Application of fusion gene RPL17-C18orf32 as benzo [g, h, i] perylene acute exposure biomarker

Abstract

The invention discloses an application of a fusion gene RPL17-C18orf32 as a benzo [g, h, i] perylene acute exposure marker, an in-vitro benzo [g, h, i] perylene acute exposure human lung bronchial epithelial cell model is established, abnormal expression of the fusion gene RPL17-C18orf32 is detected, and RPL17-C18orf32 participates in benzo [g, h, i] perylene acute exposure marker expression. The processes of cell proliferation capacity decline, apoptosis rate increase, cycle disorder, cell DNA damage and the like caused by acute exposure of benzo [g, h, i] perylene, so that the fusion gene RPL17-C18orf32 can be used as a biomarker for acute exposure of benzo [g, h, i] perylene. The invention not only provides a potential biomarker for early diagnosis for evaluating the acute exposure of the benzo [g, h, i] perylene, but also provides a new strategy for developing diseases related to the acute exposure of the benzo [g, h, i] perylene for preventing and treating the acute exposure of the benzo [g, h, i] perylene.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and more specifically, relates to the application of the fusion gene RPL17-C18orf32 as a biomarker for acute exposure to benzo[g,h,i]perylene. Background Art

[0002] Ambient air pollution is an important global public health issue with a significant impact on the world. According to the Global Burden of Disease report, air pollution is the leading cause of the global burden of disease. The environmental concentration of benzo[g,h,i]perylene (BghiP) in atmospheric polycyclic aromatic hydrocarbons is high. Studies have shown that BghiP is the PAHs with the highest exposure concentration in the Mexican atmosphere. For the first time, a study found that BghiP increased cell membrane damage and exerted toxic effects on the NL-20 human bronchial cell line by activating aromatic hydrocarbon receptors and increasing CYP4B1 expression levels. In addition, BghiP induced apoptosis of mouse oocytes by disrupting the distribution of mitochondria and increasing oxidative stress levels. BghiP increases carcinogenicity and causes lung cancer together with other compounds through synergistic effects. But overall, the current research on BghiP, especially in the identification of exposure biomarkers and mechanism research, is not in-depth enough.

[0003] Studies have found that PAHs exposure can cause DNA damage, and exogenous compounds are closely related to DNA damage and the genomic instability it causes. Genomic instability may cause chromosomal rearrangement, deletion, and translocation, causing the fusion of all or part of the sequences of two genes. Since its discovery in the 1960s, fusion genes have been widely used in clinical practice as biomarkers and drug targets for cancers such as gastric cancer, esophageal cancer, lung cancer, and nasopharyngeal cancer. In recent years, researchers have detected fusion genes in normal tissues and cells. These studies have shown that fusion genes are key molecules for maintaining cell function. Studying fusion genes helps to interpret the genetic information of the genome. In the article "Overexpression of KLHL23 protein from read-through transcription of PHOSPHO2-KLHL23 in gastric cancer increases cell proliferation", it was disclosed that the expression levels of three candidate genes PHOSPHO2-KLHL23, RPL17-C18orf32, and PRR5-ARHGAP8 in gastric cancer were significantly increased compared with normal tissues. The three genes can be used as molecular markers for the diagnosis of gastric cancer. However, there is a lack of relevant research on the toxicity mechanism of exogenous compounds, especially the research on the toxicity mechanism of RPL17-C18orf32 in exogenous compounds. Studying the role of fusion genes in the toxicity mechanism of BghiP is an important supplement to the toxicity mechanism of exogenous compounds. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide an application of the fusion gene RPL17-C18orf32 as a marker of acute exposure to benzo[g,h,i]perylene in the preparation of a product for early assessment of cell damage caused by acute exposure to benzo[g,h,i]perylene.

[0005] The second object of the present invention is to provide a reagent for detecting the expression level of the fusion gene RPL17-C18orf32 for use in the preparation of a product for evaluating cell damage caused by acute exposure to benzo[g,h,i]perylene.

[0006] The third object of the present invention is to provide a use of an agent for inhibiting the expression level of the fusion gene RPL17-C18orf32 in the preparation of a product for alleviating cell damage caused by acute exposure to benzo[g,h,i]perylene.

