The usage of human tissues was approved by the Ethics Committee of the Guangxi Medical University Cancer Hospital (No.: H2019018.v03). The experiments were undertaken with the understanding and written consent of each subject, and that the study conforms with The Code of Ethics of the World Medical Association (Declaration of Helsinki), printed in the British Medical Journal (18 July 1964).
Humane care was given during the experimental animal breeding and experimental procedures in accordance with the 3R principle of experimental animals. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). All animal treatments were approved by the Ethics Committee of the Guangxi Medical University Cancer Hospital (No.: KA190114.v02).
Human tissue collection
From April 8, 2019 to August 1, 2021, CC tissues and the corresponding PCT (defined as tissues ≥ 2 cm away from cancer tissues) from 350 cases of CC patients (250 cases of CSCC, 75 cases of cervical adenocarcinoma, and 25 cases of cervical adenosquamous cell carcinoma) were collected from the Department of Gynecologic Oncology, Guangxi Medical University Cancer Hospital. Basic information on patients was also gathered. All patients were diagnosed for the first time and had not received treatment before. Patients who received treatments before or complicated with other systemic diseases such as diabetes, cardiovascular, or cerebrovascular diseases were excluded from this study.
Isolation and culture of cells
The primary normal cervical epithelial cells (hCECs) were isolated as the prior report . Briefly, the fresh PCT was minced and digested with type I collagenase at 37 °C for 60 min. The digested mixture was then filtrated through a stainless-steel strainer (0.5-1.0mm). Tell suspension was centrifuged at 1,500 rpm for 5 min, and hCECs were collected and cultured in Dulbecco's Modified Eagle's Medium (DMEM, Thermo Fisher Scientific, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Clark, USA). The purity of hCECs was identified by > 90% positive immunofluorescence staining for cytokeratin 7 (Supplementary Figure 1A). Cells with passages > 10 were used in the following cell experiments.
We also isolated primary CAFs from cancer tissues of CSCC as previously described . Briefly, fresh cancer tissues were minced and digested with type I collagenase, and the digested mixture was filtrated through a nylon mesh of 38 um pore size. The CAFs were in the filtrate. The filtrate was centrifuged at 1000 g for 5 min, and CAFs were resuspended in DMEM supplemented with 10% FBS, and were then incubated in a fresh culture medium for 4 weeks to allow cell attachment and grow out. The purity of CAFs was identified by > 90% positive immunofluorescence staining for fibroblast activation protein (FAP) (Supplementary Figure 1B). Cells with passages > 10 were used in the following cell experiments. CSCC cell line CaSki was also used in this study. CaSki cells were purchased from the American Type Culture Collection (ATCC) and were cultured in DMEM supplemented with 10% FBS. Cells were all placed in the 37 °C incubators supplemented with 20% oxygen and 5% carbon dioxide.
Small interfering RNAs (siRNAs) for WDR5 (si-WDR5) and CXCL8 (si-CXCL8) (VectorBuilder, China) were used to knock down the target transcripts. siRNAs having no target (si-NC) were served as the control. Plasmid overexpressing WDR5 (Vector-WDR5) or CXCL8 (Vector-CXCL8) were also used to express exogenous proteins, and the empty vectors (Vector-NC) were used as the control. The transfection was conducted using Lipofectamine 3000® Transfection Reagent (Invitrogen, USA) according to the manufacturers’ instructions. The sequence of siRNAs was provided in Table 1.
Chromatin immunoprecipitation (ChIP) and ChIP-sequencing (ChIP-seq)
ChIP assays were performed as described previously . Briefly, cells were cross-linked, lysed, and sheared by sonication to produce DNA fragments with an average length of approximately 500 bp. Then, 1% of the chromatin fragments were used as the input. Chromatin was immunoprecipitated using antibodies against WDR5 (13105, Cell Signaling Technology, USA), H3K4me3 (ab8580, Abcam, USA), and MLL1 (34907, Cell Signaling Technology, USA). Normal rabbit IgG was added as the control. Then DNA fragments immunoprecipitated were purified and were further analyzed by quantitative real-time PCR (RT-qPCR). The sequence of primers used is provided in Table 1. ChIP-seq analyses were performed using the ChIP-IT High Sensitivity Kit (ab185908, Abcam, USA). The model-based analysis of the ChIP-Seq peak-finding algorithm was used to normalize ChIP against the input control.
