Tissues specimens
A total of 55 NPC tissues samples and 48 matched adjacent tissues samples were obtained by surgery from patients who suffered NPC and underwent the biopsy of the nasopharynx at The First Affiliated Hospital, Jinan University from March 2015 to March 2017. The diagnosis of NPC patients was pathologically confirmed according to the criteria of World Health Organization (WHO), and all chosen patients were informed with consent before surgery. The available samples were immediately snap frozen in liquid nitrogen and then stored at 80°C. This study was approved by the human Ethics Committee of National Engineering Research Center of Genetic Medicine.
Cell culture
Human NPC cell lines including SUNE1, HK1, CNE1 and CNE2 cells and normal nasopharyngeal epithelial cell NP69 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Cell were cultured with RPMI 1640 medium containing 10% FBS, 1% penicillin/streptomycin with 5% CO2 at 37˚C.
Cell transfection
Cell transfection was performed in SUNE1 or HK1 cells by using Lipofectamine 3000 kit. Of which, sh-TSIX, sh-NC, miR-342-3p mimics, miR-NC, miR-inhibitor, inhibitor NC and sh-AGR2 were purchased from GenePharma Co., Ltd. Suzhou. In addition, to overexpress TSIX, the entire TSIX sequence was amplified and cloned into the pcDNA3.1 vector (GenePharma) to generate the overexpressing vector pc-TSIX, with empty vector pcDNA3.1 as the negative control. The sequences as follows: sh-TSIX: 5'- CAATCTCGCAAGATC CGGTG-3'; sh-AGR2: 5'-AGA GAT ACC ACAGTC AAA CC-3'; sh-NC (AGR2): 5'-AAC AGT CGC GTT TGC GAC TGG-3'.
miR-342-3p mimics and inhibitors were obtained from Gene-Pharma (Suzhou, China) (mimic: 5-UCUCACACAGAAAUCGCACCCGU-3’; inhibitor:5-ACGGGUGCGAUUUCUGUGUGAGA-30) The NC sequence is 5′-ACAAAGUUCUGUGAUGCACUGA-3′ and 5′-ACAAAGUUCUGUGAUGCACUGA-3′
Luciferase reporter assay
The wile type (WT) or mutant type (MUT) fragments of TSIS or AGR2 containing the putative binding sites of miR-342-3p was obtained by PCR from human genome, and cloned into the pmirGLO Dual‑Luciferase miRNA Target Expression Vector (Promega Corp., Fitchburg, WI, USA). Then SUNE1 cells were co-transfected with WT or MUT TSIX/AGR2 and miR-342-3p mimics or miR-NC by using the Lipofectamine 3000 kit. After transfection for 48 h, cells were collected, lysed and the relative luciferase activity was detected by using the Dual‑Luciferase Assay system (Promega).
RNA isolation and qRT-PCR
Total RNA from tissues and cultured cells was extracted by TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription was carried out by using the M-MLV reverse transcriptase kit (Promega, Madison, USA). The quantitative real-time PCR (qRT-PCR) was performed using the Bio-Rad CFX96 Touch sequence detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA) based on the Platinum SYBR Green qPCR SuperMix-UDG reagents (Invitrogen), with GAPDH and U6 was considered as the internal reference. The relative fold changes of targets were analyzed by using 2-ΔΔCt method(27). All primers using in this study as follows: AGR2 forward: 5'-TGATGGCCAGTATGTCCC-3', reverse: 5'-CAGTCTTCAGCAACTTGAG-3'; GAPDH forward: 5'-GAGTCAACG-GATTTGGTCGT -3', reverse: 5'-TTGATTTTGGAGGGATCTCG -3'; miR-342-3p forward: 5′-GGGTCTCACACAGAAATCGC-3′, reverse: 5′-CAGTGCGTGTCGTGGAGT-3′; U6 forward: 5′-CGCGCTTCGGCAGCACATATACT-3′, reverse: 5′-ACGCTTCACGAATTTGCGTGTC-3′.
Western blot
Total protein of tissues or cultured cells was isolated by using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Approximately equal amounts of protein were separated by 12% SDS-PAGE and then transferred nitrocellulose membrane based on the wet transfer method. After blocking with 3% skim milk, the membranes were incubated with primary antibodies against AGR2 (1:1000, ab209224, abcam) and internal reference GAPDH (1:2000, ab37168, abcam) 4°C overnight. On the following day, the membranes were incubated with goat anti-rabbit IgG (1:1000, ab6721, abcam) for 1 h which was diluted with 5% skim milk. After washing, the membranes were exposed to ECL solution followed by imaging by a Bio-Rad gel imaging system (MG8600; Thmorgan Biotechnology, Beijing, China). The gray values of target proteins were quantified by IPP7.0 software.
