Ethics statement
The study was approved by the Ethics Committee of Affiliated hospital of Youjiang Medical College for Nationalities and informed written consent was gained from all patients. All animal experiments were in line with the Guide for the Care and Use of Laboratory Animal by International Committees.
Study subjects
A total of 83 patients with NPC (aged at 21 - 78 years old with the mean age of 54 years old) were collected from who received radiotherapy in Affiliated hospital of Youjiang Medical College for Nationalities from June 2012 to June 2014, of which there were 47 cases of male and 36 of female with complete clinical materials through pathological examination diagnosis. According to the International Union Against Cancer (UICC) TNM staging standard issued in 2002, there were 7 cases in stage I, 21 cases in stage II, 35 cases in stage III, and 20 cases in stage IV; and 58 cases with cervical lymph node metastasis (LNM) and 25 cases without. In the N stage, there were 25 cases of N0, 9 cases of N1, 41 cases of N2, and 8 cases of N3. Any anti-cancer treatments including radiotherapy and chemotherapy have never been adopted by all patients with NPC before biopsy. Cancer tissue was collected accompanied by a control group of normal tissue from 32 cases of chronic nasopharyngeal mucosal inflammatory tissue in patients with chronic sinusitis. Follow-up was performed for 60 months by outpatient or telephone after radiotherapy until June 30, 2019.
Hematoxylin-eosin (HE) staining
Paraffin-embedded chronic nasopharyngeal mucosal inflammatory tissues and NPC tissues were dewaxed in xylene I and xylene Ⅱ for 10 min respectively and in xylene Ⅲ if not be transparent. After the residual xylene was removed, the tissues were treated with absolute ethanol, 95% alcohol, 80% alcohol, 70% alcohol respectively for 3 min, rinsed with running water and distilled water for 2 min respectively and stained in hematoxylin solution for 5 min. Next, the tissues went on differentiation with 1% hydrochloric acid alcohol solution twice for 10 s per time, rinsed with running water, and treated with 1% eosin solution and rinsed with running water again. Finally, the tissues were dehydrated in 70% alcohol, 80% alcohol, 95% alcohol, absolute ethanol respectively for 3 min, permeabilized by xylene I, xylene Ⅱ and xylene Ⅲ for 5 min and sealed with neutral resin for observation.
Immunohistochemical staining with the streptavidin-peroxidase (SP) assay
Immunohistochemical SP method kit (Fuzhou Maxim Biotechnology Co., Ltd., Fuzhou, China) was applied to detect E-cadherin and H3K27me3 expression in tissues. Firstly, the tissues were baked for 1 h at 60°C, dewaxed in xylene for 10 min, hydrated with absolute ethanol and gradient alcohol for 3 min, and rinsed the phosphate buffered saline (PBS) 3 times for 3 min each time. Subsequently, tissue antigen retrieval was performed at 1500 mL citrate antigen retrieval solution (0.01 M, pH 6.0) at 121°C for 2 min. The tissues were incubated with 50 μL of Reagent A (endogenous peroxidase blocker) for 30 min at room temperature. Next, 50 μL of reagent B (normal non-immune animal serum) was dropped and the tissues were incubated to block non-specific antigen. After serum removing, 50 μL of primary antibody (PBS instead of primary antibody was added for the negative control (NC)) was added and incubated overnight at 4°C. After warming the next day and washing with PBS, the tissues were added with 50 μL of reagent C (biotin-labeled secondary antibody) and incubated for 30 min at room temperature. One drop of newly prepared diaminobenzidine (DAB) coloring solution was added and later washed away with tap water to stop the color reaction. And the tissues were immersed 3 min in hematoxylin dye solution, differentiated in 1% hydrochloric acid alcohol for 10 s, dehydrated for in gradient alcohol for 5 min before permeabilization, sealed with the neutral resin and observed under the microscope for analysis.
Cell selection and culture
NPC cells 6–10B, S18, CNE2, HONE1, and C666–1 were provided by Shanghai Huiying Biotechnology Co., Ltd. (Shanghai, China) and NP69, the normal nasopharyngeal epithelial cell line by Jianglin Biotechnology Co., Ltd. (Shanghai, China). Cells were cultured as required in RPMI 1640 medium (Gibco, Grand island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco) in a incubator at 37°C with 5% CO2, and medium was changed every 2 days. When cells reached 80% ~ 90% confluence, cell passage was to start. The logarithmic growth phase cells in 2 to 3 stable passages were taken to determine the expression of HOTAIR and EZH2 by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and EZH2 protein level by Western blot analysis. Subsequent experiments were performed with S18 cells with the greatest differential expression of human nasopharyngeal epithelial cell line NP69 and minimally differentiated 6–10B cells.
