Cell lines and cell culture protocols
The human CC cells lines HeLa and SiHa were purchased from the Cell Culture Collection of the Shanghai Branch of the Chinese Academy of Sciences. HeLa and SiHa cells were cultured at 37 °C and 5% CO2 in Dulbecco's Modified Eagle Medium (Gibco, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Gibco) and 2% penicillin–streptomycin (Beyotime Biotechnology, Haimen, China). On approaching 80%–90% confluence, cells were trypsinised.
Construction, packaging, and transduction of lentiviral vectors
Lentivirus GV-miRNA-101 (Genechem, Shanghai, China) was used to infect SiHa and HeLa cells at a multiplicity of infection (MOI) of 10 and lentivirus GV-EGFP (Genechem) was used as the control vector. After 72 h of transduction, cells were harvested and used for subsequent tests.
RNA isolation and quantitative reverse-transcription-polymerase chain reaction (qRT-PCR)
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and 1 µg of total RNA was retrieved using the miScriptⅡRT Kit (Qiagen, Hilden, Germany). The Bio-Rad CFX96 system (Hercules, CA, USA) with miScript SYBR Green (Qiagen) was used for qRT-PCR analysis and to determine the expression levels of the mRNA-101 and genes of interest. The qRT-PCR protocol was as follows: 95 °C for 15 min, followed by 40 cycles at 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s. After 40 amplification cycles, the melting curve was examined, the Ct value of each sample was automatically calculated, and relative mRNA expression levels were determined using the 2-ΔΔCT algorithm. The samples were analysed in triplicate and U6 was used as an internal control for miRNA amplification. miRNA-101 and U6 were reverse-transcribed (RT) with Bulge-loop miRNA qRT-PCR primers (one pair of RT primers and one pair of qPCR primers in each group) designed by Ribo Bio (Guangzhou, China).
Cell counting kit (CCK)-8 assay
The lentivirus-transduced CC cells were seeded in 96-well plates at 5 × 103 cells/well and cultured overnight at 37 °C and 5% CO2. Thereafter, 10 μL of CCK-8 solution was added into each well and incubated for 24, 48, and 72 h and the absorbance was measured at 450 nm, using a microplate reader (Bio-Rad). The control group comprised human CC cells transduced with a sham lentivirus. Cell viability was determined using the formula: cell viability = (absorbance of the test well - absorbance of the blank well)/(absorbance of the control well - absorbance of the blank well) × 100%.
Scanning electron microscopy (SEM) analysis
The same number of cells (HeLa and SiHa) was seeded on a glass coverslip (0.8 × 0.8 cm2), placed in a 48 well plate, and cultured in Dulbecco's Modified Eagle Medium at 37 °C and 5% CO2. When the cell fusion rate approached 50%, the coverslips were removed, fixed with 3.7% formaldehyde for 10 min, and washed thrice with phosphate-buffered saline (PBS), followed by sequential dehydration, air-drying, and placement on a short column. Thereafter, the slides were placed in the vacuum chamber of an SEM gold coating apparatus (plating instrument) and gold-coated at 2.5 kV, 20–25 mA for 2 min. The cells were then observed using a SEM (JEOL JSM-5800, Japan) at an accelerating voltage of 20 kV.
Immunofluorescence and confocal microscopic assays
CC cell lines (2 × 104 cells) were inoculated on a confocal plate and examined 24 h later. Cells were then fixed with 4% formaldehyde, stained with TRITC-labelled phalloidin (1:100; Solarbio, Beijing, China) at 25 °C for 30 min, followed by staining with DAPI (Solarbio) at 25 °C for 30 s. Images were acquired using the FV1000 confocal microscope (Olympus, Tokyo, Japan).
Wound healing assay
The wound healing assay was performed to evaluate the migration potential of CC cells. Briefly, human CC cell lines were inoculated onto 6-well plates (1 × 106 cells/well). Upon 70%–80% cell fusion, artificial wounds with the same width were obtained by scratching the the cell layer with sterile plastic micropipette tips. Thereafter, cell debris were eliminated by washing with PBS. Cell migration was observed and photographed at 24, 48, and 72 h after wounding and compared with the 0 h image.
Combinatorial transwell and matrigel assay
Cell invasion was assessed using matrigel invasion chambers (BD Bioscience, San Diego, CA, USA). Briefly, 3 × 104 cells were seeded in serum-free medium in the upper chamber. The medium, supplemented with 10% foetal bovine serum, was added to the lower chamber. After 24 h of culturing, the cells at the bottom of the membrane were fixed and stained with 0.1% crystal violet. Five visual fields of invasive cells were randomly selected, counted, and photographed under a microscope.
Xenograft mouse model of CC
All procedures involving mice were approved by the Experimental Animal Ethics Committee of Xinjiang Medical University and met all regulatory standards. Forty-five female (4–6 weeks old) BALB/c nude mice were divided into three groups (n = 15/group). A 0.2 mL cell suspension (5 × 106 cells) was subcutaneously injected into the right forelimb capsule. The blank group comprised mice injected with HeLa cells, the control group comprised mice injected with HeLa cells transfected with empty-load lentivirus, and the experimental group comprised mice injected with HeLa cells transfected with miRNA-101 lentivirus. The physiological features (including mental state, activity, diet, body weight, appearance, and tactile responses to touching the inoculated area) of nude mice were evaluated every 2–3 days. Tumour growth (characterised by the number of subcutaneous xenografts, the long diameter [L], and short diameter [W] of the subcutaneous xenografts that were measured with a Vernier calliper, and tumour volume [V, mm3]), was determined using the following formula: V = (L∙W2)/2. After 39 days of cell inoculation and when the diameter of the subcutaneous xenografts approached approximately 9 mm, the nude mice were euthanized and the tumour tissue was excised. The growth curve of the subcutaneous xenografts was constructed on the basis of V and inhibition rate (in %), which was determined as inhibition rate = (1 − [(average initial V of the experimental group − average final V of the experimental group)/(average initial V of the control group − the average final V of the control group)]) × 100%.
Histopathological analysis
Tumour, lung, liver, heart, kidney, and brain tissues were fixed with normal neutral formalin, embedded in paraffin, sectioned, stained with haematoxylin and eosin, and the histological characteristics of the transplanted tumour and critical organs were observed using a light microscope.
Statistical analyses
The experimental data were analysed using SPSS19.0 software (IBM Inc, Armonk, NY, USA) and all graphs were plotted using GraphPad Prism for Windows version 5.0 (GraphPad Software, San Diego, CA, USA). Continuous variables were expressed as mean ± standard deviation values and the data were assessed for normality and conformance with a normal distribution. The one-way ANOVA was performed to compare the homogeneity of the assessed differences among groups and independent samples t-test was used for between-group comparisons. Each experiment was performed in triplicate. A P-value < 0.05 was considered statistically significant. Tumour volume among groups was simultaneously analysed using a one-way ANOVA and Fisher’s least significant difference test (variance uniformity) for post hoc pairwise comparison.