RIP assay
To determine the interaction between TSIX and miR-342-3p, RIP assay was performed by using the EZ-Magna RIP kit according to the manufacturer’s instructions. In brief, cells were harvested, and the extraction was incubated with Ago2 antibody (5 μg/sample; Abcam, Cambridge, UK) with IgG was the negative control. Then co-precipitated RNAs were purified by using TRIzol reagent and the expression of TSIX or miR-342-3p was detected by qRT-PCR.
CCK-8 assay
Cell viability was evaluated by using Cell Counting Kit-8 (Dojindo, Kyushu, Japan). Briefly, approximately 1 × 103 cells were plated into 96-well plates for different times including 24h, 48h, 72h and 96h. Subsequently, 10 μl of CCK-8 solution was added into each well and incubated at 37°C for 2 h, then the absorbance was recorded at 450 nm to determine the cell viability.
Transwell assay
The Transwell assay was performed as previous described(28). Briefly, 3×104 cells with different treatments were added in the upper uncoated (migration) or 8.0-μm-pore Matrigel™-coated membranes (for invasion) with serum-free medium. Simultaneously, the lower chamber was filled with medium supplemented with 10% FBS. The chamber was cultured for 48 h to perform the migration assay and extracellular matrix gel was used for the cell invasion assay. Finally, cells were fixed by 4% paraformaldehyde and stained with crystal violet, then the cells were observed under the phase contrast microscope (×100 magnification) (Nikon) in 10 randomly fields.
Apoptosis analysis
Cell apoptosis was evaluated by using the Annexin V-FITC Apoptosis Detection Kit (Sangon Biotech). Briefly, cells with different treatments were re-suspended with 100 μL binding buffer. Then cells were double stained by Annexin V for 15 min and propidium iodide (PI) for 10 min. Afterwards, the apoptotic rate was evaluated by using BD LSRII Flow Cytometer System (BD Biosciences) and the data was quantitated with the FACSDiva Software.
EdU staining assay
To evaluate the proliferation of NPC cells with different treatments, Edu staining assay was performed using Cell-Light EdU Apollo 567 In Vitro Imaging Kit (Ribobio) as previously described(29). In brief, approximately 4 × 103 cells with different treatments in logarithmic phase were plated into 96-well plates. After 24 h, 100 μl medium containing 50 μM EdU was added into each well and incubated for 2 h at 37 °C. Then cells were fixed with 4% paraformaldehyde, and stained with Hoechst 33,342 and Apollp reaction cocktail. Images were captured by using a confocal LSM 710 (Carl Zeiss Microimaging, Jena, Germany) (×100 magnification) and EdU-positive cells were counted within each field by using ImageJ software.
Animal model
All male BALB/C nude mice (approximately 16-28g, 5-6 weeks) were purchased from The First Affiliated Hospital, Jinan University Laboratory Animal Technology Co., Ltd. and kept in a pathogen-free facility. Mice were anaesthetized with 0.1 ml/100 g pentobarbital sodium through intramuscular injection, and approximately 5 × 106 cells with different treatments were subcutaneously inoculated into the dorsal of the nude mice for in vivo xenograft assay. All mice were randomly divided into three groups: sh-NC group, sh-TSIX group, sh-TSIX + miR-342-3p inhibitor group. Tumor volume of different mice were calculated every 7 days for 28 days by the formula: volume = (length x width x width)/2. After kept 28 days, mice were sacrificed by cervical dislocation. Xenograft tumors from different groups were removed and weighted, meanwhile, the representative images of xenograft tumors were captured by a microscope. All animal producers were approved by the animal Ethics Committee of National Engineering Research Center of Genetic Medicine.
Immunohistochemistry
Paraffin-embedded tissues were deparaffinized, hydrated in ethyl alcohol and incubated with 0.3% H2O2 for elimination of endogenous peroxidase activity. After rupture of nuclear membrane with 0.1% Triton X-100 for 30 min and blockage with 5% normal donkey serum, tissue slides were incubated with primary antibody anti-Ki67 (abcam, Cambridge MA) at 4°C overnight and corresponding secondary antibody at room temperature for 1 h. Immunohistochemistry results were captured using Olympus BX 41 Microscope (Olympus Corporation, Japan) (× 200 magnification).
Statistical analysis
All data were presented as mean ± SD which were derived from at least three independent experiments. Difference between two groups was determined by Student's t‑test and difference among multiple groups was determined by one‑way ANOVA. P < 0.05 was considered to be the significant threshold.