[0007] The fourth object of the present invention is to provide a reagent for promoting the expression level of the fusion gene RPL17-C18orf32 for use in preparing a cell damage model caused by acute exposure to benzo[g,h,i]perylene.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] According to the results of cytotoxicity experiments, the present invention continuously exposed human lung bronchial epithelial BEAS-2B cells with 500 ng / mL benzo[g,h,i]perylene (Benzo[ghi]perylene, BghiP) for 2 weeks to construct an in vitro acute poisoning model. After BghiP poisoning, CCK-8 and EdU experiments were used to detect cell proliferation, flow cytometry was used to detect cell apoptosis and cell cycle, comet assay, Western blot and cell immunofluorescence assay were used to detect cell DNA damage. It was found that acute BghiP poisoning caused decreased cell proliferation ability, increased cell apoptosis rate, cell cycle disorder and cell DNA damage. RNA-seq was used to sequence and analyze BEAS-2B cells acutely poisoned with BghiP, and the abnormally expressed fusion gene RPL17-C18orf32 was screened out, and its expression level in BghiP acutely exposed cells was detected. The results showed that the expression level of the fusion gene RPL17-C18orf32 was significantly increased in both BghiP acutely exposed cells and the BghiP acute poisoning mouse model in vivo. Cell lines with knockdown and overexpression of RPL17-C18orf32 were constructed for subsequent experiments. The results showed that the fusion gene RPL17-C18orf32 was involved in the processes of decreased cell proliferation ability, increased cell apoptosis rate, cell cycle disorder and cell DNA damage caused by acute exposure to BghiP. Knocking down the expression level of RPL17-C18orf32 could alleviate the above-mentioned cell damage process.

[0010] Therefore, the present invention provides the use of the fusion gene RPL17-C18orf32 as a marker for acute exposure to benzo[g,h,i]perylene in the preparation of products for early assessment of cell damage caused by acute exposure to benzo[g,h,i]perylene, and the nucleotide sequence of the RPL17-C18orf32 is shown in SEQ ID No.1.

[0011] The present invention also provides the use of a reagent for detecting the expression level of the fusion gene RPL17-C18orf32 in the preparation of a product for evaluating cell damage caused by acute exposure to benzo[g,h,i]perylene, wherein the nucleotide sequence of the RPL17-C18orf32 is shown in SEQ ID No.1.

[0012] Furthermore, the reagent for detecting the expression level of the fusion gene RPL17-C18orf32 is a real-time fluorescence quantitative PCR primer, and the nucleotide sequences thereof are shown in SEQ ID No. 2 to 3.

[0013] The present invention also provides the use of a reagent for inhibiting the expression level of the fusion gene RPL17-C18orf32 in the preparation of a product for alleviating cell damage caused by acute exposure to benzo[g,h,i]perylene, wherein the nucleotide sequence of the RPL17-C18orf32 is shown in SEQ ID No.1.

[0014] Furthermore, the reagent for inhibiting the expression level of the fusion gene RPL17-C18orf32 is siRNA, and the nucleotide sequences thereof are shown in SEQ ID No. 4 to 6, respectively.

[0015] The present invention also provides the use of a reagent for promoting the expression level of the fusion gene RPL17-C18orf32 in preparing a cell damage model caused by acute exposure to benzo[g,h,i]perylene, wherein the nucleotide sequence of the RPL17-C18orf32 is shown in SEQ ID No. 1.

[0016] Furthermore, the cDNA sequence of the fusion gene RPL17-C18orf32 is shown in SEQ ID No.7.

[0017] Furthermore, the cell damage is one or more of decreased cell proliferation ability, increased cell apoptosis rate, cell cycle disorder or cell DNA damage.

[0018] Furthermore, the cells are lung bronchial epithelial cells, including but not limited to human, mouse, bovine, horse and other cells.

[0019] Furthermore, the product is a test kit or a test paper.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides an application of a fusion gene RPL17-C18orf32 as a marker for acute exposure to benzo[g,h,i]perylene. The present invention establishes an in vitro human lung bronchial epithelial cell model of acute exposure to benzo[g,h,i]perylene, explores the acute toxic effect of benzo[g,h,i]perylene on human lung bronchial epithelial cells and its potential mechanism of action through RNA-seq analysis, and reveals for the first time the key role of the fusion gene RPL17-C18orf32 in the cell response induced by benzo[g,h,i]perylene. Overexpression of the fusion gene RPL17-C18orf32 can lead to decreased cell proliferation ability, increased cell apoptosis rate, cell cycle disorder and cell DNA damage, etc., providing a new molecular marker for the study of the acute toxic effect of benzo[g,h,i]perylene. Therefore, the fusion gene RPL17-C18orf32 can be used as a biomarker for acute exposure to benzo[g,h,i]perylene. The present invention not only provides a new scientific basis for evaluating the health risks of acute exposure to benzo[g,h,i]perylene, but also provides a potential biomarker for early diagnosis and health monitoring of acute exposure to benzo[g,h,i]perylene, and provides a new strategy for developing prevention and treatment of diseases related to acute exposure to benzo[g,h,i]perylene. In addition, this research method can also provide reference value for toxicity research of other environmental pollutants and discovery of biomarkers, and has important application value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a test for the cell proliferation ability of BEAS-2B cells after acute exposure to BghiP. Figure 1 A in the middle is a CCK-8 experiment, and B is an EdU experiment.

