Cells and tissues
Human nasopharyngeal poorly differentiated squamous cell carcinoma cell line (SUNE-1 and HONE-1) (EBV-positive) were obtained from Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences, which was maintained in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37˚C with 5% CO2. Moreover, 20 pairs of PDNSC and paratumor tissues were collected from patients undergoing a surgical procedure at the Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University. These patients provided written informed consent prior to the operation. This study was approved by the Ethics Committee of the Integrated Hospital of Traditional Chinese Medicine, Southern Medical University.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
RNAs from clinical samples or cells were subjected to detect the mRNA expression of MYL9 mRNA using RT-qPCR analysis[25]. SuperScript III RT (Invitrogen; Thermo Fisher Scientific, Inc.) was used to synthesize complementary DNA from 1 µg of total RNA. The PCR mixture contained 1 µL of the cDNA templates, 1 × final concentration of SYBR Green (Toyobo, Osaka, Japan), and the specific primers. The sequences of the primers synthesized by IGE Co., Ltd (Guangzhou, China). All tests were conducted in triplicate on a LightCycler 480 II (Roche, Basel, Switzerland). Relative expression of MYL9 mRNA were calculated with 2−ΔΔCt method[25]. The specific primer[26] sequences were as follows: MYL9 forwards, 5’-AGTTTGTCTCGGAGCTGTGG-3’ and reverse, 5’-GGTTCGTGTTCCTCAGCGTA-3’; ABCB1 forwards, 5’-GAGGAAGACATGACCAGGTATGC-3’ and reverse, 5’-AGCTGCCAGGCACCAAAATG-3’; GAPDH forwards, 5’-CCATCTTCCAGGAGCGAGAT-3’ and reverse, 5’-TGCTGATGATCTTGAGGCTG-3’.
Transfection
The siRNA targeting MYL9 (siMYL9) and negative control (siNC) were purchased from Guangzhou IGE Co., Ltd. (Guangzhou, China). The siMYL9 sequences was 5’-GCAACATCGTCTTCAAGAA-3’. siRNAs (including siMYL9 and siNC group) were transfected into SUNE-1 and HONE-1 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol[25], respectively. This assay was performed as described in a previous study[25].
MTT assay
MTT was used to detect the effect of MYL9 expression on PDNSC cell viability at different time points (1, 2, 3 and 4 days after siMYL9 transfection). This assay was performed as described in a previous study[25].
EdU analysis
Proliferating SUNE-1 and HONE-1 cells (including siMYL9 and siNC group) were examined using the Cell-Light EDU Apollo 488 or 567 In Vitro Imaging kit (Guangzhou Ribobio Co., Ltd.) according to the manufacturers’ protocols, respectively. This assay was performed as described in a previous study[25].
Transwell assay
Cell migration assay was performed using the Transwell chambers (8 μm, 24-well insert; Corning Inc., Lowell, MA, USA). Briefly, cells (1x105) in serum-free RPMI-1640 medium were added to the upper chamber, and medium containing FBS was added to the lower chamber. Cells were incubated for 12 h. Finally, the cells which migrated into the lower chambers were fixed with methanol, stained with crystal violet and counted in six random fields[25].
Boyden assay
Cell invasion assays was performed using the Transwell chambers (8 μm, 24-well insert; Corning Inc., Lowell, MA, USA). For the invasion assay, diluted Matrigel (BD Biosciences) was used to pre-coat the insert membrane. 1x105 cells were cultured for 36 h under the same conditions. Finally, the cells which invaded into the lower chambers were fixed with methanol, stained with crystal violet and counted in six random fields[25].
Drug sensitivity test in vitro
Drug sensitivity test was determined by the MTT assay[26]. Cells were seeded in 96-well plates at a density of 2 × 103 cells/well and treated with 0, 1, 2, 4, 8µM paclitaxel (No: 33069-62-4, Shanghai yuanye Bio-Technology Co., Ltd) for 48 h. Subsequently, 20 µL of MTT (5 mg/ml; Sigma-Aldrich) was added to each well and incubated at 37 °C for 4 h. Then supernatants were removed and 150 μL of DMSO (Sigma-Aldrich) was added to measure the absorbance value (OD) of each well at 490 nm. The calculated rates were used for curve fitting and calculation of IC50. Experiments were performed three times.
Western blot
The SUNE-1 and HONE-1 cells were washed three times with ice-cold PBS and total protein was harvested using RIPA lysis buffer (Beyotime Institute of Biotechnology) containing PMSF (Bio-Rad Laboratories, Inc.) and Phosphatase inhibitors (Bio-Rad Laboratories, Inc.) (100:1:1). This assay was performed as described in a previous study[25]. The primary antibodies included MYL9 (15354-1-AP, Proteintech, Rabbit), N-cadherin (66219-1-Ig, Proteintech, Mouse), E-cadherin (60335-1-Ig, Proteintech, Mouse), Vimentin (10366-1-AP, Proteintech, Rabbit), ABCB1 (22336-1-AP, Proteintech, Rabbit), ABCG2 (27286-1-AP, Proteintech, Rabbit), and GAPDH (CW0100M, CWBIO, Mouse). The following secondary antibodies were used: HRP-conjugated goat anti-rabbit (cat. no. 6401-05; dilution 1:5000; Biovision, Inc.) and HRP-conjugated anti-mouse IgG antibody (cat. no. 6402-05; dilution 1:5000; Biovision, Inc.).
Statistical analysis
Each experiment was performed at least three times. The data were presented as the mean ± SD of more than three independent experiments. The significance between two groups were analyzed by Student’s t-test. Graphs were made using GraphPad Prism 7 (GraphPad Software, Inc.) software. All of p < 0.05 were considered as statistically significant.