Cell lines and cell culture
The human ESCC cell lines EC9706, EC109, KYSE30 and KYSE150, and a human esophageal squamous epithelial cell line Het–1A were obtained from the Chinese Academy of Sciences Committee on Type Culture Collection Cell Bank (Shanghai, China). The cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI–1640; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) at 37℃ in a 5% CO2 atmosphere.
RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
Total cellular RNA was extracted using TRIzol reagent (Invitrogen, Waltham, CA, USA). For miRNA expression analysis, qRT-PCR was carried out using the TaqMan MicroRNA Reverse Transcription kit and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The relative quantification of miR–4684–5p was performed using the 2-△△Ct method, with U6 used as an internal control. For FAM83H-AS1 and ZBTB38 expression analysis, qRT-PCR was performed using the TaqMan High-Capacity cDNA Reverse Transcription Kit and TaqMan Fast PCR Master Mix (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The relative quantification of FAM83H-AS1 and ZBTB38 was performed using the 2-△△Ct method, with GAPDH used as an internal control. The reactions were performed independently in triplicate, and the primer sequences are listed in Table 1. The RNA expression levels were analyzed as described previously [13, 21].
Name
|
Primer sequence
|
FAM83H-AS1
|
F: 5′-TAGGAAACGAGCGAGCCC-3′
R: 5′-GCTTTGGGTCTCCCCTTCTT-3′
|
miR-4684-5p
|
F: 5′-ACCAGGGGTACCTCTCTACT-3′
R: 5′-AAGGGTACGTCTCCACCGA-3′
|
ZBTB38
|
F: 5′-TGTCTTGAAGTGAGGCTCTGCTG-3′
R: 5′-AGCAAGCCTTGTGGACCAAAC-3′
|
GAPDH
|
F: 5′-GCACCGTCAAGGCTGAGAAC-3′
R: 5′-GCCTTCTCCATGGTGGTGAA-3′
|
U6
|
F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′
R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′
|
Table 1
Cell transfection
FAM83H-AS1 was cloned into empty pcDNA3.1 vectors (Invitrogen, Waltham, MA, USA) to construct FAM83H-AS1 overexpressing plasmid (p-FAM83H-AS1). The empty pcDNA3.1 vector was used as a control (vector control). Small interfering RNAs (siRNAs) targeting FAM83H-AS1 (si-FAM83H-AS1), negative control siRNAs (si-NC), miR–4684–5p inhibitors, miR–4684–5p mimics and miRNA scrambled control (control mimics) were all synthesized by GenePharma (Shanghai, China). All of the above vectors were subsequently transfected into ESCC cells by applying Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) according to the manufacturer’s manual. After 48 h of transfection, cells were collected and processed for further studies.
Cell proliferation assay
After transfection, ESCC cell viability was assessed with the CCK–8 kit (Dojindo, Kumamoto, Japan). In brief, ESCC cells (3 × 103 cells/200 μl/well) were seeded into the 96-well plates, and six replicates were set up for each sample. 100 μl of fresh medium were added to replace culture medium at the appointed time point, and 10 μl of CCK–8 solution were pipetted into each well immediately. After incubated at 37℃ for 2 h, the OD450 value were determined using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Cell migration and invasion assay
ESCC cell migration was evaluated using a transwell migration assay. 5 × 104 ESCC cells/well were resuspended in 250 μl RPMI 1640 supplemented with 1% FBS and plated onto uncoated 8-µm transwell filter inserts in 24-well plates in triplicate. The lower chambers contained 500 μl of RPMI 1640 supplemented with 15% FBS as a chemoattractant. The non-migratory cells in the upper chamber were removed with a cotton swab after incubation for 16 h, while cells on the bottom side were fixed in 100% methanol and stained with 0.5 µg/ml 4′,6-diamidino–2-phenylindole (DAPI) for 5 min. Then stained cells were counted using a fluorescence microscope (Eclipse 80i; Nikon Corporation, Tokyo, Japan) in five random fields.
A transwell invasion assay was performed to assess cell invasion ability. ESCC cells were seeded into the upper chamber of Matrigel-coated inserts containing 250 μl RPMI 1640 containing 1% FBS. RPMI 1640 containing 15% FBS was added to the lower chamber as a chemoattractant. After incubation for 48 h, cells in the upper chamber were removed, and cells on the bottom side of chamber were fixed in 70% ethanol and stained with 0.1% crystal violet for 30 min. The stained cells were lysed with 200 μl of lysis reagent. Finally, 100 μl of lysate was taken to a 96-well plate, and the absorbance was measured at 560 nm by a microplate reader (550; Bio-Rad, USA).
Glucose uptake and lactate production assay
ESCC cells were cultured in glucose-free RPMI 1640 for 16 h, and then incubated with high-glucose RPMI 1640 under normoxic conditions for an additional 24 h. Culture supernatants were then collected for intracellular glucose levels measure using a fluorescence-based glucose assay kit (BioVision, Milpitas, California, USA) according to the manufacturer’s instructions. Lactate levels were measured using a lactate oxidase-based colorimetric assay read at 540 nm according to the manufacturer’s instructions (Beyotime, Wuxi, China) and normalized to cell number.
