Clinical samples and animals
A total of 30 paired cervical cancer tissues and corresponding adjacent tissues were obtained from Harbin Medical University Cancer Hospital. Each patient signed informed consent prior to participating in the study. All procedures involving human subjects were approved by the Ethics Committee of the Harbin Medical University and were in accordance with the Declaration of Helsinki.
Sixty BALB/c nude mice were purchased from Beijing Huafukang Bioscience Co. Inc. (Beijing, China) and housed in cages with a 12 h light/dark cycle and food and water were freely available. All procedures involving animal experiments were approved by the Animal Care and Use Committee of Harbin Medical University and were in compliance with the Guide for the Care and Use of Laboratory Animals.
Cells and cell culture
Human cervical epithelial cells (HCerEpiC) and cervical cancer cells (Hela, Siha, C-33A, and CaSki) were purchased from Procell (Wuhan, China). HCerEpiC, Siha, Hela, and C-33a were cultured in minimal essential medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). Caski was cultured in Roswell Park Memorial Institute 1640 medium (Gibco) plus 10% FBS (Hyclone). All cells were maintained in an incubator set to 37°C and 5% CO2. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation, was obtained from Aladdin Reagent Company (Shanghai, China). After transfection for 24 h, 200 μmol/L PDTC was added to cells for 24 h.
Transient transfection and stable cell lines generation
Specific small interfering RNAs (siRNAs) against CLIC1 and HAT1 were obtained from JTS Scientific (Hubei, Wuhan, China). Recombinant plasmids expressing CLIC1, P300/CBP-associated factor (PCAF), E1A binding protein p300 (P300), CREB-binding protein (CBP), General Control Of Amino Acid Synthesis Protein 5-Like 2 (GCN5), Tat Interacting Protein, 60kDa (Tip60), and HAT1 were purchased from GenScript (Piscataway, NJ, USA). Recombinant plasmids expressing Flag-tagged CLIC1 or Flag-tagged mutant CLIC1 (lysine-to-arginine at position 131, CLIC1 K131R) were obtained from ViGene Biosciences (Shandong, China). They were transiently transfected into Hela or Siha cells for 48 h (except for cell proliferation assay) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). For in vivo experiments, cells with stable overexpression or knockdown of CLIC1 were selected and used.
Protein separation and western blot analysis
Total protein was extracted from cells or tumor tissues using RIPA lysis buffer (Solarbio, Beijing, China). Nuclear extracts were obtained from cells with a Nuclear Protein Extraction Kit (Solarbio). Protein concentration was determined with a BCA Protein Assay kit (Solarbio). The samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto PVDF membranes, and blocked with 5% skimmed milk. Afterward, the membranes were incubated with primary antibodies against CLIC1 (1:1000, Abcam, Cambridge, MA, USA), p-p65 (1:500, CST, Danvers, MA, USA), p65 (1:1000, CST), p-IkB (1:500, CST), IkB (1:1000, CST), PCAF (1:3000, CST), P300 (1:3000, CST), CBP (1:3000, CST), GCN5 (1:3000, ABclonal, Boston, MA, USA), Tip60 (1:3000, Abclonal), HAT1 (1:3000, Abclonal), Histone H3 (1:5000, Gene Tex, California, USA), GAPDH (1:10000, Proteintech, Wuhan, China), Ubiquitin (1:1000, Abcam), and Flag (1:1000, KangWei, Beijing, China) overnight 4°C. Protein bands were visualized by an ECL detection method (Solarbio) and quantitated by densitometry.
Cell proliferation assay
Cell proliferation ability was evaluated by Cell Counting Kit (CCK)-8 assay. Briefly, cells at a density of 4×103 were seeded in 96-well plates in quintuplicate. After treatment, the plates were incubated for 0, 24, 48, 72, 96, or 120 h at 37°C. Ten microliters of CCK-8 solution then was added to each well at indicated time points for 2 h. The absorbance value of each well was determined at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).
Cell apoptosis assay
Cell apoptosis was assessed by flow cytometry after annexin V-FITC and propidium iodide (PI) staining (Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer's instruction.
Xenograft assay
Mice were randomly divided into 12 groups (n=6). Both Siha and Hela cells with stable expression of control shRNA, CLIC1 shRNA1, CLIC1 shRNA2, CLIC1 WT CLIC1 K131R were subcutaneously injected into BALB/c nude mice. Tumor volume was measured every two days. Nineteen days later, mice were sacrificed, and the tumor tissues were collected for the following western blot analysis.
Cell migration assay
Cell migration was determined by the wound healing assay. In short, the cells were grown to confluence, and 1 μg/mL mitomycin C was added to induce growth arrest. Then 200 μL of pipette tip was used to create a scratch in the monolayer, after which the plate was washed once and replaced with the desired medium. Images were captured with an inverted phase-contrast microscope (Olympus, Tokyo, Japan).
Cell invasion assay
Cell invasion was evaluated by the transwell assay. In brief, cells at a density of 1.5×104 were seeded into transwell chambers (Corning Inc., Corning, NY, USA) coated with Matrigel. The bottom chamber was filled with 800 μL of medium containing 10 % FBS. After 48 h, cells were immobilized with 4% paraformaldehyde, stained with 0.4% crystal violet, and pictured and counted using a microscope (Olympus).
Co-immunoprecipitation (co-IP) assay
Total protein was isolated and quantified as described above. The equal amounts of cell lysates were subjected to incubation with specific primary antibodies overnight at 4°C. Afterward, 60 μL of protein A/G agarose was added for 2 h at 4°C. The immunoprecipitated complex was pelleted by centrifuging and analyzed by western blotting with indicated antibodies.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 7 (La Jolla, CA, USA). The data are expressed as mean ± SD of three independent experiments for in vitro and six animals in each group for in vivo studies. Student’s t-test was used to analyze experimental data between two groups, and one- or two-way ANOVA was used to test the difference between more than two experimental groups. Differences were considered statistically significant at ∗p < 0.05.