Cell culture and siRNAtransfection
The human cervical cancer cell line HeLa obtained from the American Type Culture Collection (ATCC) was grown in DMEM containing 10% FBS, 2 mM glutamine, 50U/ml penicillin, and 50mg/ml streptomycin at 37°C with 5% CO2. A total of 40 nM siRNA targeted against HOTAIR (siHOTAIR-I or siHOTAIR-II) or negative control siRNA (siNC) were transfected into HeLa cells using RNAi-mate. All siRNAs were purchased from GenePharma Co., Ltd., and siRNA sequences are listed in Table S1-1. HOTAIR expression levels were measured 48 h after transfection by qPCR (see below).
HeLa cells were also transfected with 40nM of siRNA targeted against YAP1 or RPL23 to silence gene expression. Again, cells were harvested 48 h after transfection, and protein expression was assessed by western blotting (see below).
MiR-200a-3p mimics, inhibitors, and negative controls were obtained from GenePharma, and their sequences are listed in Table S1-1. Cells cultured in 6-well plates were transfected miR-200a-3p mimics or inhibitors with 40nM/well according to the manufacturer’s protocol. The negative controls consisting of random sequences had no detectable effects on human cell lines or tissues.
RNA isolation and qRT-PCR
Total RNA was extracted from cultured HeLa cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. A Nanodrop 2000 spectrophotometer was used to measure the concentration of total RNA. RNA was then reverse-transcribed into first strand cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Invitrogen). Quantitative PCR was carried out using the SYBR Green PCR Master Mix (Roche) and Light Cycler 480 Real-Time PCR system (Roche). GAPDH was used as the endogenous control gene to normalize expression of the target genes. Each sample was analyzed in triplicate. The thermal cycling program consisted of 95°C for 5 min followed by 40 cycles of 95°C for 10 s, 62°C for 45 s, and 72°C for 30 s. Melting curve data were then collected to verify the PCR specificity and the absence of primer dimers. All primer sequences are listed in Table S1-2.
MiRNAs were isolated using the mirVana miRNA isolation kit (Ambion) according to the manufacturer’s protocol, and quantified using a Nanodrop 2000 spectrophotometer. Quantitative analysis of miR-200a-3p expression was performed using a Hairpin-it miRNA real-time PCR quantitation kit (GenePharma). Small nuclear RNA U6 was used as an internal control. Each sample was analyzed in triplicate.
Plasmid constructs and expression
The full-length YAP1 (NM_001130145) and RPL23 (NM_000978) cDNA were amplified by RT-PCR from total RNA isolated from HeLa cells, and inserted into the mammalian expression vector pCDNA3.1/myc-his B (Invitrogen, USA). The BamH I and XhoI restriction sites were designed in the forward and reverse primers respectively. All the sequences of primers were listed in Table S1-3.
Plasmid was transfected into HeLa cells by Lipofection 2000 (Invitrogen) according to the manufacturer's instructions. After incubation for 6 h, the medium was removed and replaced with normal culture medium for 48 hr. And the plasmid pCDNA3.1/myc-his B was used as the negative control. Proteins expression was assessed by Western blotting.
Isolation of nuclear and cytoplasmic extracts
Nuclear extraction was performed using an NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. In brief, HeLa cells were washed twice with cold PBS and centrifuged at 500 ´g for 5 min. The cell pellet was suspended in 200 μl of cytoplasmic extraction reagent I, and vortexed vigorously on the highest setting for 15 s, then incubated on ice for 10 min. Next, 11 μl CER II was added, vortexed for 5 s, incubated on ice for 1 min, and centrifuged at 16 000 ´g for 5 min. The supernatant (cytoplasmic extract) was immediately transferred to a clean pre-chilled tube. The insoluble pellet fraction, containing crude nuclei, was resuspended in 100 μl nuclear extraction reagent by vortexing for 15 s, incubating on ice for 10 min, then centrifuging at 16 000 ´g for 10 min. The supernatant (nuclear extract) was immediately transferred to a clean pre-chilled tube and used for subsequent experiments.
Wound healing assay
HeLa cells were seeded into 6-well plates and allowed to grow to 70% confluency. Cell monolayers were then wounded by scratching a plastic pipette tip (1 mm) across the plate. Cellular debris was removed by washing with PBS. The number of cells migrating into the wound surface and the average distance of migrating cells were determined under an inverted microscope at designated time points.
