Clinical tissue samples
A total of 63 samples of primary cervical cancer (CC), cervical intraepithelial neoplasia (CIN) and adjacent normal tissues were obtained with informed consent from Women’s Hospital, School of Medicine, Zhejiang University review board (Zhejiang Province, China). All tumor specimens were pathologically diagnosed and non-tumor specimens were verified according to the standard procedures. None of the patients in this study had received pre-operative adjuvant chemotherapy or radiation therapy. The clinical characteristics of all CC patients were collected and listed in Table S1.
Cell culture and chemicals
Human CC cell lines SiHa, CaSki, and Hela were obtained from Procell Life Science and Technology Co. Ltd. (Wuhan, China). HaCaT, HEK-293T, and PT67 cells were obtained from the American Type Culture Collection (ATCC). SiHa and HaCaT cells were cultured in MEM medium (Gibco). CaSki cells were cultured in RPMI-1640 medium (Gibco). HEK-293T, HeLa and PT67 cells were cultured in DMEM medium (Gibco). All culture media were supplemented with 10% fetal bovine serum (FBS, Adamas), streptomycin (100 µg/ml), penicillin (100 units/ml) and Amphotericin B Solution (0.25µg/ml). Antibodies, chemical reagents and the experimental kits are listed in additional file Table S2, S3.
RNA extraction and Real-time quantitative PCR analysis (RT-qPCR)
Total RNA of the cultured cells was isolated using TRIzol (Invitrogen, cat# 15596026) according to the manufacturer’s instructions. RNA concentrations and quality were measured using the NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA was then reverse transcribed into cDNA products using RT Master Mix (MCE, HY-K0511A) in the PCR instrument (ETC811). Real-time PCR was carried out using SYBR Green qPCR Master Mix (MCE, HY-K0501A) on the Quantstudio™ 1 Real-Time PCR Instrument (Bio-Rad Laboratories, USA). The relative expression levels of target genes were calculated by the comparative CT (2−ΔΔCT) method and normalized to β-actin or GAPDH levels. The qPCR assays were done in 3 biological replicates and statistical significance was determined using Student's t-test, and a P value of < 0.05 was considered significant. Human qPCR primer sequences were listed in Table S4 and were. available from online PrimerBank database https://pga.mgh.harvard.edu/primerbank/.
Preparation of protein lysates and western blot analysis
For total protein lysate preparation, the cervical cancer cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then lysed on ice by RIPA Lysis buffer (Solarbio, R0010) supplemented with protease and phosphatase inhibitor cocktail (MCE, HY-K0013) Cell lysates were incubated on ice for 30 min with occasionally vortexed. After centrifugation at the speed of 12 000rpm for 15min, the protein concentration of clean supernatants was quantified by BCA protein assay kit (Beyotime, P0010S).
Equal amounts of protein lysates (30 µg) were separated by SDS-PAGE gels followed by a transfer to PVDF membranes using the BioRad system. The membranes were blocked with 5% nonfat milk for 1h at room temperature (RT) and then incubated with primary antibodies overnight at 4°C. Then the membranes were washed and incubated with goat anti-rabbit or mouse horseradish peroxidase (HRP)-labeled secondary antibodies for 1h at RT. BeyoECL Moon (Beyotime, P0018FM) was used for detecting chemiluminescent signals with ImageQuant™ LAS 500 instrument. The antibody resources and dilutions used in this study were listed in Table S2.
Lentiviral transduction and establishment of TOP1 knockdown cell lines
The sequences of the sh-TOP1 plasmids used in our study were designed and synthesized by GeneChem Co., Ltd.(Shanghai, China). 293T cells were transfected with TOP1-specific shRNAs (sh-TOP1) and scrambled shRNA (sh-NC), using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. The generated viral particle supernatant was collected to transduce SiHa and HeLa cells at optimized concentrations. The transduced cells were selected by puromycin. The knockdown efficiency was confirmed by RT-qPCR and western blot analysis.
