Clinical tissue specimens
A total of 45 pairs of PTC cancer tissues were obtained with written informed consent via surgical resection at The Second Affiliated Hospital of Hebei Medical University, Shijiazhuang, Hebei, China from May 2016 to February 2020. All tissue specimens were histopathologically examined by three independent pathologists. Fresh tissue specimens were frozen in liquid nitrogen and stored at -80 °C until utilization. All the clinical samples were acquired with informed consent from the participants. The Institutional Review Board of The second hospital of Hebei Medical University reviewed and approved this study (2016-R269). The clinical studies were conducted according to the principles expressed in the Helsinki Declaration.
Cell lines and culture
The PTC cell lines TPC-1 and K1 were from American Type Culture Collection (ATCC). Cells were incubated in RPMI-1640 Medium (Thermo Fisher Scientific; Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific). All cells were kept in humidified air at 37 °C and 5% CO2.
Experimental protocol of TAT-Beclin1 peptide
TAT-Beclin1 peptide is a known autophagy inducer [27-29]. The cell permeable TAT-Beclin1 peptide was obtained from the Peptide Core at the University of Colorado Anschutz Medical Campus. The sequence of the TAT-Beclin1 peptide was RRRQRRKKRGYGGDHWIHFTANWV . In in vitro experiments, cells were treated with TAT-Beclin1 or vehicle (normal saline) at a dose of 10 μM. Tumor-bearing mice were treated with TAT-Beclin1 or vehicle via intraperitoneal injection (I.P.) at a dose of 20mg/kg/day.
Recombinant lentiviruses encoding DUSP4 were constructed by homologous recombination between the expression vector (pEX-Puro-Lv105) and cDNA in 293 cells as previously described using the lentivirus construction kit (GeneCopoeia) . The same method was used to construct and package the corresponding control vector. After 2 days, supernatants were collected, and cells were incubated in medium containing lentiviruses at a multiplicity of infection (MOI) of 25 for 2 d. The infected cells were selected using puromycin (5 μg/ml). The overexpression efficiency of viral gene was detected using qPCR analysis.
Control siRNAs or siRNAs against human DUSP4 were obtained from Thermo Fisher Scientific. The target sequences were as following:
The indicated cells were cultured in 6-well plates and then transfected with siRNAs (100 pmol/well) using RNAiMAX (Thermo Fisher Scientific) in accordance with manufacturer’s protocols. For 48 hours, the silence efficiency of siRNAs was detected using qPCR analysis.
Western blotting assays
The lysates of the indicated cells were prepared from 6-well plates, separated on 10% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (PVDF, Thermo Fisher Scientific), which were incubated with the specific antibodies against DUSP4, Beclin1, LC3B and β-actin (Cell Signaling Technology, MA, USA). Horseradish peroxidase (HRP)-linked secondary antibodies were applied to visualize the immunoreactivity under a chemiluminescence system (Omega Lum G, Aplegen, CA, USA).
Quantitative real-time PCR (qPCR) assays
The total RNA was extracted and purified by the TRIzol method. Synthesis of cDNA and qRT-PCR measurements were performed according to the manufacturer’s protocols. The pre-designed primer sequences for qPCR were as follows:
DUSP4, 5'-CTACATCCTAGGTTCGGTCAAC-3' (sense) and
GAPDH, 5'-CCTGCCTCTACTGGCGCTGC-3' (sense) and 5'-GCAGTGGGGACACGGAAGGC-3' (anti-sense). Relative expression was calculated using the 2−△△Ct method.
GAPDH was used as the internal reference. qPCR was carried out using SYBR Premix Ex TaqTM kit (TakaRa, Tokyo, Japan) and ABI7500 PCR system (Applied Biosystems, Thermo, MA, USA).
