Tissue samples and human cervical carcinoma cell lines
Human cervical cancer tumours and adjacent non-tumour tissues were obtained from Guangxi Medical University (China). The clinicopathological characteristics of the samples are summarised in Table 1. A cervical cancer tissue microarray was purchased from Shanghai Outdo Biotech Co. Ltd. (China). All patients provided informed consent for the use of their tissues before surgery. The study was approved by the Ethics Committee of the National Research Institute for Family Planning.
The human cervical carcinoma cell lines HeLa, SiHa, and C33A were purchased from the Cell Resource Center of Peking Union Medical College (Beijing, China) and cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% foetal bovine serum (FBS), 100 IU/mL penicillin, and 10 mg/mL streptomycin. All cells were maintained at 37°C in an atmosphere containing 5% CO2.
In situ hybridisation of miR-205 with a digoxigenin (DIG)-labelled LNA probe
The sections (4 μm) of cervical cancer tissues and adjacent normal cervical tissues were treated with proteinase K (20 mg/mL) for 15 min and refixed in 4% PFA for 15 min. After acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min, sections were prehybridised with hybridisation buffer (Roche, Mannheim, Germany) at 40°C for 2 h and then hybridised with a DIG-labelled LNA-miR-205 probe (5’-CAG(+A)C(+T)CCGG(+T)GGAA(+T)GA(+A)GGA-DIG-3’) at 40°Covernight. The sections were then incubated in buffer containing anti-DIG-antibody (Roche) 2 h at 37°C, followed by staining with NBT and BCIP (Promega, Madison, WI, USA). Samples were viewed under a Nikon TE 2000-U microscope (Nikon, Tokyo, Japan).
Immunohistochemical analysis of CHN1
Sections (4 μm) of cervical cancer tissues and adjacent normal cervical tissues were dewaxed and rehydrated, followed by an antigen retrieval procedure (citrate buffer, pH 6.0; 95°C heat for 15 min). For CHN1 staining, the sections were soaked in 3% H2O2 for 15 min and incubated overnight at 4°C with rabbit anti-CHN1 antibodies (12048-1-AP; 1:150; Proteintech, USA). Matched rabbit nonimmune IgG was used as a negative control. The sections were then treated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (PV-6001; Zymed Laboratories, China) and incubated for 20 min at 37°C; the proteins were visualised with 3,3’-diaminobenzidine tetrahydrochloride and counter stained with hematoxylin. Immunohistochemical analysis of CHN1 was carried out according to the “HSCORE” method ; an HSCORE of 75 or greater was considered positive. The specimens were analysed by two observers who were unaware of the patients’ clinical outcome. Discrepancies between the observers were found in <10% of the slides examined, and consensus was reached on further review.
Transfection and cotransfection
The miR-205 mimic, miR-205 inhibitor, corresponding negative control (NC), and siRNA duplex against human CHN1 were designed and synthesised by GenePharma (GenePharma Co., Ltd., Shanghai, China). Their sequences are shown in Table 2. Transient transfection was performed using lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Plasmid constructs and dual-luciferase activity assay
The 3’ untranslated regions (UTRs) of the human CHN1 gene (NM_001025201.2 ) were amplified by polymerase chain reaction (PCR) from human genomic DNA, cloned into the SbfI and NheI site of the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega), checked for orientation, and sequenced; the resulting plasmid was named pmirGLO-CHN1-wt. PCR primers used to amplify the CHN1 3’UTR are shown in Table 2. Site-directed mutagenesis of the miR-205 target site in the CHN1 3’UTR was carried out using an Easy Mutagenesis System (Transgen, China), with pmirGLO-CHN1-wt as a template; the resulting plasmid was named pmirGLO-CHN1-mut.
Next, 5 × 104 cells were seeded in each well of a 48-well plate at 24 h before transfection. For reporter assays, the cells were transiently cotransfected with 0.25 μg wild-type (WT) or mutant reporter plasmid and 7.5 pmol NC or miR-205 mimic using Lipofectamine 2000. At 48 h after cotransfection, Firefly and Renilla luciferase activities were measured consecutively using Dual Luciferase Assays (Promega) according to the manufacturer’s instructions. Three independent experiments were performed.