[0023] Figure 2 This is the detection of cell apoptosis and cell cycle in BEAS-2B cells after acute exposure to BghiP. Figure 2 A is a cell apoptosis experiment, and B is a cell cycle experiment.

[0024] Figure 3 This is the DNA damage of BEAS-2B cells after acute exposure to BghiP. Figure 3 A is the comet assay, B is the Western blot assay, and C is the cell immunofluorescence assay.

[0025] Figure 4 RNA-seq analysis of BEAS-2B cells after acute exposure to BghiP for 2 weeks. Figure 4 A is the heat map of differentially expressed genes, B is the volcano map, and C is the KEGG pathway analysis of differentially expressed genes.

[0026] Figure 5For the identification of the target gene RPL17-C18orf32. Figure 5 A is a gel electrophoresis diagram, B is Sanger sequencing, C is a schematic diagram of the RPL17-C18orf32 structure, and D is the expression level of RPL17-C18orf32 after acute exposure of BEAS-2B cells to BghiP.

[0027] Figure 6 The expression level of the fusion gene RPL17-C18orf32 in the benzo[g,h,i]perylene acute exposure and acute poisoning mouse model.

[0028] Figure 7 The RPL17-C18orf32 knockdown and overexpression cells were constructed. Figure 7 A in the figure is the knockdown effect of Si-RC-1 detected by qRT-PCR, B is the knockdown effect of Si-RC-2 detected by qRT-PCR, C is the knockdown effect of Si-RC-3 detected by qRT-PCR, and D is the qRT-PCR detection result of overexpression of RPL17-C18orf32.

[0029] Figure 8 To detect the proliferation ability of knockdown and overexpression RPL17-C18orf32 cells. Figure 8 A in the figure is the proliferation ability detected by CCK-8 after knocking down RPL17-C18orf32, B is the proliferation ability detected by CCK-8 after overexpressing RPL17-C18orf32, C is the proliferation ability detected by EdU after knocking down RPL17-C18orf32, and D is the proliferation ability detected by EdU after overexpressing RPL17-C18orf32.

[0030] Fig. 9 To detect cell apoptosis and cell cycle after knockdown and overexpression of RPL17-C18orf32. Fig. 9 A in the middle is the cell apoptosis detection of the proliferation ability of RPL17-C18orf32 knocked down, B is the cell apoptosis detection of the proliferation ability of RPL17-C18orf32 overexpressed, C is the cell cycle detection of the proliferation ability of RPL17-C18orf32 knocked down, and D is the cell cycle detection of the proliferation ability of RPL17-C18orf32 overexpressed.

[0031] Fig.10 To detect DNA damage in knockdown and overexpression RPL17-C18orf32 cells. Fig.10A in the middle is a comet assay to detect DNA damage in the case of knocking down RPL17-C18orf32, B is a comet assay to detect DNA damage in the case of overexpressing RPL17-C18orf32, C is a Western blot assay to detect DNA damage in the case of knocking down RPL17-C18orf32, D is a Western blot assay to detect DNA damage in the case of overexpressing RPL17-C18orf32, E is a cell immunofluorescence assay to detect DNA damage in the case of knocking down RPL17-C18orf32, and F is a cell immunofluorescence assay to detect DNA damage in the case of overexpressing RPL17-C18orf32. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0034] 1. Experimental Materials

[0035] 1. Cell lines, experimental animals and test substances

[0036] Human lung bronchial epithelial cells BEAS-2B were purchased from the American Type Culture Collection (ATCC). Experimental animals BALB / c mice were purchased from the Guangdong Experimental Animal Center, and all animals were approved by the Animal Ethics Committee of Guangzhou Medical University (approval number: GY2024-036). BghiP analytical standards were purchased from Shanghai Aladdin Biochemical Materials Technology Co., Ltd., China.