Immunohistochemistry was performed on paraffin-embedded tissue sections. Tissues on the sections were blocked with 5% BSA, and were then incubated overnight at 4 °C with primary antibodies for the detection of the following: WDR5 (ab178410, Abcam, USA), CXCL8 (ab106350, Abcam, USA), and FAP (ab207178, Abcam, USA). After being incubated with the secondary antibodies, slides were visualized using DAB-Substrate (Beyotime, China) and photographed using the Aperio ePathology Scanner (Leica, Germany). Protein expression was quantified by H-score, which was calculated by the formula: H-score = Pi (i), where i is the intensity of staining with a value of 1, 2, or 3 (weak, moderate, or strong, respectively) and Pi is the percentage of stained cells for each intensity in the range of 0-100%.
Cells were fixed with 4% formaldehyde and incubated overnight at 4°C with primary antibodies anti-vimentin (ab92547, Abcam, USA), anti-cytokeratin 7 (ab68459, Abcam, USA), anti-FAP (66562, Cell Signaling Technology, USA), and anti-tubulin (ab18207, Abcam, USA). Cells were then incubated with the goat anti-rabbit secondary antibody (BA1031, BOSTER, China) at 37°C for another 30 min. The nucleus was strained by 4ʹ,6-diamidino-2-phenylindole (Beyotime, China). Cells were observed by Laser Scanning Confocal Microscope (Leica, Germany) and results were analyzed by Leica Application Suite X (Leica, Germany).
Western blotting (WB)
Cells lysates containing total proteins were collected and prepared using a loading buffer. An equal amount of protein from each group was loaded into SDS-PAGE (10% gel) and separated by electrophoresis. Proteins in the gel were then transferred to a polyvinyl difluoride membrane. After immersion in quick blocking buffer (Beyotime, China) for 30 min, membranes were incubated overnight at 4 °C with the primary antibodies for the detection of the following: E-cadherin (ab40772, Abcam, USA), N-cadherin (ab76011, Abcam, USA), β-catenin (ab32572, Abcam, USA), snail (ab216347, Abcam, USA), and tubulin (ab215037, Abcam, USA). Membranes were then incubated with the secondary antibodies (ab6721, Abcam, USA) at room temperature for another 30 min. Protein bands in the membrane were visualized using ECL plus kit (Beyotime, China), and the relative expressions of target proteins were quantified using Image J (National Institutes of Health, USA).
Total RNA was extracted by the Trizol method (Thermo Fisher Scientific, USA) and were reversely transcript into cDNA using PrimeScriptTM RT Master Mix (Takara, Japan). RT-qPCR was performed on the Bio-Rad CFX96 (Bio-Rad Laboratories, USA) using SYBR Premix Ex TaqTM (Takara, Japan). The sequence of primers was provided in Table 1. The expression level of each transcript was calculated using the comparative threshold cycle (Ct), based on the using the 2-△△Ct formula.
Invasion chambers (Corning, USA) were used to perform invasion assay. Briefly, 100 ul of Matrigel (BD, USA) was used to coat the inner side of the chamber, and then cells suspended in a culture medium were loaded. DMEM containing 20% FBS was added to the lower chamber as the chemoattractant. The chambers were incubated at 37°C for 48 hours. The non-invasive cells on the upper side of the chamber were removed gently, and chambers were stained using crystal violet to locate the invaded cells. The number of invaded cells was observed and photographed under an inverted optical microscope (Olympus, Japan).
Enzyme-linked immunosorbent assay (ELISA)
Levels of growth factors [granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), CC-chemokine ligand 2 (CCL2), and transforming growth factor-β (TGF-β)] in cancer tissues and cell cultures were analyzed using ELISA kits (mlbio, China) according to the manufacturer’s protocols. The intra- and inter-assay coefficients of variation were all less than 10%. Samples were adjusted for total protein concentrations before detection to ensure that the amounts of total protein in each group were equal.
Tumor xenograft mouse model
Four-week male nude BALB/c mice (Cyagen, China) were housed in a facility with a 12 h light/dark cycle maintained at 25 ± 0.5°C and 50% to 60% humidity. The xenograft CC model was established as described before . Briefly, CaSki cells were prepared as a single-cell suspension in Matrigel, and 1×106 prepared cells were injected subcutaneously into the right axillary fossa. Mice were euthanized 14 days after modeling (pelltobarbitalum natricum, 100mg/kg, intravenous administration), and tumor lesions were enucleated for further analysis.
Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, USA). The student’s t-test was used to analyze the difference between two groups, and one-way ANOVA was used for the comparison among multiple groups. The Chi-square test was used to analyze the categorical variables. In this study, results were obtained from three independent experiments, and P<0.05 was considered to be statistically significant.