Analyze gene expression in PTC tissues and its association with clinical prognosis
The Cancer Genome Atlas (TCGA) thyroid cancer data were obtained from UCSC Xena (http://xena.ucsc.edu). A total of 59 PTC tissues and matched adjacent normal thyroid tissues were applied to compare the HOTAIR and DLX1 expression. In survival analysis, HOTAIR and DLX1 expression were obtained from 513 and 515 PTC tissues, respectively, and stratified as high or low based on the expression level with significant differences in the survival outcomes and the lowest log-rank P-value among subgroups using Cutoff Finder . HOTAIR expression in 308 TCGA PTC tissues with different genetic alternations including BRAFV600E, NRAS, HRAS mutations and RET fusion were further compared. We also utilized the TCGA data to select the putative HOTAIR suppressed genes that present a significantly negative correlation with HOTAIR expression. The correlation between HOTAIR and DLX1 expression (n = 513) was further plotted and analyzed by Pearson correlation.
Cell lines and culture conditions
The human papillary thyroid cancer cell lines including BCPAP, K1, MDA-T41 and MDA-T32 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-amphotericin B at 37 ℃ in 5% CO2.
HOTAIR knockdown using small interfering (si)RNA transfection
The HOTAIR siRNAs (Cat#N-187951-02, GE Healthcare Dharmacon) and scramble siRNA (Cat#D-001210-01, GE Healthcare Dharmacon) were used to generate the HOTAIR knockdown cells (MDA-T32 si-HOTAIR) and scramble control cells (MDA-T32 Scramble). 10 nM siRNAs with transfection reagent were added to the cells following the protocols supplied by the manufacturer. Further assays or experiments were performed at 48 h post-transfection.
Generation of constitutive HOTAIR overexpression
The sequence of HOTAIR (NR_003716) was cloned from the LZRS-HOTAIR plasmid (Addgene plasmid #26110) and inserted into the pEGFP-Lv105 vector (Capital Biosciences) as the working HOTAIR plasmid. pEGFP-Lv105 empty vector was used as a control. Then, we applied lentiviral packaging kit (Cat#3D5F03, Origene) in HEK293T cells to produce lentiviral particles. The MDA-T41 cells were transduced with HOTAIR plasmid- or empty vector-lentiviral particles in 8 µg/ml hexadimethrine bromide (Sigma)-containing growth medium to generate MDA-T41 HOTAIR-OE cells or Control cells, respectively. Then, cells were revived in normal growth medium for an additional 24–28 h with subsequent selection in puromycin-containing growth medium.
Cell proliferation assay
The Cell Counting Kit-8 (CCK-8) assay purchased from Dojindo Laboratories (Cat#TJ557, Kumamoto) was used to evaluate cell proliferation rate. All the experimental protocols were performed as described in the manufacturer’s instructions. Briefly, 1 × 103 cells per well were seeded into a 96‑well plate and cultured at 37 °C. At the indicated time points, CCK‑8 solution was added to each well with 1:9 ratio (CCK-8: media) and cells were incubated at 37 °C for a further 3 h. The absorbance at 450 nm was measured with a microplate reader (The Synergy™ HT, Bio-Tek, Taiwan).
Colony-forming unit assay
1 × 103 cells per well were seeded in a 6-well plate for 7–14 days at 37 ℃ in 5% CO2. The cells were fixed with 4% formalin for 30 min, washed by PBS and stained with 0.1% crystal violet (Cat#MKCH6258, Sigma). The colony forming units were photographed and counted using Image J.
5 × 103 cells per well were seeded in a 12-well plate to reach 80–90% confluence. Then, a small wound was created in each well with a gentle wash to remove cell fragments. Cells were cultured at 37 ℃ in 5% CO2 and images were taken under a microscope at 0 and 16 h.
Flow cytometric analysis of the cell cycle
Briefly, cells with the culture medium were harvested, washed with PBS following 1300 rpm spinning for 5 min at 4 ℃ × 3 times, fixed with ice cold ethanol (75%), washed with 1% FBS contained PBS following 1300 rpm spinning for 5 min at 4 ℃ twice, then stained with propidium iodide (PI) solution containing 50 μg/ml PI (Sigma) and 10 μg/ml RNase A (Thermo Fisher Science). Images of the cell cycle were obtained by the FACSCalibur system (BD Biosciences).