[0037] 2. Main instruments

[0038] Low-speed centrifuge (ScanSpeed), liquid nitrogen tank (Sichuan Leshan Company, China), ice machine (SANYO Company, Japan), clean bench (Thermo Fisher Scientific, USA), high-temperature constant-temperature blast drying oven (Shanghai Fuma Company, China), VORTEX-5 vortex mixer (Haimen Qilin Bell Company), water-jacketed CO2 cell culture incubator (ThermoFisher Scientific, USA), TS-2000A decolorization shaker (Haimen Qilin Bell Company), -80℃ ultra-low temperature refrigerator (Thermo Fisher Scientific, USA) Scientific Company), LS-75HD vertical pressure steam sterilizer (Jiangyin Binjiang Company, China), long-term low temperature operation platform module (Biocision Company, USA), electric constant temperature water bath (Changfeng Company, Suzhou, China), TU-100 constant temperature digital metal bath (Shanghai Yiheng Scientific Instrument Co., Ltd.), Bio-Rad universal power supply 164-5070 (Bio-Rad Company, USA), inverted biological microscope TS100-F (Nikon Company, Japan), EVOS eyepiece-free microscope (AMG Company, USA), horizontal electrophoresis tank (Bio-Rad Company, USA), ME204 analytical balance (Mettler Toledo Company, USA), refrigerator (Haier, Samsung), 5424R refrigerated centrifuge (Eppendorf Company, Germany), LX-300 mini centrifuge (Haimen Qilin Bell Company), vertical electrophoresis tank (Bio-Rad Company, USA), Synergy HT multifunctional microplate reader (Bio-Tek, USA), ultrapure water system (Millipore, USA), wet transfer tank (Bio-Rad, USA), BLT GV1500Pro fully automatic gel imaging system (Guangzhou Bo Lu Teng Company, China), laser confocal microscope SP8 (Leica, Germany), micropipette and supporting consumables (Eppendorf, Germany), HOPE-MED 8052H single concentration oral and nasal inhalation exposure system (Hepu Industry and Trade Co., Ltd., Tianjin Development Zone, China).

[0039] Example 1 Construction of in vitro model of acute exposure to BghiP and study of in vitro toxicity

[0040] 1. Experimental Methods

[0041] 1.Benzo[ghi]perylene (BghiP) poisoning

[0042] The BghiP analytical standard was dissolved in DMSO, and the BghiP concentration gradient was set to 0, 5, 50, 500 and 5000 ng / mL. After 24 hours of exposure, the appropriate exposure concentration was determined, and a time gradient exposure (0, 1, 2, 3 and 4 weeks) was performed to conduct a preliminary experiment on BEAS-2B cells. The volume fraction of DMSO was 0.1% to control the effect of the solvent on the cells. According to the results of the preliminary experiment, BEAS-2B cells with 500 ng / mL BghiP continuously exposed for 2 weeks were selected to construct an acute exposure model, and the first generation of infected cells were selected for subsequent experiments.

[0043] 2. Cell counting kit-8 (CCK-8 assay)

[0044] In a 96-well plate, 2000 cells in the logarithmic growth phase were inoculated in each well, and 5 replicate wells were set up, and the cells were cultured for 6-8 hours to allow the cells to adhere to the wall. This was the 0h group, and the subsequent cultures were cultured for 24 hours, 48 ​​hours, and 72 hours. After the culture was completed, the culture medium was replaced, and 100 μL of CCK-8 mixed solution (CCK-8 reagent and complete culture medium were prepared in a ratio of 1:10) was added to each well. After incubation for 1 hour, the OD value at 450nm was measured with an ultra-micro spectrophotometer to evaluate cell activity.

[0045] 3.5-ethynyl-2-deoxyuridine (EdU experiment)

[0046] 30,000 cells were inoculated per well in a 24-well plate, and the cells were cultured in a constant temperature incubator for 48 hours, then washed with PBS buffer and aspirated, and then EdU medium was added for 2 hours and washed. After fixing the cells for 40 minutes, they were treated with glycine solution for 5 minutes, and then washed with PBS and TritonX-100 in sequence. Finally, Apollo and Hoechst33342 staining solutions were used for staining for 40 minutes respectively, and pictures were taken under a fluorescence microscope after washing, and the cell proliferation was quantitatively analyzed using Image J software.

[0047] 4. Cell apoptosis experiment

[0048] 100,000 cells were inoculated per well in a 6-well plate and cultured for 24 hours before the experiment. The cells were digested with EDTA-free trypsin, collected and washed twice with pre-cooled PBS. 7-AAD and Annexin V-PE were added to BindingBuffer and mixed with the cells. After incubation in the dark, the apoptosis was detected by flow cytometry and analyzed by CytExpert software.