Generation of HPV E6/E7 knockdown or overexpression cells
Short hairpin RNA (shRNA) targeting HPV16 E6/E7, HPV18 E6/E7 and control were designed and synthesized by Tsingke Biotechnology (Shanghai, China). Lentiviral transduction was performed with above plasmids and lipofectamine 3000 reagents according to the manufacturer’s protocol. The efficiency of knockdown cells was verified by RT-qPCR and immunoblotting at 72 h after transfection. Sequences of these plasmids are listed in the supplementary Table S5.
Construction of retroviral vector pLXSN-vector, pLXSN-16E6, pLXSN-16E7, pLXSN-18E6, and pLXSN-18E7 were kindly provided by Dr. Laimonis A. Laimins (Northwestern University, Chicago, USA) and Dr. Frank Stubenrauch (University of Tübingen, Tübingen, Germany).As previously described[60], The individual viral plasmid was transfected into PT67 packaging cells to generate the viral particles. Then the HaCaT cells were transduced with viral supernatants and screened by G418 to generate stable cells expressing HPV16/18 E6 or E7.
Cell Counting Kit-8 (CCK8) assay
Cells (5 000 cells/well) were seeded in 96-well plates and cultured for 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h. Then 10 µL CCK8 (MCE, HY-K0301) was added into each well and incubated for 1h at 37°C. The absorbance of cells at each time course was determined by a microplate reader (Thermometer Varioskan LUX) at 450 nm wavelength. The cell viability was illustrated by GraphPad Prism 8 software. Each group was performed in 4 replicates and repeated in 3 independent biological experiments.
Wound healing assay
Cells were cultured to reach 90%-100% confluence in 6-well plates. The cell monolayers were vertically scratched with a 200 µL micropipette tip in the center of the well. The floating cells were washed away by PBS and the rest attached cells were then incubated in the serum-free medium for another 24 h at 37°C. Representative images were captured at 0h and 24 h after the injury using the microscopy imaging system. The width of baseline and wound healing region was measured by ImageJ. Cell migration rate = 100%x (width of wound at 0h–width of wound at 24 h) /width of wound at 0h. The experiment was independently repeated in triplicates.
Transwell assay
Cells (30 000) were seeded into the upper chamber of the 24-well plate (8.0 µm, Corning, USA) with serum-free medium (200 µL). The fresh medium (500 µL) containing 20% FBS was placed in the lower chamber. After 24h incubation, cells in the upper chambers were fixed using 4% PFA and stained with crystal violet. For the cell invasion assay, the upper chamber was initially coated with Matrigel (BD Bioscience, 356234) in the serum-free medium. The cells were then seeded for subsequent procedures. The representative fields were selected to be captured at 100X magnification by OLYMPUS BX53 microscope. The number of migration or invasion cells was calculated by ImageJ.
Real-time label-free cell analysis technique (RTCA)
Cells were counted to obtain a concentration of 5 000 cells/100 µL cell suspension. Prepare E-Plate 96 by adding 50 µL of medium to each well. By placing the E-Plate 96 in the RTCA Station for Scanning Plate and beginning Step 1 to detect the baseline. the E-Plate 96 was recovered and 100 µL of the prepared cell suspension was added to each well, ensuring thorough mixing. After allowing the plate to incubate for 30 min at room temperature. Returning the E-Plate 96 to the incubator on the RTCA Station and initiating the system to automatically scan the plate. The parameters of the RTCA program are cell type (SiHa, HeLa), cell number (5 000), interval (10 min), unit (min), total time (120 h), and the name of the compound (sh-NC, sh-TOP1#2, sh-TOP1#3). Then starting Step 2 to detect the cell proliferation curve.
