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 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (ZETA, USA) at 37 °C in a humidified air atmosphere containing 5% CO2. The cells were used in the logarithmic phase of growth throughout the experiment. The TC tissue microarrays were obtained from ShGnghGi Outdo Biotech CompGny (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 4℃overnight with anti-GRP78 polyclonal antibody (1:100, Santa Cruz, CA, USA). The immunoreactivity proportion were classified as follows (percentage scores): < 5% (0), 5–25%(1), 25–50% (2), 50–75% (3), and > 75% (4). The staining intensities were classified as follows (intensity scores): negative (0), weak (1), moderate (2), and strong (3). The final score was obtained by percentage scores × intensity scores. Total scores ranging from 0 to 4 were defined as low group, and total scores ranging from 5 to12 were defined as the high group.
Lentivirus-mediated siRNA Construction And Cell Transfection
The lentivirus-mediated small interfering 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, 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 blot analysis in both cells.
Tunicamycin(TM) Treatments
ARO and FRO cells were seeded in 6-well plates at 3 × 105cells/mL in a total volume of 3 mL. Post 24-hrs of inoculation, different concentrations of TM(0–1 µg/mL) was added and the cells were cultured for another 72 hours until day. A parallel untreated culture with same passage number as adapted cultures was maintained as control cultures, the level of ER-stress was further confirmed by western blot analysis in both cells.
Western Blot Assay
The western blot were performed as described previously[20]. The antibodies used for the western blot 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 plate, pre-incubated for 24 h and then incubated for 0 h, 24 h, 48 h, 72 h or 96 h (5% CO2, 37 °C). At which point, 10 µL CCK-8 solution was added to each well and cells were further incubated for one to three hours. The absorbance (OD value) at 450 nm was measured with a microplate reader.
Transwell Migration Assays
Transwell chambers were used for migration assays in vitro. as described previously[12], 5 × 104 cells were seeded per well in the upper chamber (8 µm pore size, Corning, USA). After incubation for 24 h at 37 ˚C, the wells were washed with PBS three times and then fixed with methanol for 1 h and 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 by a light microscope (Olympus BX51, Olympus) at 2009 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)[35]. The functional roles of differentially expressed genes (DEGs) were revealed by determining the transcriptional profiles acquired using RNA-seq and by GO and KEGG enrichment analyses. We used the GO database to analyze the functional enrichment of DEGs, focusing on the pathways associated with these genes that were enriched in the terms 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 of MMP13 and CLO1A1 genes
Differential expression of MMP13 and CLO1A1 genes between 512 TC tissue and 337 normal tissue was acquired from GEPIA (http://gepia.cancer-pku.cn/)[35]. In addition, we further explored 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 mean ± SD. Differences with p < 0.05 were considered statistically significant.