[0049] 5. Cell cycle assay

[0050] 100,000 cells were inoculated per well in a 6-well plate and cultured for 24 hours before the experiment. The cells were digested and collected, washed with pre-cooled PBS, and fixed with 70% ethanol for at least 2 hours. After washing and centrifugation, RNase A and PI staining working solution were prepared, the samples were stained and incubated in the dark for 30 minutes. Finally, flow cytometry was used to detect and analyze the cell cycle using FlowJo software.

[0051] 6. Comet assay

[0052] Wash the slides and dry them. Prepare the first layer of gel containing normal melting point agarose in a 58°C metal bath, drop it on the slide, let it stand and then store it. Prepare the second layer of gel containing low melting point agarose in a 40°C metal bath, add the cell suspension and drop it on the first layer of gel, let it stand and solidify. Immerse the slides in 4°C cell lysis solution for 1 hour in the dark, then put them in the electrophoresis tank, add pre-cooled electrophoresis buffer, and perform electrophoresis at a constant voltage of 25V for 30 minutes. After neutralization, stain with NA-Red staining solution, dry it, take pictures under a fluorescence microscope, and analyze DNA damage indicators with CSAP software.

[0053] 7. Western blot experiment

[0054] After the cells grew to 70% confluence, they were washed with pre-cooled PBS and lysed with RIPA lysis buffer containing PMSF for 30 minutes. The lysate was collected and centrifuged, and the supernatant was stored at -80°C. The protein concentration was determined using a BCA kit. The sample was mixed with the reagent and incubated at 37°C for 30 minutes, and the OD value at 562nm was measured. The protein sample was mixed with SDS-PAGE loading buffer and denatured at 98°C for 5 minutes and stored at -20°C. After preparing the SDS-PAGE gel, the protein sample and marker were electrophoresed and transferred to a PVDF membrane after constant voltage electrophoresis. After treating the PVDF membrane with blocking solution, the primary and secondary antibodies were added respectively, and TBST was used to wash after each incubation. Finally, the results were scanned and analyzed using a near-infrared imaging system.

[0055] 8. Cell Immunofluorescence Experiment

[0056] Add 10 μL of complete culture medium and cell slides to a 24-well plate, then add 1 mL of a suspension containing 10,000 cells and culture for 24 hours. After washing with PBS, fix with 4% fixative for 20 minutes, then wash with PBS and 0.5% Triton X-100 in sequence. After blocking for 1 hour, add the primary antibody solution and keep overnight at 4°C. The next day, wash with PBS and add the fluorescent secondary antibody solution, and protect from light for 1 hour. After washing with PBS, add DAPI staining, wash and dry. Invert the cell slide on a glass slide and store it at 4°C for one month after sealing. Finally, use a confocal microscope to observe and count the number of cell foci.

[0057] 2. Experimental Results

[0058] (1) Effects of acute exposure to BghiP on cell proliferation

[0059] The results of CCK-8 and EdU experiments are as follows Figure 1 As shown, acute exposure to BghiP caused a significant decrease in the proliferation ability of BEAS-2B cells.

[0060] (2) Effects of acute exposure to BghiP on cell apoptosis and cell cycle

[0061] The experimental results of cell apoptosis and cell cycle are as follows Figure 2 As shown, acute exposure to BghiP increased the apoptosis rate of BEAS-2B cells and blocked the cell cycle progression.

[0062] (3) Effects of acute exposure to BghiP on cellular DNA damage

[0063] Comet assay, Western blot and cell immunofluorescence assay results are shown in Figure 3 As shown, acute exposure to BghiP caused DNA double-strand breaks in BEAS-2B cells, leading to DNA damage and triggering genomic instability.

[0064] Example 2 RNA-seq sequencing and fusion gene screening and identification

[0065] 1. Experimental Methods

[0066] 1. RNA sequencing (RNA-seq)

[0067] The infected cells (BEAS-2B cells were exposed to 500 ng / mL BghiP for 2 weeks to construct an acute infection model, and the first generation of infected cells were selected) were sent to Beijing Biomarker Biotechnology Co., Ltd., and the company provided the sequencing results.

[0068] 2. Total RNA Extraction

[0069] When the cells grow to 70% confluence, wash with PBS and add Trizol reagent to collect the cells, transfer to EP tubes after pipetting. Add chloroform to the Trizol suspension, shake vigorously, let it stand and centrifuge, and aspirate the supernatant into a new EP tube. Next, add isopropanol, mix well, let it stand, and retain the RNA precipitate after centrifugation. Wash the RNA precipitate with pre-cooled 75% ethanol and anhydrous ethanol, dry it, dissolve it with enzyme-free water, and store it at -80℃ for later use.

