Cell culture and treatment
Human CC cell lines (SiHa, CaSki, ME-180, C4-1) and human normal cervical cell line (Ect1/E6E7), from ATCC (Rockville, Maryland), were allowed to grow under 37 °C and 5% CO2 in the DMEM (Invitrogen, Carlsbad, CA). The 1% antibiotics and 10% FBS, both from Invitrogen, were acquired for purpose of cell culture. Besides, 20 mmol/l of LiCl, 10 mM of DMSO and 20 nM of C646 were all purchased from Sigma Aldrich (St. Louis, MI) to treat SiHa and CaSki cells.
The total RNA from cultured cells were extracted with Invitrogen TRIzol reagent, then 1 μg of total RNA was prepared to synthesize cDNA. Expression levels of target genes were monitored through qRT-PCR with SYBR R Premix Ex TaqTM II (Takara, Shiga, Japan), processed by the 2−ΔΔCt method and normalized to U6 or GAPDH.
The designed shRNAs were produced by Genepharma (Shanghai, China) to silence EGFR-AS1, CBP and ACTN4 employing the transfection kit Lipofectamine 2000 (Invitrogen). Negative control (sh-NC) was also obtained. The pcDNA3.1/EGFR-AS1, pcDNA3.1/ACTN4 and empty pcDNA3.1 vectors, along with miR-2355-5p mimics and NC mimics, were all acquired from Genepharma for 48 h of transfection.
Cells in six-well plates (1000 cells/well) were cultivated for 14 days. Next, generated colonies were fixed with 4% paraformaldehyde and stained by 0.1% crystal violet. After washing in PBS, clones were counted.
Cells in 24-well plates were transfected with designed plasmids and plated on the sterile coverslips. EdU assay kit (Ribobio, Guangzhou, China) was used in light of direction. Cell nucleus was double stained by EdU and DAPI, and then subjected to fluorescence microscopy (Olympus, Tokyo, Japan).
Flow cytometry analysis
Annexin V-FITC/PI Apoptosis kit was acquired from Invitrogen for measuring apoptotic cells. 2 × 105 cells were collected and added into the binding buffer. 15 min later, samples were assayed using FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).
Cultured CC cell apoptosis was also measured by TUNEL assay. Cells in 6-well plates were cultured on coverslips and fixed by 4% paraformaldehyde. In situ cell death detection kit (Minneapolis, MN) was used as per manual. Apoptotic CC cells were observed under fluorescence microscopy.
Total protein samples were separated with 10% SDS-PAGE and shifted to PVDF membranes. Following blocking in 5% nonfat milk, membranes were probed with primary antibodies (1:2000; Abcam, Cambridge, MA) and appropriate HRP-tagged secondary antibodies (1:5000; Abcam). After culturing in TBST solution, samples were quantified by ECL Prime Western Blotting Detection reagent (GE Healthcare, Chicago, IL).
Transwell invasion analysis
Invasion assay was implemented using the 8-mm pore size Transwell chambers coated with matrigel (Corning, Corning, NY). Lower chamber was filled with complete medium; cells in serum-free medium were added to upper chamber. After 24 h, fixed cells were treated with 0.1% crystal violet dye and observed under light microscope.
Primer sequences of EGFR-AS1 promoter were specifically designed as followed. Forward: GATGGTGGGTGGAAAGGGAG (Start: 778); Reverse: CCTTTCGAATGGGCAGGAGT (Start: 1022). With ChIP kit (Millipore, Billerica, MA), ChIP assay in CC cells were conducted as designed. After the DNA and protein underwent cross-linking, the chromatin was fragmented by ultrasonic and immunoprecipitated with the specific antibody to H3K27ac or CBP. IgG antibody acted as control. qRT-PCR was used for detecting the relative DNA enrichment.
With PARIS™ Kit, the cytoplasmic and nuclear fractions were severally isolated from processed CC cells in line with user guide (Ambion, Austin, TX). The isolated RNAs were assayed by qRT-PCR.
RNA FISH assay was implemented using the EGFR-AS1-FISH probe synthesized by Ribobio as instructed by supplier. After staining nuclei with Hoechst, fluorescence microscopy was employed.
RNA pull down assay
The protein extracts from cultured CC cells were mixed with the EGFR-AS1 biotin probe or EGFR-AS1 no-biotin probe, and then incubated with magnetic beads for 1 h. RNA enrichment in pull-downs was finally monitored.
Cell extracts from CC cells were acquired and cultivated with the magnetic beads conjugated to specific antibodies including human Ago2 and normal control IgG. Immunoprecipitated RNAs were purified for qRT-PCR analysis.
Luciferase reporter assay
The wild-type and mutated EGFR-AS1 or ACTN4 fragments covering miR-2355-5p binding sites were obtained and inserted to the pmirGLO reporter vector (Promega Corporation, Madison, WI). The formed EGFR-AS1-WT/Mut and ACTN4-WT/Mut reporter vectors were co-transfected to SiHa and CaSki cells with indicated transfection plasmids. 48 h later, luciferase reporter assay system (Promega) was applied. Besides, TOP/FOP-flash reporter vectors were available to assay the Wnt/β-catenin signaling activity as guided by manufacturer (Addgene, Cambridge, MA).
Averaged results of more than 3 independent experiments were used, and data were all exhibited as SD. Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA) was employed to analyze data by t test, one-way or two-way ANOVA, with p value below 0.05 as the threshold.