br Fresh frozen GC tissues and corresponding normal gastric
Fresh-frozen GC tissues and corresponding normal gastric epithelial tissues were collected from 38 patients who underwent tumor surgical resection at The First Aﬃliated Hospital of Soochow University from May 2015 to May 2018. The study was approved by the ethics com-mittee of The First Aﬃliated Hospital of Soochow University and in-formed consents were provided by all patients. The normal human gastric epithelial mucosa cell line (GSE-1), and human GC cell lines (SGC-7901, BGC-823, MKN-28, AGS, MGC-803, and MKN-45) were all purchased from the Culture Collection of Chinese Academy of Sciences (Shanghai, China). All KX2-391 were maintained and stored following the instructions obtained from their providers. Briefly, MGC-803, BGC-823, MKN-28, SGC-7901 cells were maintained in RPMI-1640 medium GES-1 and HEK-293T cells were maintained in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA). AGS and DMEM/F12 medium cells were cultured in DMEM/F12 medium (ThermoFisher; Cat. No. 11330-057). All medium were supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine, penicillin and streptomycin (Gibco BRL, NY, USA). All cell lines were cultured in humidified air supplemented with 5% CO2 at 37 °C.
2.2. circRNAs expression profile analysis
Two gene expression profiles (GES100170 and GSE83521) of GC were downloaded from the Gene Expression Omnibus database (GEO, http://www.ncbi.nlm.nih.gov/geo). GSE100170 consisted of 10 sam-ples, including 5 normal samples and 5 GC samples. GSE83521 include 12 samples, consisting of 6 normal mucosa tissue samples and 6 GC tissue samples. GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/), an online analysis tool, was used to compare the diﬀerentially expressed genes (DEGs) of GES100170 and GSE83521 in our study. The adjusted P values were used to correct for the occurrence of false positive results via the Benjamini and Hochberg false discovery rate method.
2.3. RNase R resistance analysis of circRNAs
The circFAT1(e2) from MGC-803 cells was treated with RNase R (4 U/mg, Epicenter) or actinomycin D and incubated for 30 min at 37 °C. Then, the treated RNAs were reverse transcribed with specific primers and detected by quantitative real-time PCR (qRT-PCR) assay.
2.4. RNA extraction and quantitative real-time PCR (RT-PCR) assay
The total RNA of the GC tissues/cell lines and corresponding normal tissues/cell lines were extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. After RNA quantification and quality examination by NanoDro2000c (Thermo Scientific, Waltham, USA), 2 μg of total RNA were reverse transcribed to cDNA via BestarTM qPCR RT kit (#2220, DBI Bioscience, China). Then, the quantitative polymerase chain reaction (RT-PCR) assay was conducted using BestarTM qPCR MasterMix (#2043, DBI Bioscience, China) on an ABI7500 according to the protocols obtained from the manufacturers. The primers used in this study are listed in Table 1. All primers were designed and purchased from Sangon, China; the mRNA expression of miR-548g was normalized to U6, and the mRNA expression of EGFP, c-Met, and CDC25A were normalized to Cancer Letters 442 (2019) 222–232
2.5. Fluorescence in situ hybridization (FISH)
In situ hybridization was carried out using specific probes to the circFAT1(e2) sequence. Biotin-labeled specific RNA probes were tran-scribed from circFAT1(e2) PCR fragments using the biotin-labeling mix and RNA polymerase (Roche, China) according to the instructions provided by manufacturers. After grown to the exponential phase, MGC-803 cells were fixed using 4% formalin. Tissues were cut into 4 μm sections and then also fixed with 4% formalin. Cells and tissues were then hybridized in hybridization buﬀer with biotin-labeled probes specific to circFAT1(e2). Signals were measured by a tyramide-con-jugated Alexa 488 fluorochrome TSA kit. The results were obtained by using a Laser Scanning Confocal Microscope (Leica, Germany) at 400 x magnification.