The microarray datasets including GSE59652 and GSE143224 available online were downloaded from the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI). Differentially expression analysis of GSE59652 was performed to screen differentially expressed lncRNAs between laryngeal cancer tissues and non-tumorous tissues by using the limma package of R language based on the criteria “P Value< 0.05 and |fold change| > 1.” The top 10 upregulated and top 10 downregulated lncRNAs were used to generate a heat-map by R language.
A total of 90 patients with laryngeal cancer were recruited in this study at Beijing Chaoyang Hospital of Capital Medical University from between 2011.01 and 2016.01. 90 matched cancerous and noncancerous tissues were tested by real-time qPCR. 90 pairs of tumor and adjacent normal tissues were obtained by surgical excision, and the histopathological diagnosis for each sample was confirmed by two pathologists independently. All participants have written to obtain their infirmed consents. All producers were approved by the human Ethics Committee of Beijing Chaoyang Hospital of Capital Medical University. The clinicopathological characteristics of 90 laryngeal cancer patients were shown in Table 1.
Human laryngeal carcinoma cell lines TU212, TU177 and AMC-HN-8 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HEK293T cells for the luciferase reporter assay were purchased from the Institute of Biochemistry and Cell Biology (Shanghai). All cells were cultured in DMEM Medium (Gibco, USA) supplementing with 10% fetal bovine serum (FBS) (Invitrogen), and 1% penicillin/streptomycin at 37°C with 5% CO2.
MiR-384 mimics, miR-384 inhibitor and corresponding negative controls miR-NC and inhibitor NC were purchased from RiboBio (Guangzhou, China). To overexpress POU2F1, the cDNA sequence of POU2F1 were amplified by PCR and then cloned into Expression Vector pcDNA3.1 (Invitrogen, CA, USA) to generate oe-POU2F1. Meanwhile, the empty vector pcDNA3.1 (oe-NC) was used as the negative control. Cell transfection for siRNAs and plasmids into TU212 and TU177 cells was performed by using Lipofectamine 2000 kit (Invitrogen, USA) according to the manufacturer’s instructions. After transfection for 48 h, cells were harvested and used for subsequent experiments.
The construction of stable cell lines with downregulation of DUXAP8
The short hairpin RNA targeting DUXAP8 (sh-DUXAP8) and negative control sh-NC were inserted into pLKO.1 lentiviral vector and then transfected into TU212 and TU177 cells with a lentiviral gene delivery system as previously described. Subsequently, cells were treated with 1 μg/ml puromycin for 2 weeks to screen the stable cell lines. And the expression of DUXAP8 was determined by qRT-PCR analysis. The sequences used in this study as follows: sh-DUXAP8: 5′- GCAGCATACTTCAAATTCACAGCA -3′; sh-NC: 5′-UUCUCCGAACGUGUCACGUTT-3′.
Total RNA of tumor tissues and cultured cells was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Approximately 1.2 μg of total RNA was reversely transcribed into cDNA by a Reverse Transcription Kit (Takara, Dalian, China). Then qRT-PCR assay was performed by using the SYBR Green (Takara) based ABI 7500 Detection System (Applied Biosystems, Foster City, CA, USA) with GAPDH and U6 as the internal references. The relative expression change of targets was analyzed by the 2-ΔΔCt method. The primers used as follows: DUXAP8 forward: 5′- AGGATGGAGTCTCGCTGTATTGC -3′; reverse: 5′- GGAGGTTTGTTTTCTTCTTTTTT -3′; POU2F1 forward: 5′- GCGAAGCTTGTTAAAATATTCAAAATGGCGGAC -3′; reverse: 5′- GATTGCTCCTCCTACAGGCAC -3′; GAPDH forward: 5′‐AGGGCTGCTTTTAACTCTGGT ‐3′, reverse: 5′‐ CCCCACTTGATTTTGGAGGGA ‐3′; U6 forward: 5′- GCTTCGGCAGCACATATACTAAAAT -3′, reverse: 5′- CGCTTCACGAATTTGCGTGTCAT -3′.