Gene expression analysis
Total RNA was extracted from cells using TRIzol reagent and cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit (Cat#00984365, Thermo Fisher Science). SensiFAST SYBR No-ROX mix (Cat#98005, Bioline) was used for running quantitative real-time PCR in a LightCycler 480 (Roche). The ΔCT values of target genes were normalized to the ΔCt of stably expressed reference transcripts (GAPDH). The primer sequences used are listed in Additional file 1: Table S2.
Protein extraction and western blot
Total cells are washed with ice-cold PBS and whole cell extracts were prepared using protein lysis buffer. The Pierce™ bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Science) was used to quantify protein amount of the samples. All immunoblots are standardized to the same amounts of proteins per well. Then, proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Western blotting analysis was subsequently performed via incubating the membranes with primary antibodies against to proteins of interest under appropriate dilution. Then, horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence assay were applied to visualize the signal. Band intensities are determined via using an UVP GelStudio™ PLUS Imager (Analytik Jena, Germany). The primary antibodies against CDK1 (1:1000, Cat#9116), CDK2 (1:1000, Cat#2546), CDK4 (1:1000, Cat#12790), CDK6 (1:1000, Cat#13331), Cyclin A2 (1:1000, Cat#91500), Cyclin B1 (1:1000, Cat#4135), Cyclin D1 (1:1000, Cat#55688), Cyclin E1 (1:1000, Cat#4129), p-AKT (Ser473) (1:1000, Cat#9271S), AKT (1:3000, Cat#9272S) and PTEN (1:1000, Cat#9552) were purchased from Cell Signaling Technology. Other primary antibodies used here including DLX1 (1:1000, Cat#PA5-28899, Thermo Fisher Science) and β-actin (1:10000, Cat#NB600-501, Novus). The complete photos of western blotting on these proteins were presented in the Additional file 1: Figure S4.
Chromatin Immunoprecipitation (ChIP)
We used the EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation kit (Cat#17–10086, Millipore) to perform ChIP experiments as the standard protocols. Briefly, in vivo cross linking was conducted via adding 1% formaldehyde to the cells (1 × 106 per ChIP) for 10 min. Sonication (2*10 bursts of 30 s ON/OFF at high-level output in the sonicator (XL2015, Misonix)) of the cross-linked chromatin was performed to generate < 500 bp DNA fragments with confirmation via running electrophoresis on the agarose gel stained with DNA VIEW (Cat#TT-DNA01, TOOLS). 1% of chromatin supernatant was collected as the input. Then, antibodies with ChIPAb + H3K27me3 (Cat#17–622, Millipore), ChIPAb + EZH2 (Cat#17–662, Millipore) or mouse IgG (Cat#12-371B, Millipore) were used to perform immunoprecipitation. For binding the antibody/antigen/DNA complex, magnetic protein A/G Beads (Cat# CS204457, Millipore) were applied. Then, ChIP samples were going through washing, reversal of cross-linking, and ChIP DNA isolation. The eluted ChIP DNA was used as the template for real-time qPCR using SYBR Green. To assess the occupancy of H3K27me3 and EZH2 on the DLX1 gene, the primers target on DLX1 promoter region were designed as listed in Additional file 1: Table S2.
Immunohistochemistry staining of normal thyroid and papillary thyroid cancer tissues
The paraffin embedded sections of normal thyroid tissues (n = 22) and papillary thyroid cancer tissues with different stages (n = 52 with stage I, n = 20 with stage II, n = 15 with stage III and n = 3 with stage IVA) were purchased from US Biomax, Inc (Cat#TH8010a, Cat#TH208, Cat#TH961). Immunohistochemistry (IHC) staining of DLX1 was performed as the standard protocol including deparaffinized, rehydrating, antigen retrieval, immunohistochemical staining using the antibody against DLX1 (Cat#PA5-28899, Thermo Fisher Science), dehydrating and stabilizing with mounting medium and viewing the staining under the microscope. The staining intensity from 0 to 3 was scored, with 3 referred as the section with maximum intensity, while 0 indicated negative. The percentage of staining was estimated, and 100% staining was scored as 1. The IHC scores were calculated by staining intensity*staining percentage and assessed by an independent pathologist.
Graphpad Prism 6.0 and SPSS version 22 were used for data analysis. The details of statistical methods were described in the figure legends.