2.1 Cell Culture
HPV16-positive (CaSki and SiHa cells) cervical cancer cells and HEK293T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone Laboratories, Inc., Logan, uT, USA), and supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) with 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C with 5 % CO2
2.2 miRNA transfection, siRNA interference and adenoviral infection
The miR-23b-3p mimic, anti-miR-23b-3p, and negative control were purchased from Shanghai GenePharma, Co., Ltd. (Shanghai, China). The E6, E7 siRNA and the corresponding negative control siGL3 were purchased from Biomics Biotechnologies, Co., Ltd. (Jiangsu, China). Their sequences are shown in Table 1. SiHa and CasKi cells were transfected using Lipofectamine® 2000 (Invitrogen, CA, USA) according to the manufacturer's protocol. After 6-8 h of transfection, the medium was replaced with fresh growth medium.
Recombinant adenovirus AdICAT and the corresponding negative control AdRFP were kindly donated by Dr Tongchuan He (university of Chicago Medical Center, Chicago, IL, USA). SiHa and CasKi cells were infected with AdICAT and AdRFP with polybrene (Sigma-Aldrich). The medium was replaced with fresh medium after 8 h of cultivation. Fluorescence was then observed after 36 h.
2.3 Cell counting kit-8 (CCK8) assay
A Cell Counting kit-8 assay kit (CCK-8; Beyotime Institute of Biotechnology, Haimen, China) was used to evaluate cell proliferation ability. 24 h after transfection, cells were resuspended and plated into the 96-well plate at a density of 3000 cells/well in 100 µl complete DMEM with 10% FBS. Cells were cultured in an incubator at 37˚C with 5% CO 2, 10 µl of CCK-8 reagent was added into each well of the plate at 12, 24, 48, 72 and 96 h. Subsequent to incubation at 37˚C for another 2 h, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
2.4 Wound healing assay
The transfected SiHa and CasKi cells were seeded in a 6-well plate and incubated at 37°C until they were at least 90% confluent. A wound field was made using a sterile 10-μl tip among cells in each well. After washed with phosphate-buffered saline (PBS) three times, 2 mL of serum-free DMEM was added. Wound healing was observed under an inverted phase contrast microscope, and images were captured at 0, 24 and 48 h after the wound was made. The wounding healing rate was evaluated as: (W1–W2)/W1× 100 %, where W1 is the 0h wound width and W2 is the 24 h and 48 h wound width. This assay was independently repeated three times.
2.5 Transwell migration and invasion assays
The cell migration and invasion assays were designed in Transwell chambers (24-well Transwell chambers, 8-µm pore size; Corning, Inc., Corning, NY, USA). In the migration assay, 5x104 transfected cells were suspended in serum-free medium and added into the upper chamber. In the invasion assays, 8 x 104 transfected cells in serum-free medium were seeded into the upper chamber of the Transwell after diluted Matrigel was added. Medium containing 20% FBS was added into the lower chamber and used as a chemoattractant. After 24 h incubation at 37˚C of a 5% CO2 atmosphere, the Transwell chambers were taken out, fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 10 min at room temperature. The cells on the upper surface of the chamber that did not pass through were carefully removed using a cotton swab. Then the cells were counted and photographed by Inversion Microscope, and each group was counted for 5 fields of view. The ImageJ software was used to cell count. Each assay was carried out in triplicate.
2.6 RNA isolation and real-time quantitative RT-PCR analysis (RT-qPCR)
Total RNA from cervical cancer cells were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA (1 μg) was used for cDNA synthesis using an RNA reverse transcription amplification kit (TaKaRa). A real-time quantitative polymerase chain reaction (RT-qPCR) assay was performed to evaluate mRNA and mature miRNA expression using the SYBR-Green PCR Master Mix (TaKaRa). The target gene expression was normalized to the level of GADPH or U6. Primers used in this study were shown in Table 1
2.7 Western Blot Analysis
48 h post-transfection, proteins were extracted from cells using a radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentrations were measured using a BCA Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China). After denaturation in boiling water, equal amount of proteins was loaded, separated by 8-15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes. The membranes were blocked with 5% bovine serum albumin at 37°C for 2 h. The membranes were washed with TBST and incubated at 4°C overnight with respective primary antibody against E-cadherin, N-cadherin, Vimentin (1:1000; Cell Signaling Technology, Danvers, MA, USA), PCNA, Cyclin D1 (1:1000; Wanleibio Co., Ltd., Beijing, China), ICAT (1:1,000; Abcam, Cambridge, UK), and β-actin (1:1000; Santa Cruz Biotechnology, CA, USA). After that, the membrane was washed using TBST for 3 times/10 min and incubated with a horseradish peroxidase (HRP)-labeled secondary antibody (1:5,000; Beijing Zhongshan Golden Bridge Biotechnology) for 1 h at 37˚C. Next the membrane was washed with TBST for 3 times/10 min. This was followed by treatment with HRP-conjugated secondary antibody (1:5000; Beijing Zhongshan Golden Bridge Biotechnology) for 1 h at 37 ◦ C. The protein bands were visualized with the Supersignal West Pico Chemiluminescent substrate kit (Millipore, Billerica, MA, USA) and β-actin was used as an internal control. All experiments were performed in triplicate.
2.8 Bioinformatics analysis and dual luciferase reporter assay.
According to the results obtained from the online prediction system, TargetScan (http://www.targetscan.org), miRBase (http://www.mirbase.org/), and miR-23b-3p was predicted as miRNAs that could potentially regulate ICAT. It was shown in the databases that there was a potential binding sequence in the 3’-untranslated region (UTR) of ICAT mRNA that matched with miR-23b-3p. The sequence fragments in the 3’-UTR of target gene ICAT that complementarily bind to miR-23b-3p and mutant (mut) ICAT 3’-UTR sequence fragments were synthesized and cloned into a pGL6 vector. For the dual-luciferase reporter assays, HEK293T cells were first cultured in a 24-well culture plate (10×104 cells/well). Then, the correctly sequenced luciferase reporter plasmids WT or MUT were respectively co-transfected with miR-23b-3p mimic or inhibitor and pRL-TK plasmid coding Renila luciferase into the HEK-293T cells. The cells were collected at 48 h after transfection to determine the luciferase activity using a Dual-Luciferase reporter gene assay kit (Promega, Madison, WI, USA), adhering to manufacturer’s protocol. Firefly luciferase activity was normalized against the activity of the Renilla luciferase gene.
2.9 Statistical Analysis
Data were expressed as the mean ± standard deviation of at least three independent experiments. GraphPad Prism 8.3.0 software (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analysis. Data were analyzed by using Student’s t-test or ANOVA to assess the statistical significance. P<0.05 was considered to indicate a statistically significant difference.