Cell grouping and transfection
S18 and 6–10B cells (1 × 105 cells/well) in logarithmic growth phase were seeded into 24-well cell culture plates and transfected until cells reached 80% confluence in accordance with lipofectamine 2000 kit instructions (Invitrogen, Carlsbad, California, USA). Each transfection sequence was diluted in 3 mL serum-free RPMI 1640 medium (both from Shanghai GenePharma Co., Ltd., Shanghai, China, with a final concentration of 50 nM), mixed and incubated. Next, 600 μL lipofectamine 2000 was diluted with 3 mL serum-free RPMI 1640 medium, mixed and incubated. The above-mentioned two were mixed together and incubated and then added to the cell culture well. After 6-hours culture at 37°C, 5% CO2 with saturated humidity, cells were cultured in RPMI 1640 medium containing 10% FBS for subsequent experiments rather than the medium containing transfection solution.
S18 cells were divided into 7 groups, including Blank-S18 group (cells without any treatment), sh-NC group with S18 cell line transfected with poorly expressed HOTAIR vector NC, sh-HOTAIR group with S18 cell line transfected with poorly expressed HOTAIR vector, sh-Ctr group with S18 cell line transfected with poorly expressed EZH2 vector NC, sh-EZH2 group with S18 cell line transfected with poorly expressed EZH2 vector, sh-HOTAIR + overexpression (OE)-Ctr group with S18 cell line transfected with poorly expressed HOTAIR vector and then transfected with over-expressed EZH2 vector NC and sh-HOTAIR + Oe-EZH2 group with S18 cell line transfected with poorly expressed HOTAIR vector and then transfected with over-expressed EZH2 vector.
Seven groups were separated from 6–10B cells, including Blank–6–10B group without any treatment for 6–10B cell line, Oe-NC group with 6–10B cell line transfected with over-expressed HOTAIR vector NC, Oe-HOTAIR group with 6–10B cell line transfected with over-expressed HOTAIR vector, Oe-Ctr group with 6–10B cell line transfected with over-expressed EZH2 vector NC, Oe-EZH2 group with 6–10B cell line transfected with over-expressed EZH2 vector, Oe-HOTAIR + sh-Ctr group with 6–10B cell line transfected with over-expressed HOTAIR vector and transfected with poorly expressed EZH2 vector NC, Oe-HOTAIR + sh-EZH2 group with 6–10B cell line transfected with over-expressed HOTAIR vector and transfected with poorly expressed EZH2 vector.
3-(4,5-Dimethylthiazol–2-yl)–2,5-diphenyltetrazolium bromide (MTT) assay
S18 and 6–10B cells in logarithmic growth phase were inoculated into a 96-well plate at 200 μL cell suspension per well with the cell concentration adjusted to 2 × 105 cells/mL. With 24-h culture, 200 μL RPMI 1640 containing 10% FBS was replaced after cell adherence, and 20 μL of 5 mg/mL MTT solution was added to each well after 0 h, 24 h, 48 h, and 72 h respectively. Then cells were incubated for 4 h at 37℃, centrifuged and washed with PBS for 3 times with the supernatant discarded. Next, 150 μL of dimethyl sulphoxide (DMSO) was added per well and placed on the shaking table for 10 min. The optical density (OD) values of the wells were measured at a wavelength of 492 nm on a M450 microplate reader (Bio-Rad, CA, USA) when the blue crystals were sufficiently dissolved.
Colony formation assay
S18 and 6–10B cells were detached with 0.25% trypsin, and stained with trypan blue to count the number of viable cells. Then, RPMI 1640 medium containing 10% FBS was applied to dilute the cell suspension and cells were inoculated and evenly dispersed into a 6 cm cell culture dish at 1000 cells/10 mL, which was cultured in an incubator at 37℃ with 5% CO2 for 12–15 days. Next, cells were separated from culture solution, rinsed with PBS twice and fixed with 10 mL of 4% paraformaldehyde for 20 min. Finally, departed from paraformaldehyde, the cells were treated with crystal violet staining solution which was washed away 30 min later and dried at room temperature. And the culture dish was photographed under a microscope and the number of cell colonies was counted.
Flow cytometry
First, the transfected S18 and 6–10B cells in each group were incubated with 100 μL of propidium iodine (PI)-Rnase A without light exposure and analyzed by flow cytometry to compare the DNA content in each cell phase. Second, cells were detached, centrifuged, washed twice with PBS, suspended with pre-cooled 75% ethanol and fixed at –20℃ overnight. Third, cells were centrifuged again with the supernatant discarded, washed twice with PBS and resuspended in 450 μL of PBS per sample. Last, cells were treated with 100 μL of Rnase A at 37°C and stained with 400 μL of PI at 4℃ without light exposure for 30 min and cell cycle status was measured and analyzed by BD flow cytometry (BD Biosciences, Mountain View, CA, USA). Cell apoptosis was detected by flow cytometry with the supplement of 5 μL of Annexin-V FITC and 10 μL of PI.
Hoechst33342 staining
The S18 and 6–10B cells were inoculated in 24-well plates at 2 × 104 per well after detachment. When the cell reached 70% confluence, they were cultured for 12 h with serum-free medium and fixed with 1 mL of 4% paraformaldehyde at 20°C for 20 min. After that, the cells were stained with 1 mL of Heochst33342 dye per well at 37℃ in an incubator. At last, the cells were recorded and photographed under an inverted fluorescence microscope.