[0070] 3. Reverse transcription of RNA

[0071] Take 1.8 μL RNA sample to test the concentration and quality. The OD260 / OD280 ratio of qualified samples should be between 1.8 and 2.0. TM The Reverse Transcription System kit reverse transcribes RNA into cDNA. First, mix the RNA sample with Oligo(dT)Primer and Random Primer, denature at 70°C and let stand at 4°C. Then, prepare a mixture containing Rnasin Ribonuclease Inhibitor, PCR Nucleotide Mix, GoScript ReverseTranscriptase, MgCl 2 The reaction mixture of , Reaction Buffer and Nuclease-Free Water was added to the denatured RNA mixture and reverse transcription was performed according to the program of 25℃, 42℃ and 70℃. After completion, the cDNA samples were stored at -20℃.

[0072] 4.PCR

[0073] use qPCR Master Mix Kit detects the presence of fusion genes. Mix enzyme-free water, forward and reverse primers, qPCR Master Mix and sample cDNA, a total of 20μL reaction system. Place the mixture in a thermal cycler, first perform reverse transcription at 50℃ for 10 minutes and pre-denaturation at 95℃ for 2 minutes, then perform 40 cycles of denaturation at 95℃ for 15 seconds, annealing at 60℃ for 1 minute, and extension at 72℃ for 1 minute, and finally extend at 72℃ for 10 minutes to complete amplification. The primers are as follows:

[0074] Fusion gene RNA RPL17-C18orf32:

[0075] Forward primer: 5'-TCTCCCTGCCACATTGAGAT-3' (SEQ ID No. 2)

[0076] Reverse primer: 5'-AGGAATGCACACCATTTTCC-3' (SEQ ID No. 3)

[0077] 5. Agarose gel electrophoresis and Sanger sequencing

[0078] Weigh 2.0g agarose powder, add 100mL 1×TBE electrophoresis buffer, and heat in a microwave oven until completely dissolved. Add NA-Red dye, mix well, pour into the mold, insert the comb, and let stand at room temperature for 30 minutes. After pulling out the comb, put the gel into the electrophoresis tank, add 1×TBE buffer, and connect the hole to the black pole. Mix the DNA loading buffer with the RT-PCR product and add it to the well, and add DNA Marker on both sides. Electrophoresis at a constant voltage of 100V for 50 minutes, then observe and take pictures with a UV Tray ultraviolet excitation plate. Confirm the length of the fusion gene fragment by agarose gel electrophoresis, and send the PCR product to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing.

[0079] 6. Quantitative reverse transcript-polymerase chain reaction (qRT-PCR)

[0080] use qPCR Master Mix Kit, prepared according to 20μL reaction system: Mix enzyme-free water, C×R, forward and reverse primers, qPCR Master Mix and sample cDNA. The mixture was placed in a 7500PCR instrument and pre-denatured at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing / extension at 60±3°C for 1 minute. Melting curve analysis was then performed. -ΔΔCt The differential expression fold was calculated based on the GAPDH internal reference gene, ΔCt = Ct target gene - Ct internal reference gene, ΔΔCt = ΔCt experimental group - ΔCt control group. The differential expression fold of the target gene in the experimental group compared with the control group = 2 -ΔΔCt The gene primers were synthesized by Guangzhou Aiji Biotechnology Co., Ltd. and the primers were designed as follows:

[0081] GAPDH:

[0082] Forward primer: 5'-ACAGTCAGCCGCATCTTCTT-3' (SEQ ID No. 8)

[0083] Reverse primer: 5'-GACTCCGACCTTCACCTTCC-3' (SEQ ID No. 9)

[0084] RPL17:

[0085] Forward primer: 5'-AGAGGTTCCAATCTTCGTGTTCACTT-3' (SEQ ID No. 10)

[0086] Reverse primer: 5'-ACCTGCCAACTCCACCATTGTAAC-3' (SEQ ID No. 11)

[0087] C18orf32:

[0088] Forward primer: 5'-GCAGCCAACTCCACCTGACTTC-3' (SEQ ID No. 12)

[0089] Reverse primer: 5'-CCAGCAAGCCAGATAGCACTCTT-3' (SEQ ID No. 13)

[0090] 2. Experimental Results

[0091] (1) RNA-seq sequencing analysis

[0092] RNA-seq was used to perform mRNA sequencing analysis of the BEAS-2B cell Control and BghiP groups. The results are as follows Figure 4 As shown, compared with the Control group, 199 differentially expressed genes were detected in the BghiP group, of which 63 were upregulated and 136 were downregulated. Among them, 22 chimeric RNAs were identified and screened by STAR-Fusion.

