American Type Culture Collection (ATCC; Manassas, VA, USA) was the institute provided cell lines used in these study on 4th, Nov, 2018. Cell lines used in these study including: HeLa (Cat: CCL-2), C-33a (Cat: HTB-31; Cat: CRL-2615), SiHa (Cat: HTB-35), HCC94 and End1/E6E7 (normal cervical cell line). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Carlsbad, CA, USA) added with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin, which were then maintained in a humidified air at 37 °C with 5% CO2. The culture medium was refreshed every 2 days. Cell lines used in this study had been authenticated by STR cell identification on 13th, Nov, 2018. Cell lines were not infected by mycoplasma. All experiments were performed with mycoplasma-free cells. Cells were not contaminated when referring to NCBI.
All plasmids including siRNAs against circAGFG1 containing si/circAGFG1#1 (reduced circAGFG1 expression by 87% in HeLa cells and by 84% in SiHa cells), si/circAGFG1#2 (reduced circAGFG1 expression by 81% in HeLa cells and by 79% in SiHa cells), si/circAGFG1#3 (reduced circAGFG1 expression by 75% in HeLa cells and by 71% in SiHa cells), pcDNA3.1/circAGFG1 (increased circAGFG1 expression to about 114 fold change in HeLa cells and to about 108 fold change in SiHa cells), pcDNA3.1/RAF1 (increased RAF1 expression to about 123 fold change in HeLa cells), miR-370-3p mimic (increased miR-370-3p expression to about 104 fold change in HeLa cells and to about 111 fold change in SiHa cells) and miR-370-3p inhibitor (reduced miR-370-3p expression by 79% in HeLa cells and by 72% in SiHa cells) were constructed. All plasmids and their negative controls were purchased from GenePharma (Shanghai, China) and transfected into CC cells with Lipofectamine® 2000 (Invitrogen) under recommended direction.
Total RNA extraction was conducted with TRIzol reagent (Invitrogen), which were reverse-transcribed into cDNA by utilizing PrimeScript RT Reagent Kit (Takara, Dalian, China) routinely. qRT-PCR were carried out by using TB Green Premix Ex Taq (Takara) on the Bio-Rad CFX96 system (Bio-Rad, CA, USA). Quantification of circRNA and mRNA and miRNA was made by referring to GAPDH or U6. Relative gene expression was determined with the use of 2–ΔΔCT method. Special primers are listed as follows:
F: 5′- CTTCAGGAACGAGGTGGCTGTT-3′.
R: 5′- TGCTGCCTTCACACCACTGAGT-3′.
Cell viability and proliferation assays
1 × 103 CC cells were maintained in 96-pore plates for 1 d and grown for extra 0, 24, 48, 72 and 96 h. Cell Counting Kit-8 (Bosterbio, Wuhan, China) was utilized to evaluate cell viability following the supplier’s protocol at each time point. After 4 h of culture, a spectrophotometric plate reader (BioTek, VT, USA) was applied to measure the optical density at 450 nm for detection of cell viability.
CC cells (200 μL, 2 × 104/mL) were immobilized by 70% alcohol and subsequently cultured with 50 μM EdU (5-Ethynyl-2′-Deoxyuridine) labeling solution (Invitrogen) for 2 h at 37 °C. The fluorescent intensity of EdU was examined at 550 nm through applying Cell Light EdU DNA imaging kit (Invitrogen). Cells were cultivated using 5 μg/mL Hoechst 33342 for 0.5 h for DNA staining. The visualization and photograph of immunostainings were implemented with a fluorescent microscope (Olympus inverted microscope IX71).
Flow cytometry analysis
Apoptosis of indicated cells was measured using flow cytometry analysis in accordance with a previous study .
Transwell migration assay
Cells with a density of 1 × 105 cells/hore were seeded in 6-well plates, suspended in 200 μL serum-free medium and planted into the upper chambers without Matrigel mixture. 500 μL medium with 10% FBS was added into the lower chambers (BD BioCoat, MA, USA) as attractants. After 24 h, cells in the upper chambers were removed. Migrated cells in the lower chamber were fixed by ethanol and stained with crystal violet, which were subsequently photographed and calculated by a light microscope (Olympus Corporation, Tokyo, Japan).
Fluorescence in situ hybridization (FISH)
Specific Cy3-labeled circAGFG1 probe and FITC-labeled miR-370-3p probe were designed and synthesized via RiboBio (Gangzhou, China) at 37 °C for whole night, and dyed utilizing DAPI obeying the guidebooks of the supplier. Slides were photographed with a fluorescence microscope (Leica, Wetzlar, Germany).
Luciferase reporter assay
Binding sequences of circAGFG1 and RAF1 3′UTR for miR-370-3p as well as mutant versions (circAGFG1-WT, RAF1-WT; circAGFG1-MUT, RAF1-MUT) were synthesized and subcloned into luciferase reporter vector pGL3 (Promega, Madison, WI, USA). Then these vectors were co-transfected with NC/mimic or miR-370-3p mimic into HeLa and SiHa cells. For affirming the specificity of miR-370-3p and circAGFG1, we constructed the full length of circABCC2, circLRP6, circSCAF11 and circAGFG1 into luciferase reporter vector pGL3 to observe the changes of these luciferase activities by miR-370-3p mimics. As for the regulation of miR-370-3p and circAGFG1 on the luciferase activity of RAF1, RAF1-WT or RAF1-MUT was co-transfected with NC/mimic, miR-370-3p mimic, miR-370-3p mimic + pcDNA3.1 or miR-370-3p mimic + pcDNA3.1/circAGFG1 into CC cells. The luciferase activities were measured by Dual Luciferase Assay Kit (Promega) according to the manufacturer’s directions.
RNA immunoprecipitation (RIP)
Under the manufacturer’s protocols, RIP was performed with Magna RIP kit (Millipore). Transfected cells were lysed in RNA lysis buffer and cell lysates were incubated with magnetic beads with anti-Argonaute2 (Ago2) or anti-IgG (both from Millipore) at 4 °C for 4 h. After the beads were washed, the immuno-precipitated RNAs were purified and detected by qRT-PCR.
RNA pull-down assay
G-50 Sephadex RNA Columns (Roche) was employed to purify biotinylated RNA synthesized in vitro with the help of T7 RNA polymerase. Biotinylated miR-370-3p sense was named as bio-miR-370-3p-WT probe, and biotinylated miR-370-3p antisense was named as bio-miR-370-3p-MUT probe. Transfected cells were lysed with lysis buffer and incubated with streptavidin-coated magnetic beads to pull down the biotin-labeled RNA complex. Human Ago2 or normal mouse IgG antibody (Millipore) were used. The beads were washed and the precipitates were purified with TRIzol (Takara). The abundance of circAGFG1 was determined with qRT-PCR.
Western blot analysis
Extracted proteins were separated by 10% SDS-PAGE, and then transferred onto PVDF membrane (Bio-Rad). After being blocked with 5% skimmed milk, the membranes were incubated with primary antibodies against RAF1, p-RAF1, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2 and GAPDH at 4 °C. After overnight incubation and then incubated with secondary antibodies at room temperature for 2 h. All antibodies were purchased from Abcam (Burlingame, CA, USA). In the end, bands were measured with Immobilob™ Western Chemiluminescent HRP Substrate (Millipore).
All independent experiments were conducted for three times. SPSS 19.0 software (IBM Corporation, Armonk, NY, USA) was responsible for data analyses. Data of three experimental results were exhibited as the mean ± standard deviation (SD). Student’s t-test and one-way ANOVA were two statistical methods for comparison of difference between two or more groups. P<0.05 is a symbol that indicates statistical significance.