Matrigel cell invasion assay
Transwell chambers (Corning, 8.0 μm pore size) coated with Matrigel (BD Biosciences) were used to measure the invasiveness of cancer cells. In brief, 2×105 cells were plated in the upper chamber in serum-free media, and medium with 10% FBS was added to the bottom chamber. After 48 h incubation, the bottom of the chamber insert was fixed in methanol for 15 min and stained with Giemsa stain. Invading cells were photographed and counted under a microscope. Each membrane was divided into four quadrants and an average from four quadrants was calculated.
Protein extracts (10 μg) prepared with RIPA lysis buffer were resolved on a 12% SDS-PAGE gel, and transferred to an Immobilon-P PVDF transfer membrane (Millipore) by electro-blotting. After blocking with 5% non-fat milk, membranes were incubated overnight at 4°C with a 1:1000 dilution of the following primary antibodies: rabbit anti-YAP1 polyclonal, rabbit anti-RPL23 polyclonal, rabbit anti-mutant p53 polyclonal, rabbit anti-TBP (TATA binding protein) polyclonal, or mouse anti-GAPDH polyclonal. Blots were then incubated with peroxidase-conjugated IgG (ABclonal) diluted for 1 h at room temperature and then developed using a Super Signal West Pico kit (Pierce). Immunoblots were scanned using an Image Scanner. Blot densitometry analysis was performed using Image J software. All analyses were performed in triplicate.
Potential YAP1 miRNA binding sites were predicted by computer-aided algorithms on the TargetScan Human (Release 7.0) (http://www.targetscan.org/vert_61/) (56), and PicTar (pictar.mdcberlin.de/) (57). The downstream transcriptional regulator of YAP1 was predicted using the Cistrome Data Browser (http://cistrome.org/db/)(58), which is a data portal for chromatin immunoprecipitation (ChIP)-Seq and chromatin accessibility data in humans.
Assay of luciferase activity
Site-directed mutagenesis was used to change the binding sites of miR-200a-3p in the 3¢UTR of YAP1. The sequences of primer were shown in Table S1-4. For the reporter assay, a total of 20 nM miR-200a-3p mimics or control miRNA were co-transfected with 0.1μg pGL3-YAP1-3¢UTR or pGL3-YAP1-3¢UTR-mut into HeLa cells using Lipofectamine2000 (Invitrogen). Cells were harvested 48 h post-transfection using lysis buffer. Luciferase activities in cell lysates were determined using the Dual-Luciferase Reporter Assay System (Promega).
The ChIP assay was performed using ChIP Assay Kit, following the manufacturer’s instructions. Briefly, 1×107 HeLa cells were cross-linked with 1% formaldehyde for 10 min at 37°C. Then, cells were scraped and resuspended with ice-cold PBS containing protease inhibitor cocktails (Pierce). Cells were then lysed and sonicated to shear DNA to an average length of 200–1000 bp. All procedures were performed on ice. Lysates were immunoprecipitated with an anti-YAP1 or anti-H3K27me3 antibody at 4ºC overnight. Immunoprecipitation with an irrelevant normal IgG was used as a negative control. Immune complexes were then isolated with Protein A/G Sepharose beads at 4ºC for 1 h. After washing, DNA fragments contained in immune complexes were purified and amplified by PCR. The sequences of primer were shown in Table S1-5.
HeLa cells were grown on sterile glass-bottomed culture dishes, fixed in 4% formaldehyde for 30 min, and permeabilized in 1% FBS, 0.2% Triton-X100 on ice for 5 min. After washing, cells were blocked with 1% BSA for 1 h at room temperature, and then incubated with antibody at a dilution of 1:200 overnight. They were then incubated with Dylight 488-conjugated IgG (H+L) at a dilution of 1:100 for 2h in the dark, then stained with DAPI for 15 min. Subsequently, cells were thoroughly washed three times with PBS and examined using an LSM 710 laser scanning confocal microscope (Zeiss, Germany).
Four-week-old female athymic BALB/c mice were purchased from Vital River Laboratories. For xenograft models, 2 ´106HeLa cells transfected with siNC or siHOTAIR were subcutaneously injected in the right flank of BALB/c nude mice (n=5 per group). Three weeks later, mice were sacrificed, tumors were homogenized, and proteins were extracted for western blotting. All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Institute of Wuhan University of Science and Technology.
Statistical analysis was performed using SPSS standard version 13.0 software. The independent Student’s t-test was used to compare continuous variables between two groups. Data were expressed as means ± SD from at least three independent determinations. Values of P<0.05 (or P<0.01) were considered statistically significant.