Bioinformatics analysis
The potential target miRNAs of FAM83H-AS1 were predicted via computational algorithms, including TargetScan (http://www.targetscan.org/vert_72/) and miRDB (http://mirdb.org/). The highest-ranked predicted potential target of FAM83H-AS1 was miR–4684–5p. To identify potential genes targeted by miR–4684–5p, we also used the TargetScan and miRDB. From the list of target genes obtained, all genes likely to contribute to ESCC progression were extracted.
Luciferase reporter assay
The partial sequences of FAM83H-AS1 or ZBTB38 3′-UTR, which contains the putative miR–4684–5p-binding site, were amplified by PCR and constructed into the pmirGLO Luciferase vector (Promega, Madison, WI, USA) to generate wild-type FAM83H-AS1 reporter (FAM83H-AS1-WT) or ZBTB38 reporter (ZBTB38-WT). The GeneArt™ Site-Directed Mutagenesis System (Thermo Fisher Scientific, Waltham, MA, USA) was used to produce mutant-type FAM83H-AS1 (miR–4684–5p target site-mutation FAM83H-AS1, FAM83H-AS1-MUT) reporter or mutant-type ZBTB38 (miR–4684–5p target site-mutation ZBTB38 3′-UTR, ZBTB38-MUT) reporter. All constructs were verified by DNA sequencing. Subsequently, the luciferase reporter and control mimic or miR–4684–5p mimic were co-transfected into ESCC cells. 48 h after transfection, luciferase assays were performed using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) following the manufacturer’s instructions.
RIP assay
RIP was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) following the manufacturer’s instructions. In brief, 1 × 107 ESCC cells transfected with miR–4684–5p mimic and control mimic were lysed in the RIP lysis buffer containing a protease inhibitor cocktail. Next, the ESCC cell supernatant was incubated with the RIP buffer containing a magnetic bead conjugated with antibodies against human AGO2 or the control normal mouse IgG. Then the protein and DNA in the RIP complex were removed using RNase-free DNase I and Proteinase K. Then, the immunoprecipitated RNA complex was isolated and subjected to qRT-PCR to detect the enrichment of FAM83H-AS1 or ZBTB38.
RNA pull-down assay
ESCC cells were transfected with biotin-labeled wild type miR–4684–5p (Bio-miR–4684–5p-WT), mutated miR–4684–5p (Bio-miR–4684–5p-MUT) or antagonistic miR–4684–5p probe (GenePharma, Shanghai, China). 48 h after transfection, cells were harvested and incubated with a specific lysate buffer (Ambion, Austin, TX, USA) for 10 min. Then, the cell lysates were mixed with M–280 streptavidin magnetic beads (Invitrogen, Waltham, CA, USA) for 3 h at 4℃. The pull-down products were subjected to qRT-PCR for FAM83H-AS1 expression.
In vivo xenograft experiments
The animal experiments were approved by the Animal Care and Use Committee of Taizhou University School of Medicine and performed in accordance with the relevant guidelines and regulations of the committee. BALB/c athymic nude mice (female, 4 weeks old) were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and randomly divided into three groups (Blank control, si-FAM83H-AS1 and si-NC; n = 5 each). A total of 1 × 107 EC9706 cells (trancfected with si-FAM83H-AS1 or si-NC, untreated cells as blank control, respectively) were subcutaneously injected into one flank of each nude mouse. Tumor volume was calculated using the formula (Length×Width2)/2. Tumors were harvested and weighed after 24 d.
Western blotting analysis
Cells were collected and lysed using RIPA Lysis Buffer (Beyotime, Wuxi, China) supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Roche, Pleasanton, CA, USA). The protein concentration was determined using a BCA Protein Assay Kit (Beyotime, Wuxi, China). Approximately 50 µg of protein extract was separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to polyvinylidene fluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany) and incubated with specific antibodies. The primary antibodies against ZBTB38 (1:1000, catalog no.ab231263; Abcam, Cambridge, MA, USA), LDH-A (1:1000, catalog no.ab125683; Abcam, Cambridge, MA, USA) and GLUT1 (1:1000, catalog no.ab15309; Abcam, Cambridge, MA, USA) were used. Following extensive washing, membranes were incubated with a horseradish peroxidase-conjugated goat polyclonal anti-rabbit IgG secondary antibody (1:2000, catalog no.7074; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Immunoreactivity was detected by enhanced an chemiluminescence system kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and visualized using an LAS–4000 imaging system (Fujifilm Holdings Corporation, Tokyo, Japan). GAPDH (1:1000, catalog no.ab181602; Abcam, Cambridge, MA, USA) served as a loading control.
Statistical analysis
All data are presented as the mean ± standard deviation (SD). The statistical analyses were performed using SPSS 18.0 software (IBM, New York, USA). Differences between groups were analyzed using Student’s t-test (two groups) or one-way ANOVA (multiple groups). P<0.05 was considered statistically significant.