Total protein of cultured cells was extracted by using by using RIPA lysis buffer (Beyotime Institute of Biotechnology). Approximately equal amounts of protein were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore). After blocking with 5% skim milk, the membranes were incubated with primary antibodies POU2F1 (#8157S, 1:1000, CST, USA), GAPDH (ab181602, 1:5000, Abcam, UK) overnight at 4°C. Of which, GAPDH was considered as the internal reference. Then the membranes were exposed to HRP‐conjugated secondary antibody at room temperature for another 1 h. ECL chromogenic substrate was used for quantification by densitometry (Quantity One software; Bio-Rad).
To determine the cellular localization of DUXAP8, cytosolic and nuclear fractions of TU212 cells were separated by the Nuclear/Cytosol Fractionation Kit (BioVision, Milpitas, CA, USA). Then, RNA from the cytosolic and nuclear fractions was extracted by using TRIzol reagent, and the expression level of DUXAP8 in cytoplasm and nucleus was evaluated by qRT-PCR method.
CCK-8 assay was performed by using a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Gaithersburg, MD). In brief, 1,000 TU212 and TU177 cells were seeded into 96-well plates overnight. And 10 ul of CCK-8 reagent was added to each well at 0, 24, 48, 72 and 96 h for another 2 h. The absorbance at 450 nm was detected with a microplate reader.
Colony formation assay
For colony formation assay, 1,000 TU212 and TU177 cells /well were seeded into 6-well plates and cultured for two weeks. Subsequently, cells were fixed with 4% paraformaldehyde and stained with crystal violet, then colonies were photographed and counted under a light microscope.
EdU staining assay
EdU staining assay was performed by using an EdU Apollo DNA in vitro kit (RIBOBIO, Guangzhou, China) according to the manufacturer’s instructions. Briefly, cells were incubated with 100 μl of 50 μM EdU/well at 37 °C for 2 h, and then visualized by using a fluorescence microscopy.
Luciferase reporter assay
Starbase v2.0 was applied to predict the potential binding sites between DUXAP8 and miR-384, as well as miR-384 and POU2F1. To determine the interactions among them, the wild type (WT) and MUT type (MUT) 3'-UTR of DUXAP8 and POU2F1 were amplified by PCR and inserted into psiCheck2 luciferase reporter vector. Then luciferase reporter plasmids were co-transfected with miR-384 mimics or miR-NC into HEK 293T cells. After transfection for 48 h, cells were lysed and relative luciferase activity was measured by using a dual luciferase reporter system.
RNA immunoprecipitation (RIP) assay
RIP assay was carried out by using a Thermo Fisher RIP kit (Thermo Fisher Scientific, MA, USA). In brief, the whole lysates of TU212 cells were incubated with magnetic beads conjugated to anti-Ago2 antibody or the negative control anti-IgG (Abcam) overnight at 4°C. On the next day, 30 μL of magnetic beads were added and incubated for another 2 h at 4°C, and immune-precipitated RNA was purified by TRIzol reagent and used for qRT-PCR analysis.
Immunohistochemistry (IHC) assay
To evaluate the cell proliferation in vivo, tumor tissues from mice were paraffin-embedded and cut into 5 µm-thick sections. Hematoxylin and eosin (H&E) staining was used to select the representative areas as previously described. Then antibody against Ki-67 (1:400, Abcam, ab15580, USA) was used to detect the proliferation. The staining positivity was quantified in three different high-power fields of each section.
Xenograft tumor model
BALB/c nude mice (approximately 4 weeks old) were purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China). 2 × 106 TU212 cells stably transfected with sh-DUXAP8 or sh-NC were subcutaneously injected into the mice to construct the in vivo model as previously described (N = 6). Tumor volume was calculated with the following formula: V (mm3) = (L × W2) / 2 every week for 4 weeks. On the day 28, mice were sacrificed through cervical dislocation and tumor weight was evaluated. All animal experiments were approved by the animal Ethics Committee of Beijing Chaoyang Hospital of Capital Medical University.
All data were presented as mean ± SD derived from at least three independent times. Statistical analysis was performed by using SPSS v.20.0 (Abbott Laboratories, Chicago, IL, USA). Difference was determined by Student’s t test (two groups) or one-way ANOVA (among three or more groups). Kaplan–Meier method was used to compare the survival difference of laryngeal cancer patients based on the expression median of DUXAP8. Spearman correlation analysis was used to evaluate the correlation between groups. P < 0.05 was considered as the significant threshold.