, after ZC3H12A was knocked down, BRCA1, as a key protein for homologous recombination repair, was recruited to the damage site, indicating that ZC3H12A may affect DNA damage repair.

[0080] Immunofluorescence analysis was performed on HeLa cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours after exposure to 4 Gy ionizing radiation. The cells were stained with DAPI (blue), dsDNA (green), and 53BP1 (red) and observed under a confocal microscope. Data are mean ± SD, n ≥ 50. ** P = 0.0089 (unpaired t-test). The results are shown in Figure 9. Under 4 GyIR 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, dsDNA increased, further proving that ZC3H12A affects DNA damage repair.

[0081] Immunofluorescence analysis was performed on HeLa cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours after exposure to 4 Gy ionizing radiation. Cells were stained with DAPI (blue), dsRNA (green), and 53BP1 (red) and observed under a confocal microscope. Data are mean ± SD, n ≥ 50. **** P < 0.0001 (unpaired t-test). The results are shown in Figure 10. Under 4 GyIR 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, dsRNA increased, indicating that ZC3H12A knockdown led to increased genomic instability.

[0082] Immunofluorescence analysis was performed on HeLa cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours after exposure to 4 Gy ionizing radiation. Cells were stained with DAPI (blue), DNA-RNA (green), and 53BP1 (red) and observed under a confocal microscope. Data are mean ± SD, n ≥ 50. **** P < 0.0001 (unpaired t-test). The results are shown in Figure 11. Under 4GyIR conditions, after ZC3H12A knockdown, 53BP1, 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 knockdown affects DNA damage repair and leads to increased genomic instability.

[0083] Immunofluorescence experiments were performed on Hela cells and U2OS cells exposed to 8 Gy of 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). The results are shown in Figure 12. Changes in ZC3H12A expression were detected in HELA and U2OS cells before and after irradiation with 8 Gy irradiation. The results indicate that ZC3H12A expression increases after ionizing radiation. Radiotherapy, as a means of inducing DNA damage, also promotes increased ZC3H12A expression. Therefore, it is suggested that ZC3H12A may affect DNA damage repair.

[0084] Example 4 Plate cloning

[0085] Experimental purpose: To demonstrate the effect of ZC3H12A on the sensitivity of small cell radiotherapy.

[0086] Experimental methods:

[0087] 1. Plate approximately 120,000 cells per well of a 12-well plate.

[0088] 2. After overnight, transfect siRNA for 48 hours.

[0089] 3. Immediately after 4 Gy ionizing radiation, cells were trypsinized and centrifuged at 800 RPM for 5 minutes.

[0090] 4. Count the cells, dilute and add 2 ml of culture medium to a six-well plate, and seed 1500 cells per well.

[0091] 5. After 14 days, discard the waste solution, wash three times with PBS, and fix with 4% paraformaldehyde for 15 minutes.

[0092] 6. Incubate with crystal violet stain at room temperature for 30 minutes, rinse with water, and let dry.

[0093] Experimental results:

[0094] HeLa cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours were subjected to plate cloning assays at 1 Gy, 2 Gy, and 4 Gy ionizing radiation. The number of cell colonies was counted 14 days later. The survival fraction was calculated as the number of cell colonies at each dose divided by the number of cell colonies at 0 Gy. Data are mean ± SD, N = 3. * P=0.01, *** P=0.0007, ***P = 0.0004 (unpaired t-test). The results are shown in Figure 13. Comparison of the number of clones in the ZC3H12A knockdown and NC groups in HELA cells at different ionizing radiation doses showed that after ZC3H12A knockdown, the number of clones decreased more significantly than that in the negative control group, indicating that ZC3H12A knockdown can increase the radiosensitivity of cells.

[0095] A plate cloning assay was performed on 446 cells transfected with negative control (siNC) and ZC3H12A knockdown (siZC3H12A) for 48 hours after exposure to 1 Gy, 2 Gy, and 4 Gy ionizing radiation. The number of cell colonies was counted 14 days later. The survival fraction was calculated as the number of cell colonies at each dose divided by the number of cell colonies at 0 Gy. Data are mean ± SD, N = 3. ** P=0.0015, * P=0.0285, * P = 0.0232 (unpaired t-test). The results are shown in Figure 14. Comparison of the number of clones in the ZC3H12A knockdown and NC groups in NCI-H446 cells at different ionizing radiation doses showed that after ZC3H12A knockdown, the number of clones decreased more significantly than that in the negative control group, indicating that ZC3H12A knockdown can increase the radiosensitivity of cells.

[0096] Example 5: Separation of subcellular proteins and Western blot analysis

[0097] Experimental purpose: To detect the recruitment of ZC3H12A to various cell components after IR.

[0098] Experimental methods:

[0099] 1. Treat cells with 8 Gy of ionizing radiation.

[0100] 2. Use the ThermoScientific Subcellular Protein Fractionation Kit to separate the cell components.

[0101] 3. Perform protein electrophoresis on the separated subcellular proteins.

[0102] 4. Electrophoresis at 80 v for 30 min, 120 v for 1 h. Electrotransfer at 400 mA for 40 min.

[0103] 5. Block with 5% skim milk for 1 hour and incubate with 1 antibody at 4°C overnight.

[0104] 6. Incubate with secondary antibody at room temperature for 1 hour and then expose for detection.

[0105] Experimental results:

[0106] Western blotting was performed on the 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. The results are shown in Figure 15. Under 8Gy IR conditions, the various components of HELA cells were fractionated. The expression of ZC3H12A was detected at different time points after IR. After IR, the expression of ZC3H12A in total protein increased, and the expression of ZC3H12A in chromatin also showed an increase. However, the expression level of ZC3H12A in the cytoplasm remained almost unchanged. Therefore, after exposure to ionizing radiation, ZC3H12A may be recruited to chromatin to play a role.

Claims

1. 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.

2. A marker for diagnosing DNA damage during radiotherapy or chemotherapy of small cell lung cancer according to claim 1, characterized in that: The nuclease is preferably ZC3H12A.

3. A method for screening markers for diagnosing DNA damage during radiotherapy or chemotherapy of small cell lung cancer, the method comprising the following steps: S1. Construction of nuclease siRNA library; S2. small cell lung cancer cells are transfected with the siRNA library, and then irradiated to block cell division; 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.

4. A method for screening markers for diagnosing DNA damage during radiotherapy or chemotherapy of small cell lung cancer according to claim 3, characterized in that: In step S1, a nuclease library including DNA exonuclease and RNA exonuclease is constructed.

5. The method for screening markers for diagnosing DNA damage during radiotherapy or chemotherapy of small cell lung cancer according to claim 3, characterized in that: In step S2, the radiation treatment is performed at 4 Gy-8 Gy.

6. A diagnostic kit for diagnosing DNA damage during radiotherapy of small cell lung cancer using the marker according to claim 1, wherein the diagnostic kit comprises a diagnostic reagent and instructions.

7. A diagnostic kit for diagnosing DNA damage during radiotherapy of small cell lung cancer using the marker according to claim 1 according to claim 6, characterized in that: When one or more genes of 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.

8. Use of the marker according to claim 1 in the preparation of a detection reagent for diagnosing DNA damage during radiotherapy of small cell lung cancer.

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