Patients and plasma samples
We collected plasma samples from 30 patients with locally advanced stage ESCC who received radiotherapy or chemoradiotherapy at the Cancer Hospital, Chinese Academy of Medical Sciences from 2007 to 2010. None of the patients had undergone surgery. Eligibility criteria were as follows: age between 18 and 70 years, Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≤ 1 and no history of other malignances. All patients provided written informed consent and the study was approved by the Institutional Review Boards of Cancer Hospital, Chinese Academy of Medical Sciences.
MiRNAs isolation
Total RNA, which included the miRNA fraction, was extracted from serum samples using the mirVana PARIS Kit (Ambion, USA) and from cultured cells using Trizol reagent (Invitrogen) according to the manufacturers’ instructions. The purity and concentration of all RNA samples were evaluated according to their 260/280 nm absorbance ratios, which were determined using a NanoDrop 2000C spectrophotometer (NanoDrop Technologies, Rockland, DE, USA).
MiRNAs microarray and qRT-PCR analyses
Expression levels of the miRNAs were determined using an AB TaqMan Human MicroRNA Array (Applied Biosystems), which included probes for 748 mature human miRNA sequences. Expression of miR-323a-3p was determined by the stem-loop qRT-PCR method. Briefly, complementary DNA (cDNA) was prepared using 2 ug total RNA as template and synthesized using a SuperScriptTM First-Strand Synthesis System for RT-PCR Kit (Invitrogen, USA) based on the specific stem-loop RT primers for miR-323a-3p or U6 (RiboBio, China). U6 small nuclear RNA was used as an internal control for miR-323a-3p. For quantification of FMR1 mRNA, total RNA was reverse transcribed into cDNA using a First-Strand cDNA Synthesis Kit (Promega, USA) using FMR1-specific primers. The FMR1 primer sequences were as follows: forward, 5′-CGGCAAATGTGTGCCAAAGA-3′; reverse, 5′-ATGTGCTCGCTTTGAGGTGA-3′. The qRT-PCR was performed on an ABI Prizm 7300 Sequence Detection System (Applied Biosystems) using Light Cycler DNA Master SYBR Green I Mix (Roche, Switzerland) according to the manufacturer’s instructions. All qRT-PCR reactions were performed in triplicate. The expression levels of miR-323a-3p and FMR1 mRNA were quantified using the 2-ΔΔCt method and normalized to internal control levels.
Bioinformatic prediction of miRNA target genes
The predicted target genes of miR-323a-3p were investigated using the iterative algorithms of TargetScan (http://www.targetscan.org/), miRbase (http://microrna.sanger.ac.uk/), and miRDB (http://mirdb.org/).
Cell Culture
Seven ESCC cell lines, KYSE-30, KYSE-140, KYSE-150, KYSE-180, KYSE-450, KYSE-510, and YES-2, were cultured in RPMI-1640 medium (Gibco BRL, USA) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, USA) at 37°C in humidified air containing 5% carbon dioxide. Throughout the experiments, the cells were used in the logarithmic phase of growth.
Cell transfection
We selected KYSE-30, KYSE-150, and YES-2 cells for the functional studies. Synthesized miR-323a-3p mimics, inhibitor, mimic negative control (NC), and NC inhibitor were purchased from RiboBio (Guangzhou, China). Cell transfections were performed at a concentration of 50 nM using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. Total RNA and protein were harvested 48 h after transfection and used for qRT-PCR and western blot analysis, respectively, as described.
Cell proliferation, migration, and invasion assays
Cell proliferation analysis was performed using transfected cells. The cells were seeded into 96-well plates at approximately 3 × 103 cells per well in 200 μL of medium. At 0, 24, 48, and 72 h post seeding, 100 μL 5% MTS reagent (Promega, USA) in phosphate-buffered saline (PBS) was added to each well and incubated for 2 h. Absorbance was then read at a wavelength of 490 nm using a microplate reader (Bio-Rad, USA).
