Patients and follow-up
A total of 60 ESCC patients (37 males and 23 females, 62.4 +/- 5.6 years old) who were diagnosed as ESCC through histopathological exam between May 2015 and August 2015 were enrolled in this study. This study was approved by the Ethics Committee of General Hospital of Eastern Theater Command. All patients were diagnosed for the first time. This study excluded the factors that can potentially affect the expression of the target gene of the present study, such as initiated therapy within 3 months prior to admission, other clinical disorders and history of malignancies, from the 60 ESCC patients. All patients signed the informed consent. The 60 patients were grouped into stage I or II (n = 32), and III or IV (n = 28). All patients were followed up for 5 years from the day of admission to record survival. Follow-up was performed through a monthly manner and all patients completed the follow-up or died of ESCC during follow-up.
ESCC tissue acquisition and ESCC cells
ESCC and adjacent (within the area 3 cm around tumors) paired non-tumor tissue samples were collected from the 60 ESCC patients through fine needle aspiration. All tissue samples were confirmed by histopathological exam. Tissue samples were kept in liquid nitrogen prior to the subsequent assays. ESCC cell model used in this study was human ESCC cell line KYSE450 (Laboratory of Birth Defects and Reproductive Health, China). Cells were cultivated in medium composed of 10% FBS and 90% RPMI-1640 medium under the conditions of 5% CO2, 37 °C and 95% humidity to reach about 80% confluence prior to the subsequent assays.
CircRIMS and miR-613 were overexpressed in KYSE450 cells by transfecting KYSE450 cells (106) with either 1 μg CircRIMS expression vector or 50 nM miR-613 mimic. Mimic of miR-613 and negative control (NC) miRNA were obtained from Genecopoeia (Guangzhou, China). The expression vector of CircRIMS was constructed with pcDNA3.1 expression vector obtained from Invitrogen (Shanghai, China). NC experiments were performed by transfecting empty vector or NC miRNA into the same number of cells. In all transfections, untransfected cells were the control (C) cells. The transfected cells were kept for 48 h in fresh medium to perform the following experiments.
Total RNAs were isolated from tissues and cells using Ribozol (VWR). RNA samples were digested with DNase I (Invitrogen) until to remove genomic DNA. RNA integrity analysis was performed through 5% urea-PAGE gel electrophoresis.
StaRT Reverse Transcription kit (AnyGenes) was used to prepare cDNA samples through reverse transcriptions (RTs) using RNA samples as template. PowerTrack SYBR Green Master Mix (Rhenium) was used to perform qPCRs to analyze the expression of CircRIMS. The endogenous control of CircRIMS was 18S RNA. The expression of mature miR-613 was analyzed using All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia) by adding poly (A), followed by RTs and qPCRs with poly (T) as reverse primer. Ct values of target genes were normalized to endogenous controls using 2−ΔΔCT method.
Methylation-specific PCR (MSP)
Quick Genomic DNA Extraction Kit (Clinisciences) was used to isolate genomic DNA from KYSE450 cells with transfections. EZ DNA Methylation-Gold™ Kit (ZYMO RESEARCH) was used to convert genomic DNA samples. The methylation of miR-613 was analyze through routine PCRs and MSPs, which were all performed using 2x Taq mix (Invitrogen).
Cells with transfections were cultivated in a 96-well plate with 4,000 cells in 0.1 ml medium per well. Cells were cultivated under the aforementioned conditions for 48 h, followed by incubation with BrdU (10 mM) for 48 h. After that, cells were fixed and incubated with peroxidase-coupled anti-BrdU-antibody (Sigma–Aldrich) for 48 h. After washing, peroxidase substrate was used to incubate the cells for 2 h. Cell proliferation was analyzed by measuring OD values at 450 nm.
Heml 1.0 software was used to plot heatmaps to present the differential expression of CircRIMS and miR-613 in paired tissue samples. ANOVA Tukey’s test was used to compare differences among multiple cell transfection groups. Correlations were analyzed by Pearson’s correlation coefficient. With the median expression levels of CircRIMS in ESCC tissues as the cutoff value, the 64 ESCC patients were grouped into high and low (n = 32) level groups. Survival curves of both groups were plotted and compared by log-rank test. P < 0.05 was considered as statistically significant.