Cell proliferation analysis
To assess the cell proliferation, cell counting Kit-8 (CCK-8) assays were carried out using the CCK-8 kit (Dojindo, Kumamoto, Japan). For CCK-8 assay, the indicated cells were plated into 96-well plates with 2,500 cells/well. Following the indicated time, all cells were incubated with 10 μl CCK-8 reagent. After 2 h incubation, the optical density at 450 nm (OD450) was measured using Varioskan Flash reader (Thermo Fisher Scientific).
Cell death analysis
To evaluate the cell death, trypan blue exclusion assays were performed as previously described19. The cells failing to exclude the presented blue-dye were defined as the dead cells. The total death rate (%) = number of dead cells/(number of living cells + number of dead cells) × 100%.
The indicated cells were seeded on 6-cm dishes and fixed using 4% paraformaldehyde (PFA). After perforated with 0.5% Triton-100, cells were blocked using 1% bovine serum albumin (BSA) and incubated with the specific antibody against LC3B (Cell Signaling Technology) at 4°C overnight. Subsequently, cells were stained with ﬂuorochrome-labelled secondary antibody for 30 min and then counterstained with DAPI for 10 min. Ultimately, the cells were observed and counted under the fluorescence microscopy (Olympus IX81, Tokyo, Japan). The cells with more than five LC3-puncta were considered positive cells .
Cell migration assays
The migratory ability of cells was evaluated by Transwell assay. For Transwell assay, the indicated cells suspended in serum-free DMEM along with 1mg/ml mitomycin C (aimed to inhibit cell proliferation) were seeded onto the upper chamber of the Transwell. DMEM containing 20% FBS was added into the lower chamber. After 36 h of incubation, the cells that migrated to the lower surface of the inserts were fixed, stained with 1% crystal violet, and photographed. The migration levels were determined by counting the number of stained cells.
6-week-old male athymic BALB/c nude mice (20–22.5 g) were purchased from Animal center of Gem Pharmatech Co., Ltd (Nanjing, China). LV-DUSP4-transduced and corresponding control K1 cells were inoculated subcutaneously on the ventral side of the right rib at the density of 5x106 cells per mouse (8 mice per group). According to the application of TAT-Beclin1 peptide and the transduced lentiviruses, the above tumor-bearing mice were divided into four groups: 1) LV-Cont group; 2) LV-Cont+TAT-Beclin1 group; 3) LV-DUSP4 group; 4) LV-DUSP4+TAT-Beclin1 group. The tumor volumes in the four groups of mice were measured every three days to observe the growth of tumours. The shortest diameter (A) and the longest diameter (B) were measured with a caliper to determine the tumour volume. The volume was calculated using the formula V = (A2 × B)/2. After 30 days, all mice were anesthetized with isoflurane (2%, Inhalation anesthesia), and then sacrificed via cervical dislocation. All mice were considered dead when their hearts and breathings stopped. Their tumours were removed and weighted. These experimental protocols were approved by the Institutional Animal Care and Use Committee of The Second Affiliated Hospital of Hebei Medical University (2016-R269). The mice were housed in a specific pathogen-free facility with barrier in which the room temperature was 20~30℃ and the humidity 60~80% and fed a SPF mouse chow and sterile water.
Immunohistochemistry (IHC) assessment
The tissue sections were prepared, incubated overnight at 4°C with primary DUSP4 antibody (1:100), and then visualized by the PV-9000 DAB detection kit in accordance with manufacturer’s protocols. The sections were observed under the IX81 microscope. DUSP4 staining was graded semi-quantitatively. Staining intensity was graded as 1 (no stain), 2 (weak stain), 3 (clear stain), or 4 (strong stain). The total immunoreactivity score was obtained by multiplying the intensity and abundance (expressed as a fraction).
All statistical analyses were performed using the GraphPad Prism Software 6. For comparisons, Wilcoxon rank sum test, one-way ANOVA test or Student’s t-test were performed as indicated. Tukey test was used for Post-Hoc Multiple Comparisons. Pearson chi-square test were performed for correlations. P < 0.05 was considered statistically significant.