Cell migration and invasion assays were analyzed using Transwell chambers (Costar, USA) and Matrigel matrix (BD Biosciences, USA). Briefly, 1 × 105 transfected cells in 200 μL serum-free medium were seeded into the upper chamber with 100 μL of 2% Matrigel for migration or without Matrigel for invasion assays. The lower chambers were filled with 600 μL RPMI-1640 containing 20% FBS. The chambers were then incubated at 37 °C with 5% CO2 for 8–20 h. The cells that migrated or invaded the membrane of the insert were fixed, strained, and analyzed. Cells in 10 random microscopic fields (×100 magnification) were counted for each insert.
Western blot analysis
Cells were lysed with cold lysis buffer consisting of 1% NP-40 supplemented with a complete protease inhibitor tablet (Sigma, USA) for 30 min on ice. Equivalent amounts of total-protein extracts were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The blots were blocked with 3% bovine serum albumin (BSA) for 1 h and the incubated with primary antibodies against fragile X mental retardation protein (FMRP, 1:500; Proteintech, China) and β-actin (control, 1:5000, Santa Cruz, USA) at 4 °C overnight. After washing with PBS three times, the membranes were incubated with their corresponding secondary antibodies at room temperature for 1 h. The membranes were washed again and then incubated with the chemiluminescence substrate. Photographs were taken using an ImageReader LAS-4000 (Fujifilm, Japan) and analyzed using Image-Pro Plus 6.0 software.
Plasmid construction and luciferase activity assays
The FMR1 3′-UTR target site sequence and sequence with an eight-nucleotide mutation in the miR-323a-3p target site were synthesized and cloned downstream of the luciferase gene in the pEGFP-C1 luciferase vector (Generay, China). The resulting vectors were named FMR1-3′-UTR-WT and FMR1-3′-UTR-Mut, respectively. Cells were seeded into 12-well plates and co-transfected with the constructed plasmids, internal control Renilla luciferase plasmid (pRL-SV40), and mimic or mimic NC using Lipofectamine 2000 (Invitrogen, USA). After 48 h, cells were harvested and luciferase activity measured using a Dual Luciferase Assay Kit (Promega, USA) and a Synergy H1 hybrid reader (Biotek, USA). Results were normalized to Renilla luciferase activity and the data expressed as relative luciferase activity. Experiments were performed in triplicate on three separate occasions.
Retroviral infection
The lentivirus for miR-323a-3p was purchased from GeneChem. The miR-323a-3p lentivirus was prepared and used to transfect cells according to the manufacturer’s protocol.
Analysis of in vivo tumorigenicity
BALB/c nu/nu male mice, 4 weeks old, were purchased from Vital River for the in vivo tumorigenicity study. All experimental procedures were approved by the Institutional Animal Welfare Guidelines. Mice were injected subcutaneously in the hind legs with 2 × 106 cells in 0.2 mL. The size of the tumors was determined based on caliper measurements of subcutaneous tumor masses. Tumor volumes were calculated according to the formula 4/3πr1 × r22 (r1 > r2). Each experimental group included six mice and the experiments were repeated three times. In addition, mice were injected via tail veins with 1 × 106 cells in 0.2 ml.
Statistical analysis
Differences between groups were estimated using the Student’s t test and the Chi-square test. OS was defined as the time between the date of diagnosis and death. Local recurrence-free survival (LRFS) was defined as the time between the date of diagnosis to local or regional lymph node recurrence, last follow-up, or death. Survival data were analyzed using the Kaplan-Meier method and compared with the log-rank test. All experiments were performed at least three times. For the expression of miR-323a-3p, relative qualification (RQ) > 1 was regarded as overexpression and RQ ≥ 2 or ≤ 0.5 were considered significantly different. Data are expressed as mean ± SEM. Differences between groups were analyzed by two-tailed Student's t-test. Statistical significance is indicated by * P < 0.05, ** P < 0.01, and *** P < 0.001 versus the relevant control. The data were analyzed using SPSS 19.0 software or Prism 7.0 software (GraphPad).