Colony formation assay
800 cells/well from each group were seeded in 6-well plates. Then cells were observed during the incubation process until the majority of the individual clones contained more than 50 cells, indicating completion of the cloning process. Which usually took about 14 days to complete. The supernatant of the cell culture was discarded, and the cells were washed with PBS. For cell fixation, 4% paraformaldehyde was added to each well, and the plates were allowed to stand at room temperature for 30 min. Then cells were washed again with PBS. After washing with PBS, a crystal violet stain solution was added to each well and stained at room temperature for 20 min. After which the stained cells were washed several times with PBS. Cells were air dried, and 6-well plates were photographed using a digital camera. Based on the photographs taken, the individual clones were counted.
Co-immunoprecipitation (Co-IP)
Cells were lysed on ice for 30min using IP lysis buffer (Beyotime, P0013) along with protease and phosphatase inhibitor cocktails. Lysates were quantified for protein concentration using the BCA Protein Assay Kit. The total number of lysates was then divided into the input control group (50 µL lysates processed with loading buffer), IP group and IgG group. The latter two groups were incubated with the specific antibodies according to the manufacturer's instructions overnight. Followed by addition of beads of 20 uL protein A&G sepharose (Beyotime, P2055) with rotation for 2 h at 4°C. Beads of the mixture were washed three times with the IP lysis buffer and the immunoprecipitated protein complexes were resuspended in 2× SDS-PAGE protein sample loading buffer (Beyotime, P0288) for further analysis by western blotting.
Immunofluorescence
Cells (2 × 105) were seeded in 35 mm glass-bottom dishes (Ibidi). Cells were fixed with 4% PFA (Biosharp, BL539A) for 15 min, permeabilized in PBS containing 0.1% Triton X-100 for 15min and then blocked for 1h at RT, followed by incubated with the primary antibodies overnight at 4°C (TOP1 1:200, cGAS 1:200) or (BRCA1 1:200, γH2AX 1:500). The following day, Cells were washed 3 times with PBS, and incubated with the secondary antibodies (488 donkey anti-Mouse 1:2000, 555 donkey anti-Rabbit 1:500) in the dark for 1h. After washes, the cells were mounted with DAPI containing mounting medium (Solarbio, S2110), and visualized using a laser scanning confocal microscope (Nikon A1R). Images were subsequently processed using NIS-Elements Viewer 4.20 software, and the colocalization analysis with endosome markers was performed based on the Fiji/ImageJ.
Comet assay (Single Cell Gel Electrophoresis Assay)
Comet slides were pre-coated with cells (avoid light), mixed with molten low melting agarose (LMA) at 1:10 ratio, and placed in 4°C lysis solution overnight. Then the slides were incubated in cold neutral electrophoresis buffer for 30min followed by electrophoresis for 45 min at 21 V using Comet Assay® ES unit. Slides were placed in DNA precipitation solution for 30 minutes and washed with 70% ethanol. Dried slides (at 37°C) were then stained with SYBR Gold dye in dark (1:10000, Invitrogen, S11494) and visualized using an OLYMPUS BX53 microscope at 496/522 nm excitation/emission wavelength. The comet tail length was quantified using CASP software analysis.
Xenograft formation assay
Female BALB /c nude mice (4–5 weeks old), purchased from Changzhou Cavens Laboratory Animal Co.Ltd (Changzhou, Jiangsu, China), were housed under specific pathogen-free (SPF) conditions (25°C and 50% humidity with a 12/12 light cycle) with regular food supplies. All the mice experiments were carried out in accordance with standard guidelines of the Institutional Animal Care and Use Committee of Chongqing Medical University. After being randomly divided into sh-NC and sh-TOP1 groups (n = 5/group), the mice received the indicated subcutaneously injection of CC cells (2 × 106/mouse) at a 1:1 mixture of media and Matrigel (BD Biosciences). Tumor length (L), width (W), and weight were measured using a caliper and an electronic scale. Tumor volumes (mm3) = 0.5× L × W2.