Total RNA extraction and qRT-PCR experiments were performed for analysis of gene expression as follows. Briefly, RNA was isolated using TRIzol reagent (Invitrogen). For cDNA synthesis, approximately 2 µg of total RNA was used for reverse transcription with oligo-(dT)18 primers using moloney murine leukaemia virus (M-MLV) reverse transcriptase (TaKaRa Bio, Otsu, Japan). The specific forward primer for miR-205 was designed by GenePharma based on the miRNA sequence from the miRbase database.
qRT-PCR was performed with an ABI Prism 7700 Sequence Detector System (PE Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq II (TaKaRa Bio) and specific primers for each gene. To control for uniform amount of input RNA template, mRNA and miRNA expression results were normalised to the expression level of the internal control gene GAPDH or U6 snRNA, respectively. Thermal cycling conditions were as follows: an initial activation cycle at 95°C for 30 s, followed by 40 cycles of denaturation (95°C for 10 s), annealing, and amplification (60°C for 30s). The final amplification products were verified by agarose gel electrophoresis for treatment samples and negative controls. The primer sequences are shown in Table 2. Each sample was assayed in triplicate. To compare the expression levels among different samples, relative quantification was achieved using the 2-ΔΔCt approach, in which ΔΔCt is the calibrated Ct value.
Western blot analysis
Total protein lysates were obtained using RIPA lysis buffer supplemented with 1 mM PMSF, protease inhibitor cocktail, 1 mM Na3VO4, and 10 mM NaF (Sigma Aldrich, St. Louis, MO, USA). The protein concentrations in extracts were determined by colorimetric BCA protein assays (Thermo Scientific, USA). Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on 10% Tris-glycine gels (Amresco, Solon, OH, USA) and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 1 h at RT with TBST (50 mM Tris-HCl, 150 mM NaCl, and 0.1% [v/v] Tween-20) containing 5% (w/v) nonfat dried milk and were subjected to immunoblotting with antibodies to CHN1 (12048-1-AP; Proteintech) and β-actin (CoWin, Beijing, China).
Cell proliferation assay
The proliferation of HeLa, SiHa, and C33A cells was estimated with a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan) according to the manufacturer’s instructions following transfection with miR-205 mimic, NC, miR-205 inhibitor, or inhibitor NC, with five wells for each treatment. The experiment was repeated three times, and the results are described as the ratio of the absorbance at 450 nm for the miR-205 mimic or inhibitor to that of the corresponding control.
Flow cytometry analysis
Cell apoptosis was analysed using flow cytometry analysis with an Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen). Samples containing 5 μL Alexa Fluor 488 annexin V and 1 μL of 100 μg/mL propidium iodide (PI) were assayed to determine the phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane. After incubation for 15 min at in a light-protected area, the specimens were quantified by flow cytometry (BD Biosciences, San Jose CA, USA). Each treatment was repeated twice, and the experiment was repeated three times.
In vitro migration and invasion assays
HeLa, SiHa, and C33A cells were transfected with the miR-205 mimic, NC, miR-205 inhibitor, or inhibitor NC. Transfected cells were harvested and subjected to the following assays at 48 h after transfection. For migration assays, the transfected cells (0.5 × 106 cells/mL) were seeded in the top of a chamber containing a membrane with 8.0-μm pores (Corning Costar Corp., Cambridge, MA, USA). Following a 12–18h incubation period, cells that passed through the membrane were fixed and stained with hematoxylin. Cells were scraped and removed from the top of chamber. Membranes were mounted on cover slides, and cells were counted. Cell migration was quantified by counting the number of cells passing through the pores from five different randomly selected fields of view per sample at 100× magnification under a microscope. For invasion assays, Matrigel (BD Biosciences) diluted to 1 mg/mL in serum-free cold cell culture medium was added to the top of a chamber containing a membrane with 8.0-μm pores and incubated at 37℃ overnight until the matrigel solidified. Analysis was then carried out as described above.
Data are expressed as means ± SEMs. The statistical significance of the quantitative data was assessed by paired Student’s t-tests, and clinical correlations were analysed by Pearson chi-square test. P < 0.05 was considered to be statistically significant.