Totally 53 clinical ESCC and adjacent noncancerous tissue samples (> 2.0 cm from the tumor edge) were obtained during surgery at the Hunan Cancer Hospital of Central South University from January 2015 and December 2016. No patients underwent preoperative therapy. The samples underwent snap freezing in liquid nitrogen and stored at -80°C. The collection and utilization of tissue samples had approval from the ethics committee of Hunan Cancer Hospital, conforming with current regulations. Each participant provided signed informed consent. The clinicopathologic features of all patients with ESCC are listed in Table 1.
Human ESCC cells (TE-1, Eca109, KYSE30, KYSE150, KYSE410 and KYSE510), and human normal esophageal epithelial HET-1A cells were obtained from the Institute of Cell Research, Chinese Academy of Sciences (Shanghai, China). KYSE30 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS, Hyclone, USA) and 1% PenStrep (100 U/mL Penicilium and 100 µg/mL Streptomycin), the other cell lines were cultured in RPMI 1640 containing 10% FBS and 1% PenStrep. All cells were maintained in a humid atmosphere containing 5% CO2 at 37°C.
Lentivirus construction and cell transfection
For stable overexpression of LIPH-4 and Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), LIPH-4 and IGF2BP2 expression plasmids were constructed by inserting the related cDNA sequences into the Ubi-MCS-SV40-EGFP-IRES-puro lentiviral vector (Genechem, China). For stable knockdown of LIPH-4 and IGF2BP2, siRNA constructs for LIPH-4 or IGF2BP2 were obtained by cloning the DNA sequences targeting LIPH-4 or IGF2BP2 into the hU6-MCS-CBh-gcGFP-IRES-puromycin plasmid, which were introduced in a lentiviral vector (Genechem, China). For lentivirus transfection, cells were incubated with viral particles overnight with 10 µg/mL polybrene (Sigma-Aldrich, USA). Then, the specimens were incubated for 14 days with 2 µg/ml puromycin to establish stable transfectants. MiR-216b-5p mimics and inhibitors were provided by Hanbio (Shanghai, China) and transient transfection utilized lipofectamine 3000 (Invitrogen, USA) as directed by the manufacturer. The sequences of siRNA were listed in Supplementary Table 1.
Cell counting Kit-8 (CCK-8) assay
ESCC cells underwent seeding in a 96-well plate at 2×103/well and incubated for 4 days. Daily, 10 µl of CCK-8 solution was supplemented per well and incubated for 1 h at 37°C before absorbance measurement at 450 nm.
Colony formation assay
Totally 103 ESCC cells underwent seeding into a 35-mm dish and incubation for 14 days. Cell colonies were fixed with 4% formalin for 15 min and stained with 0.1% crystal violet. Colonies with > 50 cells were imaged by light microscopy and counted with Image J.
5-ethynyl-2’-deoxyuridine (EdU) assay
The EdU assay was carried out with EdU Cell Proliferation Kit with Alexa Fluor 555 (Epizyme, China) as directed by the manufacturer. Briefly, ESCC cells were cultured in a 24-well plate overnight. Then, the EdU reagent was added for 2 h at 37°C, followed by cell fixation (4% formalin), permeabilization (0.5% Triton-X-100, Sigma) and Hoechst 33342 counterstaining. A fluorescence microscope was utilized for analysis.
Flow cytometry assay
Cells underwent culture in 6-well plates for 24 h culture, followed by overnight fixation at 4°C in 70% ethyl alcohol. The specimens were assessed with Cell Cycle and Apoptosis Analysis Kit (Beyotime, China) as directed by the manufacturer. Cell cycle analysis was carried out with a FACS Calibur flow cytometer (BD Biosciences, USA).
Cell culture was performed in 6-well plates for 24 h, and the apoptotic rate was examined with the Annexin-VFITC apoptosis detection kit (BD, USA) according to the inserted protocol. Analysis was performed with a FACS Calibur flow cytometer.
RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA extraction from tissue and cell samples was carried out with TRIzol (Invitrogen, USA) following the kit’s protocol. LncRNAs and mRNAs were assessed with a SYBR Green PCR Kit (Takara, Japan), using GAPDH as an internal control gene. A miDETECT A Track Kit (RiboBio, China) was utilized to detect microRNAs, with U6 for normalization. The 2−△△Ct method was utilized for data analysis. Primer sequences were listed in Supplementary Table 2.
Cytoplasmic and nuclear fractions were obtained with a PARIS Kit (Invitrogen, USA) as directed by the manufacturer, and qRT-PCR was performed to quantitate LIPH-4 mRNA, with U6 and GAPDH as nuclear and cytoplasmic controls, respectively.
Total protein was obtained from cell samples with the RIPA buffer (Beyotime, China) supplemented with protease inhibitor cocktail tablet (Roche, Switzerland). Western blot was carried out with rabbit antibodies targeting IGF2BP2 (1:5000, Abcam, UK), cyclinD1 (1:1000, Abways, china), AKT (1:1000, Abways, china), p-AKT (1:1000, Abways, china), and GAPDH (1:5000, ZENbio, China) antibodies. HRP-conjugated IgG (1:5000, Beyotime, China) was utilized as secondary antibodies. The ECL-Plus reagent (Millipore, USA) was utilized for development. GAPDH was used for normalization.
4-5-week-old female BALB/c nude mice (n = 6/group) were housed under specific pathogen-free conditions. To establish the xenograft model, stably transfected cells (5×106 in 0.2 ml PBS containing 10% Matrigel (BD Biosciences, USA) were injected by the subcutaneous route into the mouse armpit. Each tumor was measured every 3 days with digital calipers to derive tumor volume as length × width2/2. Euthanasia was performed at 4 weeks, and the final volume and weight of each tumor were assessed. The tumors were paraffin embedded, followed by hematoxylin and eosin (H&E) staining or immunohistochemistry (IHC). Experiments involving animals had approval from the Animal Ethics Committee of Hunan cancer hospital, following The Guidelines for the Care and Use of Laboratory Animals.
Luciferase reporter assay
Bioinformatics tools were utilized for predicting potential miR-216b binding sites of LIPH-4- and IGF2BP2-3ʹ-UTR (Starbase v2.0, RegRNA2.0, miRcode and microRNA.org). Human 293T cells were co-transfected with 160 ng of empty pmirGLO-LIPH-4-wt/mut or pmirGLO-IGF2BP2-wt/mut and 5 pmol miR-216b mimic or miR-NC utilizing Lipofectamine 3000 (Invitrogen, USA) strictly following the manufacturer’s recommendations. After 48 h of incubation, Dual-Luciferase Reporter Assay System (Promega, USA) was utilized for luciferase activity assessment in triplicates experiments.
RNA immunoprecipitation (RIP) assay
An EZ Magna RNA immunoprecipitation Kit (Millipore, USA) was applied as directed by the manufacturer. In brief, KYSE510 cells underwent lysis with RIP lysis buffer. The resulting cell lysates were added to magnetic beads linked to anti-Ago2 antibodies (Millipore, USA) or control anti-IgG for overnight incubation at 4°C. The immunoprecipitated RNA was isolated and assessed by qRT-PCR.
Data are mean ± standard deviation from three assays or more, performed independently. SPSS 18.0 (SPSS, USA) and GraphPad Prism 6 (GraphPad, USA) were utilized for data analysis. The Pearson chi-square test was utilized to assess associations of LIPH-4 expression with clinicopathological variates. Kaplan–Meier curve analysis was used to assess survival, and the log-rank test was used for comparisons. Differences between groups were analyzed by Student' s t-test. P < 0.05 was deemed statistically significant.