Cell culture
K-1, TPC-1 and 293T were purchased from Cell Bank in Chinese Academy of Sciences (Shanghai, China). 10% FBS DMEM (100 units/mL penicillin, 100 µg/mL streptomycin) was used to culture cells in humidified 5% CO2 environment at 37 ºC.
Lentivirus generation
Lentivirus was generated as previously described. Briefly, 20 µg plasmids with targeting sequence was co-transfected with psPAX2 (10 µg) and pMD2.G (µg) into 293T cells using the standard calcium phosphate transfection method. Lentivirus supernatant was collected with 0.45 filter after 48 hours and infected TC cells. 5 µg/ml puromycin was used to select shRNA infected TC cells.
CircRNA construction
Circ-FLNA nucleotides with 1 kb upstream and 200 bp downstream were amplified from the gene locus, and cloned into PWPI (Pme I + Pac I digested) using the Gibson ligation system at 50 ℃ for 1 hour. Gene mutation was performed using site-directed mutagenesis method as previously described.
Real time qPCR
Isolation of total RNAs was done with trizol reagent following the standard RNA extraction protocol. Two µg total RNAs were subjected to reverse transcription using superscript III transcriptase (Invitrogen). RT product with 200 dilutions was used to perform real time qRT-PCR in Bio-Rad CFX96 machine with SYBR green to determine the expression levels of interested genes. 18 s ribosome RNA was utilized to normalize gene expression.
RNase R digestion: RNase R digestion was done with 2 µg total RNAs in 10 µl reaction at 37 0C for 1 hour: 0 unit or 20 units of RNase R (Epicentre), 1 × RNase R buffer, 1 unit murine ribonuclease Inhibitor (NEB). Treated RNAs was used to perform reverse transcription to make cDNA. Then real time PCR was performed as aforementioned.
Western blotting: After PBS wash, TC cells were lysed by RIPA buffer and proteins were collected. 20 µg proteins were loaded for electrophoresis in 10%-12% SDS-PAGE gel. Then proteins were transferred to PVDF membrane and blocked with 10% BSA for 1 hour. Incubation with specific primary antibody was performed at 4 ºC for at least 16 hours. Next day, the membrane was washed and incubated with 1:2500 HRP conjugated secondary antibody for 1 hour at room temperature. After extensive TBST wash, blots were visualized using enhanced chemiluminescence (Thermo Fisher Scientific).
Wound healing assay
TC cells with gene manipulations were scratched using a pipette tip. Images were captured at 0, 24 hours. Experiment was done in triplicate and quantification was gained using the Image J software.
Transwell invasion assay: Transwell inserts (Corning) were pre-coated with 1:8 diluted matrigel (BD Bioscience) before use. PTC cells with gene manipulations were collected and loaded into the inserts at the density of 1 × 105/well. 0% FBS medium in the lower chamber was used as chemoattractant to attract cells. 36 hours later, the invading cells in inserts were fixed with cold methanol and stained with 0.5% crystal violet. Images were taken by inverted microscope (Olympus) and quantifications of invading cells were done with ImageJ.
Chromatin Immunoprecipitation Assay (ChIP)
TC cells were cross-linked with 4% formaldehyde for 10 minutes. Then cells were collected and sonicated to 300–500 bp DNA fragments. Cell lysates were pre-cleared with protein A-agarose, and followed by the incubation with TR4 primary antibody (IgG was used as negative control). The next day, TR4-DNA fragments were intensively washed with high salt buffer. Proteins were digested with proteinase K and DNA fragments were extracted for RT-PCR. Specific primer sets were designed to amplify a target sequence within the promoter of FLNA and RT-PCR products were examined by agarose gel electrophoresis.
Xenografted mouse model: 2 × 106 K-1 cells were subcutaneously implanted into 6-week male nude mice with 1:1 matrigel (Corning). Tumor size was measured weekly by caliper. 8 weeks later, mice were sacrificed and tumors were removed, washed, fixed for IHC staining.
Immunohistochemical staining (IHC): Tissues (human and mice) were fixed in 10% (v/v) formaldehyde and embedded in paraffin. After hydration and antigen retrieval in boiling citrate buffer (pH 6.0), sections were treated with 3% peroxidase in methanol for 15 minutes and blocked with 10% goat serum PBS buffer for 30 minutes. The slides were incubated with 1:100 diluted TR4 or MMP9 primary antibody at 4℃ overnight. Next day, 1:100 diluted biotin-labeled secondary antibody was used to incubate slides for 30 minutes, and followed by 30 minutes incubation with streptavidin (PK-4000, Vectastain, Burlingame, CA.). The protein signal was determined by DAB staining.
Statistical Analysis
All statistical analyses were performed using GraphPad Prism software. Data were presented as mean ± SE. Differences were analyzed with the one-way ANOVA test, and significance was set at P < 0.05.