Cell culture and TC tissues
The undifferentiated ATC cell lines ARO and FRO were initially obtained from the Chinese Academy of Medical Science. All cell lines were maintained in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, ZETA, USA) at 37 °C in a humidified atmosphere containing 5% CO2. The cells in the logarithmic phase of growth were used throughout the experiment. The TC tissue microarrays were obtained from Shanghai Outdo Biotech Company (Shanghai, China). Each array contained TC tissues and adjacent TC tissues from a total of 62 cases.
Immunohistochemical analysis (IHC) and pathology scores
As described previously[18], IHC staining was performed according to the manufacturer’s instructions. The tissue microarrays were incubated at 4ºC for overnight with anti-GRP78 polyclonal antibody (1:100, Santa Cruz, CA, USA). The immunoreactivity proportion was classified based on percentage scores as < 5% (0), 5%–25%(1), 25–50% (2), 50–75% (3), and > 75% (4). The staining intensities based on intensity scores were classified as negative (0), weak (1), moderate (2), and strong (3). The final score was obtained by multiplying the percentage scores with the intensity scores. The total scores that ranged from 0 to 4 were defined as low group, and those that ranged from 5 to12 were defined as high group.
Lentivirus-Mediated siRNA Construction and Cell Transfection
The lentivirus-mediated small interference RNA for GRP78 (siGRP78) and the negative control RNA (siNC) were designed and synthesized by Genepharma (Shanghai, China). The GRP78 siRNA sequence was: F: 5-GGUACUGCUUG
AUGUAUGUTT-3, R: 5- ACAUACAUCAAGCAGUACCTT 3. The control siRNA sequence was: F: 5-UUCUCCGAACGUGUCACGUTT-3, R: 5-ACGUGACACGUUCGGAGAATT-3. For transfection experiments, the cells of ARO and
FRO were plated in 6-well plates and siRNAs were transfected using LipofectamineTM 2000 reagent (Invitrogen, USA) according to the manufacturer’s protocol for 24 hours. The efficiency of gene silencing was further confirmed by western blotting in both the cell lines.
Tunicamycin (TM) treatments
ARO and FRO cells at a density of 3 x 105cells/mL in a total volume of 3 mL were seeded in 6-well plates. After inoculation after 24 hours, different concentrations of TM (0–1μg/mL) were added and the cells were cultured for another 72 hours. A parallelly untreated culture with same passage number as that of adapted cultures was maintained as control cultures, and the level of ER stress was further confirmed by western blotting in both the cells.
Western blot assay
The cells were collected, the total protein was prepared in RIPA buffer (medium, Beyotime, China), and then the cytoplasmic proteins were obtained using the Nuclear and Cytoplasmic Protein Extraction Kit (P0028, Beyotime, China). Western blotting was performed as described previously[20]. The antibodies used for western blotting were PERK, XBP1s and CLO1A1 (Abcam, USA), GRP78 and MMP13 (Santa Cruz, CA, USA), and β-actin (Beyotime Institute of Biotechnology Jiangsu, China).
Cell proliferation
Cell counting kit-8 (CCK-8, Dojindo laboratories, Kumamoto, Japan) was used to measure cell proliferation. Cells were inoculated in 96-well plates, pre-incubated for 24 h and then for 0 h, 24 h, 48 h, 72 h or 96 h (5% CO2, 37 °C). At each time point, 10 μL CCK-8 solution was added to each well and the cells were further incubated for one to three hours. The absorbance (OD value) was measured using a microplate reader at 450 nm.
Transwell migration Assays
Transwell chambers were used for conducting migration assays in vitro as described
previously[12]. The cells at a density of 5x104 cells per well were seeded in the upper chamber (8 μm pore size, Corning, USA). After incubation for 24 h at 37 ºC, the wells were washed with PBS thrice, fixed with methanol for 1 h and then stained with 1% crystal violet for 30 min. The cells on the upper surface of the filter were scraped off, and the cells on the lower surface of filters were counted under a light microscope (Olympus BX51, Olympus) at 200X magnification in ten randomly selected fields. The experiment was repeated independently three times.
GO Enrichment, KEGG Pathway analysis of RNA-seq data
RNA‐sequencing data of GRP78 and si-GRP78 in ARO and FRO cells were tested by BGI (Shenzhen, China)[36]. The functional roles of differentially expressed genes (DEGs) were determined by the transcriptional profiles that are acquired using RNA-seq and GO and KEGG enrichment analyses. The GO database was used to analyze the functional enrichment of DEGs, focusing on the pathways associated with these genes that were enriched in the biological process, molecular function and cellular component. The KEGG pathway database was used to determine the enrichment of DEGs.
GEPIA database for differential expression analysis ofMMP13 and CLO1A1 genes
Differential expression of MMP13 and CLO1A1genes between 512 TC tissues and 337 normal tissues was acquired from the GEPIA (http://gepia.cancer-pku.cn/)[35]. In addition, the differential expression of MMP13 and CLO1A1 genes in terms of pathological stage (I-IV) was analyzed among the TC patients.
Statistical analysis
Statistical significance was calculated with GraphPad Prism Software using one-way ANOVA with Tukey’s post-test and Student’s t-test. Data are presented as means±SD. The values with p<0.05 were considered to be statistically significant difference.