Patient samples and Ethical statement
LSCC tumorand non-tumor samples were collected from 30 patients between 2014 and 2021 at the Ruijin Hospital and Ninth hospital and Shanghai Pudong Gongli Hospital, Shanghai, China. This study was approved by the Human Research Ethics Committee of Pudong Gongli Hospital.
LSCC cells line (Hep2, NH8, TU686, TU212) and cell line (Hela) werepreserved by the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Hep2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco company, USA) with 10% Fetal Bovine Serum (FBS, Gibco). Hep2 cells were incubated in a humidified incubator with 37°C temperature and 5% CO2.
Animals were obtained from the Institute of Zoology, Chinese Academy of Sciences. Animal experiments were performed on basis of the guidelines for the care and utilization of experimental animals of the Institute of experimental animals. The following study protocol has been approved by the IRB of the medical center. Animal care and treatment were conducted on basis of the guidelines for the care and utilization of experimental animals of the NIH. At the end of the experiment, the animals were killed by intraperitoneal injection of excess pentobarbital sodium (4%, 200 mg/kg; Sigma, Shanghai, China). The lung of each group was then collected for further analyses.
Co-IP assay was performed following the manufacturer’s instructions (Thermo Scientific)3.
RNA Extraction and Quantitative Real-time PCR (qRT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and then was reversetranscribed to cDNA using PrimeScript Reverse Transcriptase system (Promega, Madison, WI, USA). QRT-PCR was performed to quantify IL-6 mRNA level with the SYBR Green PCR core Reagent kit (Applied Biosystems, Foster city, CA, USA). GAPDH was used as the endogenous reference. Data were analyzed by using the comparative Ct method. Specificity of resulting PCR products was confirmed by melting curves. The primers were designed using Primer Express v 2.0 software (Applied Biosystems, Foster City, CA, USA). The primers used in this assay were: ALG3: forward, 5′-CACCTTCTGGGTCATTCACAGG-3′ and reverse, 5′-GTGTCACCCTGCAGTTGGGTATAGT-3′; GAPDH: forward, 5′-CTCCTCCACCTTTGACGCTG-3′ and reverse, 5′-TCCTCTTGTG CTCTTGCTGG-3′.
Western blot analysis
TU686and NH8 cells were treated with the treatment of AURKA inhibitor (VX680)for corresponding time. Cells were lysed with RIPA buffer (Pierce, Rockford, USA) to extract the protein (The inhibitor was preserved by Selleck Chemicals company, Houston, TX, USA). And the concentration was measured with the BCA Protein Assay Kit. Proteins with an equal amount (100 μg/sample) were electrophoresed by 10% SDS-PAGE for 2 h and transferred onto0.22um PVFD membranes (Millipore, MA, USA). Membranes were incubated with primary antibodies (anti-p-AURKA (1:2000, Cell Signaling Technology), anti-p-ALG3 (1:2000, Cell Signaling Technology)and GAPDH (1:5000, Abcam).) overnight at 4°C. After membranes were incubated with secondaryantibody (1:5000, Cell Signaling Technology) for 2 h, the proteins were visualized using enhanced chemiluminescence detection system (Bioscience, Piscataway, NJ, USA).
CCK8 assay and Plate colony formation assay Cells
96-well plates seeded 2×103 cells in 100 ul of DMEM. CCK8 (10 ul) was added to every well. Cells were incubated for 2 h and OD450 absorbance values were measured. Cells were seeded into 6-well plates at 1×103 and 2×103 cells/well and cultured for 3 weeks with DMEM. Then cells were washed twice with PBS and stained with crystal violet for 30 min. Finally, cell colonies in every well were counted.
In Vitrocell migration and invasion assays
2×105treated cells after overnight starvation were plated in the coated filters in 100 μl of serum-free medium. And 600μl of medium containing 10% FBS was added to the lower chamber. The insert chambers’ membrane was coated by Diluted Matrigel (BD Biosciences) for measuring the cells invasion. All cells were counted under a high-power objective (10x) in random fields. For migration assays, the upper chamber membranes were plated on top of uncoated (Matrigel-free) filters.
In vivo metastasis
4-week-old male immunodeficient mice maintained by the animal resources facility of the medical school of Shanghai Jiaotong University. Animal care and experiments are performed following the "guidelines for the care and utilization of experimental animals" and "principles for the utilization and care of vertebrates", and are approved by the ethics committee of experimental animals of the Medical College of Shanghai Jiaotong University. The average volume and bodyweight of the four groups of mice were similar. The experimental animals were grouped according to the randomization formula. Researchers were not aware of the group allocation at the different stages of the experiment during the allocation, the conduct of the experiment, the outcome assessment, and the data analysis. Mice was randomly assigned to 5 groups (five in each group).Cells were inoculated via tail vein into the mice. After six months, counting pulmonary metastatic nodules by H&E staining.
Statistical analyses were performed using SPSS 13.0 software. The relationship between the ERp19 expression level and clinicopathologic parameters were calculated with the Pearson χ2 test. Significant differences between groups were determined using the student t test. Survival data analysis was performed using the Kaplan–Meier and log-rank tests (GraphPad Prism software v6.0).