2.1 Cell culture
HN4 cells, a human HNC cell line, were purchased from KINDU (Shanghai, China) and cultured in high glucose DMEM containing penicillin‒streptomycin solution and 10% fetal bovine serum (FBS). The human nasopharyngeal epithelial cell line (NP69 cell) was obtained from Fenghui Biological Research (Changsha, Hunan) and cultured in RPMI 1640 medium containing penicillin‒streptomycin solution and 10% FBS. The cells were cultured in a 37 ℃ humidity-controlled incubator with 5% CO2.
2.2 Quantitative real time-polymerase chain reaction (qRT-PCR) analysis
Total RNA was extracted from cell or tissue samples with TRIzol reagent (Invitro-gen, USA). The first strand of complementary DNA (cDNA) of FUT6 was transcribed by plus all-in-one 1st Strand cDNA Synthesis SuperMix (JinAn protein, Shanghai). The SYBR green PCR mixture was detected by a Light Cycler 480 machine (Roche, USA). The first cDNA of lncRNA PART1 was transcribed using the LNRCUTE lncRNA first-strand cDNA Kit (Tiangen, Beijing). PCR amplification was performed following the procedures of 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The SYBR green PCR mixture was detected by a QuantStudio3 ma-chine (ABI, USA). The relative quantitative expression of lncRNA PART1 and FUT6 was calculated using the 2−∆∆CT method. The GAPDH was used as an internal reference.
LncRNA PART1-forward:5’-CTCTGGAAAGCTGAAAGGGCT-3’; LncRNA PART1 -reverse: 5’-TGTCCTTTTCCCCTCCGACA-3’; FUT6-forward: 5’-GCCTTTTAACAAACCCATAGCT-3’; FUT6-reverse:5’-GTTGTACATGACCTCTCGGTG-3’; GAPDH-forward: 5’-GGGGTCATTGATGGCAACAATA-3’; GAPDH-reverse: 5’-ATGGGGAAGGTGAAGGTC-3’.
2.3 Cell transfection
The target plasmid of lentiviral vector pCDH-FUT6-MCS-EF1-COPGFP - T2A-PURO or the control plasmid pCDH-CMV-MCS-EF1-COPGFP-T2A-PURO (purchased from General Biology, Anhui) and the package plasmid pSPAX2 and pMD2.G were mixed according to the ratio of pCDH - FUT6-MCS-EF1-COPGFP -T2A-PURO or pCDH-CMV-MCS-EF1-COPGFP-T2A-PURO: pSPAX2: pMD2.G = 4:3:1. The mixed plasmids were transfected into 293T cells with Lipofectamine 3000. After 48 h, the supernatant of cells containing lentivirus was collected. The obtained lentivirus and polybrene (Biyuntian, Shanghai) were added to HN4 cells. Forty-eight hours later, puromycin was added to obtain FUT6-overexpressing cells. Biomics (Shanghai, China) chemically synthesized two small interfering RNAs targeting lncRNA PART1 (si-lncRNA PART1#1 5-CTGTATGAATC GCCA TGAA GACT-3, si-lncRNA PART1#2 5-CCGCTAAACTGGACATTTCAAGA-3). Specific knockdown of lncRNA PART1 was achieved by transfection of lncRNA PART1 siRNA (200 nM) into HN4 cells using Lipofectamine 3000 and Opti-MEM (Invitrogen; Thermo Fisher Scientific). The expression levels of lncRNA PART1 in the total RNA of the transfected cells were determined by qRT‒PCR.
2.4 Cell proliferation assay
HN4 cells were cultured in 96-well plates at a density of 2.0×105/cm2 in a 37 ℃, 5% CO2 humidity-controlled incubator for 24 h. Cell viability was assessed with the cell count kit-8 (CCK-8) (Vazyme, Nanjing, China). A 10% volume of solution was added to the cells and incubated at 37 ℃ for 1 h. The amount of formazan dye generated was directly proportional to the number of viable cells, which was detected at an absorbance wavelength of 450 nm and quantified by an automatic microplate reader.
2.5 Immunohistochemistry
The 5 µm thick tissue sections were dewaxed, dehydrated and subjected to antigen repair. The reagents were added dropwise according to the reagent instructions (ZSGB-BIO, Beijing, Universal SP Kit (Mouse/Rabbit Streptomyces Ovalle in biotin assay system, SP-9000)), stained with DAB solution followed by hematoxylin solution, and finally sealed with neutral gum. The tissues were observed turning a brown‒yellow color under the Olympus microscope, and quantitative immunohistochemical analysis was performed with Image-Pro Plus.
2.6 Transferase-mediated dUTP nick-end labeling (TUNEL) assay
The cells were plated on a polylysine-coated slide, and then 4% paraformaldehyde was added to fix the cells for 30 min at 4 ℃. After that, according to the instructions of the reagent (Vazyme, TUNEL FITC Apoptosis Detection Kit, A111-01), the cells were stained with a dye. Subsequently, the TUNEL-positive cells were analyzed by fluorescence inverted microscopy (Olympus, Japan), and the apoptosis rate was calculated by Image-Plus 6 software.
2.7 Flow cytometry
After digesting the cells with trypsin, the cells were washed twice with PBS and centrifuged at 2000 rpm and 4 ℃ for each wash. Then, 70% precooled ethanol was added to the cells and fixed at 4 ℃ overnight. Subsequently, the fixed cells were stained according to the instructions (C1052, Biyuntien, China), and the cell cycle was tested by flow cytometry (BD/BD FACSCanto II, USA). Data on cell fluorescence were collected to obtain the formation of a univariate karyotype histogram around the fluorescence area signal, and the percentage of nuclei in G1, S and G2/M phases were analyzed by FlowJo software.
2.8 Migration assay
Cells were seeded on 6-well plates until the cells in the wells were confluent, and then the monolayer was scratched with the tip of a sterilized pipette. The medium was then changed to medium without fetal bovine serum. Twelve hours later, the migration of cells along the scratched edges was photographed with a microscope. The results were represented as (S0 − St)/S0, where S0 represents the edge area scratched at the beginning, and St represents the edge area scratched after 12 h.
2.9 Xenograft mouse model of HNC tumor growth
Four-week-old male athymic BALB/C nude mice were purchased from Guangdong Yaokang Biotechnology Co.LTD. The FUT6-overexpressing HN4 cell lines and the control HN4 cell lines were separately mixed with biological matrix adhesive (Corning, USA) at a ratio of 1:1, and then the mixture was subcutaneously injected into the legs of nude mice at a concentration of 5×106 M. Tumor growth was recorded every three days. Four weeks later, the nude mice were sacrificed under isoflurane anesthesia. After size and weight measurements, tumors and organs of nude mice were collected and embedded for further study.
2.10 Statistical analysis
Statistical analysis with t tests was performed using GraphPad Prism V.8 software (GraphPad Software, San Diego, California) to calculate the mean ± SEM of the indicated number of samples. A two-sided value of p < 0.05 was considered statistically significant.