[0093] (2) Screening of target fusion gene RPL17-C18orf32

[0094] The results are as follows Figure 5 As shown, the fusion gene RPL17-C18orf32 (SEQ ID No. 1) showed a bright band in BEAS-2B cells, and Sanger sequencing results showed that the fusion gene RPL17-C18orf32 was fused from the parental genes RPL17 and C18orf32. The results of qRT-PCR experiments showed that the expression level of the fusion gene RPL17-C18orf32 in the BghiP group of BEAS-2B cells was significantly increased.

[0095] Example 3 Effects of Acute Exposure to Benzo[g,h,i]perylene on RPL17-C18orf32 in Mice

[0096] 1. Experimental Methods

[0097] 1. Experimental animals and groups

[0098] Sixteen 3-4 week old SPF grade BALB / c mice, half male and half female, were purchased and quarantined to adapt to the environment for one week. They were randomly divided into a control group and a benzo[g,h,i]perylene poisoning group, with 4 males and 4 females in each group.

[0099] 2. Setting of exposure conditions

[0100] According to the conversion formula for internal and external exposure doses in respiratory toxicology, 500 μg / m 3 As the concentration for mice to be exposed to. The mice were in good condition after adapting to this concentration for one week, so this concentration was selected as the final exposure concentration, and a DMSO solvent control group was set up. At the same time, the exposure conditions for in vivo acute inhalation toxicity experiments were set with reference to international standards and guidelines (OECD TG 412), and the exposure time and frequency were set to 28 days, 6h / day, and 7d / week. The growth status of the mice was also observed during the exposure period.

[0101] 3. Collection of mouse lung tissue and extraction of total RNA from lung tissue

[0102] After the exposure, the mice were killed by cervical dislocation and the mouse lung tissues were collected and stored at -80°C for future use. About 20 mg of lung tissue was added with 1 mL of Trizol solution and fully ground by a tissue grinder. The subsequent extraction of total RNA from mouse lung tissue was consistent with the extraction method of total cell RNA in Example 2.

[0103] 4. Detection of the expression level of RPL17-C18orf32 in mouse lung tissue

[0104] After total RNA was extracted from the lung tissue of the mouse model, reverse transcription and qRT-PCR experiments were performed according to the experimental method in Example 2 to detect the expression level of RPL17-C18orf32.

[0105] 2. Experimental Results

[0106] The expression level of TVP23C-CDRT4 in mouse models Figure 6 As shown, the expression level of TVP23C-CDRT4 was significantly increased in mice acutely exposed to benzo[g,h,i]perylene compared with the Control group.

[0107] Example 4 Construction of fusion gene RPL17-C18orf32 knockdown and overexpression cell lines

[0108] 1. Experimental Methods

[0109] 1. RNA interference

[0110] In a 6-well plate, 200,000 cells were inoculated into each well and divided into Si-1, Si-2, Si-3 groups, negative control NC group and Control group. medium, and the other groups were in Opti- After adding siRNA to the culture medium and incubating, mix with LipofectamineTM2000, add to the well plate and culture for 24 hours, then replace with complete culture medium and culture for another 24 hours. Guangzhou Ruibo Biotechnology Co., Ltd. provides siRNA kits, the sequence is as follows:

[0111] siRNA target sequence:

[0112] Si-RPL17-C18orf32-1(Si-RC): 5'-GCCCAGAAGAAAAAGTTGA-3'(SEQ ID No.4)

[0113] Si-RPL17-C18orf32-2 (Si-RC-2): 5'-CCAGAAGAAAAAGTTGAGG-3' (SEQ ID No. 5)

[0114] Si-RPL17-C18orf32-3 (Si-RC-3): 5'-AAAGTTGAGGAGCTCAAGC-3' (SEQ ID No. 6)

[0115] 2. Stable lentiviral transfection

[0116] The virus was dissolved and the MOI was set to 200. A blank control group (BghiP), a negative control group (Vector) and an overexpression group (OE-RC, the nucleotide sequence is shown in SEQ ID No.7) were set up, and each group was repeated twice. 40,000 cells were inoculated in each well of a 24-well plate, and the virus was infected after 24 hours of culture. The required virus volume was calculated according to the virus titer and mixed with HiTransGP solution. The culture medium was discarded, washed with PBS and then the virus infection solution was added. After 24 hours of infection, the culture medium was replaced, puromycin was added and cultured continuously for 2 weeks, green fluorescence was observed under a microscope and the infection effect was detected by RT-qPCR. The vector was constructed by Shanghai JiKai Gene Co., Ltd.