Immunohistochemical (IHC) analysis
Tumor tissues were fixed in 4% paraformaldehyde (PFA), dehydrated in alcohol with gradient percentages, and embedded in paraffin. The paraffin sections (5 µm) were cut into slides, dewaxed in xylene, hydrated, as well as blocked for endogenous peroxidase activity (Beyotime, P0100B). Slides were processed for antigen retrieval (pH 9.0) using heat-induced EDTA solution (Solarbio, C1034). Slides were incubated in blocking buffer (Beyotime, P0260) at 37°C in a humidified chamber, followed by incubation with diluted primary antibodies at 4°C overnight. Then with a secondary antibody conjugated to HRP (zsbio, PV-6000). DAB substrate solution (zsbio, PV-8000) was applied to the tissue sections to visualize the staining color of the antibodies. The slides were also immersed in hematoxylin for 1–2 min to make counterstain slides. The sections were finally dehydrated through a series of ethyl alcohol, cleared with xylene, and mounted with neutral resin. Images of the antibody staining were immediately captured using OLYMPUS BX53 microscope.
Hematoxylin and eosin (HE) staining
Tissue sections were dewaxed and hydrated prior to staining of the nucleus with Hematoxylin Solution approximately 90sec (Solarbio, G1120). Slides were then washed with water before the final Eosin Y Aqueous Solution staining for 10-15sec, dehydrated with dimethylbenzene and mounted with resinous medium in a routine order.
Serum IFNβ analysis
Blood samples were collected into non-heparinized tubes and kept still at RT for 30min until clot formation. The clear serum was then centrifuged for 15min at 1000g and assayed for endogenous levels of IFNβ protein by Human IFNβ ELISA Kit (Proteintech, KE00187). The IFNβ protein was captured by IFNβ antibody pre-coated onto the microwells. were After extensively washing, wells were incubated with a second biotinylated antibody. The streptavidin-HRP reagent and the tetramethyl-benzidine substrate (TMB) were then added to the wells for color development, and the reaction was stopped with sulfuric acid prior to measuring the absorbency of 450 nm bound proteins on a microplate reader (Thermometer Varioskan LUX).
RNA-sequencing
Total RNA was extracted using TRIzol reagent (Invitrogen, 15596026) in accordance with the manufacturer's instructions. The RNA-sequencing was performed at Novogene Co. Ltd (Beijing, China) using Illumina Novaseq platform. Feature Counts v1.5.0-p3 was used to assess the quality of raw and post-alignment sequencing reads. These reads were then mapped to human GRCh38 genome model using Hisat2 v2.0.5. Differentially expressed genes (DEGs) were carried out using edgeR in knockdown groups compared with control groups. Duplicate biological specimens were included in each group. Pathway enrichment analysis was performed with Metascape. The threshold criteria set as adjusted P (FDR) < 0.05 and |log2fold changes (FC)| > 1 for identification of significant DEGs. Raw sequencing data will be provided upon being requested for the reviewers.
Bioinformatic analysis
Gene Expression Profiling Interaction Analysis (GEPIA) or the Kaplan Meier plotter online databases were used to determine the relative expression levels of TOP1 in human CC and control tissues, and association of TOP1 expression with CC survival. Tumor Immune Estimation Resource (TIMER) was conducted to analyze the correlation between TOP1 and cGAS in CC samples.
Statistical analysis
For each experiment, statistical analysis was performed using SPSS 22.0 and GraphPad Prism 8.0 software. Data were presented as mean ± SD. Significant differences were analyzed by two-tailed unpaired Student’s t-test between two independent groups, one-way ANOVA test or Spearman’s rank-order correlation test for comparisons among more than two groups. P-value less than 0.05 was considered statistically significant. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, ns indicates no significance. The RNA-sequencing and animal experiments were performed using biological replicates as indicated in the figure legend and corresponding methods section. Western blot, qPCR, Co-IP and IHC staining analyses along with other experiments were performed at least 3 times.