[0117] 2. Experimental Results

[0118] We selected RNA interference to construct the fusion gene RPL17-C18orf32 knockdown cell line (Si-RC), and selected lentiviral stable transfection to construct the fusion gene RPL17-C18orf32 overexpression cell line (OE-RC) for subsequent experimental studies, such as Figure 7 As shown, qRT-PCR experiments confirmed that the fusion gene RPL17-C18orf32 knockdown and overexpression cell lines were successfully constructed.

[0119] Example 5 Functional verification of the fusion gene RPL17-C18orf32

[0120] 1. Experimental Results

[0121] The experimental method is the same as in Example 1.

[0122] 2. Experimental Methods

[0123] (1) Effects of knockdown and overexpression of RPL17-C18orf32 on the proliferation of acutely exposed BghiP cells

[0124] like Figure 8 As shown, the proliferation ability was increased after knocking down RPL17-C18orf32, and the proliferation ability was decreased after overexpressing RPL17-C18orf32. The results of CCK-8 and EdU experiments both showed that RPL17-C18orf32 promoted the decrease in the proliferation ability of BEAS-2B cells induced by acute exposure to BghiP.

[0125] (2) Effects of knockdown and overexpression of RPL17-C18orf32 on apoptosis and cell cycle of acutely exposed BghiP cells

[0126] like Fig. 9 As shown, after knocking down RPL17-C18orf32, the cell apoptosis rate decreased, and the proportion of cells in the G0 / G1 phase increased. After overexpressing RPL17-C18orf32, the cell apoptosis rate increased, and the proportion of cells in the G0 / G1 phase decreased. The experimental results of cell apoptosis and cell cycle showed that RPL17-C18orf32 promoted the increase of apoptosis rate and blocked cell cycle process of BEAS-2B cells induced by acute exposure to BghiP.

[0127] (3) Effects of knockdown and overexpression of RPL17-C18orf32 on DNA damage in acutely exposed BghiP cells

[0128] like Fig.10 As shown, knockdown of RPL17-C18orf32 reduced DNA damage, while overexpression of RPL17-C18orf32 aggravated DNA damage. The results of comet assay, Western blot and cell immunofluorescence experiments all indicated that RPL17-C18orf32 promoted the DNA damage process of BEAS-2B cells induced by acute exposure to BghiP.

Claims

1. Use of the fusion gene RPL17-C18orf32 as a marker for acute exposure to benzo[g,h,i]perylene in the preparation of a product for early evaluation of cell damage caused by acute exposure to benzo[g,h,i]perylene, characterized in that: The nucleotide sequence of RPL17-C18orf32 is shown in SEQ ID No.

1.

2. Use of a reagent for detecting the expression level of the fusion gene RPL17-C18orf32 in the preparation of a product for evaluating cell damage caused by acute exposure to benzo[g,h,i]perylene, characterized in that: The nucleotide sequence of RPL17-C18orf32 is shown in SEQ ID No.

1.

3. Use of an agent for inhibiting the expression level of the fusion gene RPL17-C18orf32 in the preparation of a product for alleviating cell damage caused by acute exposure to benzo[g,h,i]perylene, characterized in that: The nucleotide sequence of RPL17-C18orf32 is shown in SEQ ID No.

1.

4. Use of an agent for promoting the expression level of the fusion gene RPL17-C18orf32 in preparing a cell injury model caused by acute exposure to benzo[g,h,i]perylene, characterized in that: The nucleotide sequence of RPL17-C18orf32 is shown in SEQ ID No.

1.

5. The use according to any one of claims 1 to 4, characterized in that: The cell damage is one or more of decreased cell proliferation ability, increased cell apoptosis rate, cell cycle disorder or cell DNA damage.

6. The use according to any one of claims 1 to 4, characterized in that: The cells are lung bronchial epithelial cells.

7. The use according to claim 2, characterized in that: The reagents for detecting the expression level of the fusion gene RPL17-C18orf32 are real-time fluorescence quantitative PCR primers, and the nucleotide sequences thereof are shown in SEQ ID No. 2-3.

8. The use according to claim 3, characterized in that: The reagent for inhibiting the expression level of the fusion gene RPL17-C18orf32 is siRNA, and the nucleotide sequences thereof are shown in SEQ ID No. 4 to 6.

9. The use according to claim 4, characterized in that: The cDNA sequence of the fusion gene RPL17-C18orf32 is shown in SEQ ID No.

7.

10. The use according to any one of claims 1 to 3, characterized in that: The product is a test kit or a test paper.

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