Cell migration assay
The Transwell chamber with a polycarbonate membrane pore size of 0.8 μm was placed in a 24-well culture plate. Suspensions of S18 and 6–10B with a density of 3 × 105 cells/mL in serum-free medium were added into the upper chamber at 200 μL/well and 0.5% bovine serum albumin (BSA) was used to maintain the upper osmotic pressure. The medium containing 10% serum was added into the lower chamber at 600 μL/well and incubated in a 5% CO2 incubator at 37°C. After 24 h, the upper chamber was fixed in 4% paraformaldehyde for 20 min, air-dried and stained in 0.1% crystal violet dye for 30 min, decolorized twice with water, and the residual cells in the upper chamber were scraped off with a cotton swab.When the chamber was air-dried, five fields of view were randomly selected under the microscope for photographing, and the cells were counted by ImageJ image processing software for statistical analysis.
Cell invasion assay
The Matrigel-coated Transwell chamber was put into a 24-well culture plate, suspensions of each group of S18 and 6–10B with a density of 3 × 105 cells/mL in serum-free medium were prepared and added into the upper chamber at 200 μL/well with the osmotic pressure maintaining. And the medium with 10% serum was added to the lower chamber that were incubated at 37°C in a 5% CO2 incubator. After 48 hours, the upper chamber was fixed with 4% paraformaldehyde, air-dried, stained with crystal violet dye, decolorized, photographed, counted, and statistically analyzed.
Tumorigenesis in nude mice
Seventy clean BALB/c nude mice (Hunan SJA Laboratory Animal Co., Ltd., Hunan, China), weighing 20–25 g, were raised adaptively for 7 h in line with the rules of animal protection and use with room temperature of 25 ± 2°C, relative humidity 65% ~70%, 12 hours of light and 12 hours of dark cycle, good ventilation, hygienic cages and free drinking water. A total of 0.1 mL of S18 and 6–10B cells (4 × 107 cells/mL) in the logarithmic phase was subcutaneously injected into the back of the nude mice, and the tumor length and short diameters of nude mice were recorded by vernier calipers on the 3rd, 6th, 9th, 12th, 15th, 18th and 21st day respectively. On the 21st day, the weight of the anatomical tumor tissue was measured after euthanasia of the nude mice.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
The total RNA in NPC tissue, chronic nasopharyngeal mucosal inflammatory tissue and S18, 6–10B cells were extracted by Trizol (Takara Co., Ltd., Dalian, China). The instructions of the All-in-One First-Stand cDNA Synthesis Kit (GeneCopoeia, Maryland, USA) was applied to perform reverse transcription of RNA into cDNA. Designed primers including HOTAIR, EZH2 and E-cadherin were synthesized by Shanghai Genechem Co., Ltd. (Shanghai, China). The mRNA expression of each gene was detected by a real-time PCR kit (Takara), and detected using a real-time PCR instrument (ABI 7500, ABI, Foster City, CA, USA) with glyceraldehyde phosphate dehydrogenase (GAPDH) as an internal reference. The 2-ΔΔCt referred to the calculation of the relative expression of each target gene. Each experiment was repeated 3 times.
Western blot analysis
Total proteins were extracted from tissues and cells of which the protein concentration was measured by bicinchoninic acid test kit instructions (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China). Briefly, proteins were separated by 10% polyacrylamide gel (Boster), transferred to polyvinylidene difluoride (PVDF) and blocked with 5% BSA. The membrane was incubated with the primary antibodies EZH2 (1:500, Abcam, Cambridge, UK), H3K27me3 (1:1000, Millipore, Billerica, MA, USA), E-cadherin (1:1000, BD Biosciences) and GAPDH (1:2000, Jackson Immuno Research, Grove, Pennsylvania, USA) at 4°C and the horseradish peroxidase (HRP)-labeled secondary antibody (1:2000, Abcam). Images were obtained using an Odyssey two-color infrared fluorescence scanning imaging system. The grayscale values of the bands were measured using the Quantity One image analysis software. The discrepancy between the target bands and internal reference bands was used to compare the differences between the groups.
RNA-binding protein immunoprecipitation (RIP)
RIP was guided under the instructions of RIP kit (Millipore). S18 and 6–10B cells were resuspended in complete RIP lysate, and incubated on ice. The magnetic beads were triturated, suspended and washed with RIP wash buffer. The sterile enzyme-free eppendorf tubes were labeled as Anti-EZH2, Anti-H3K27me3 and IgG. The magnetic beads were supplemented with RIP wash buffer (100 μL) for resuspension, and added with 5 μg of the target antibody to incubate, centrifuged and snipped the supernatant. Then the sample was added with 0.5 mL of RIP wash buffer and placed on the magnetic separator to discard the supernatant. Each tube was added with 900 μL of RIP immunoprecipitation buffer and cell RIP lysate was centrifuged at 14,000 rpm for 10 min at 4°C, and then resuspended in RIP wash buffer. The supernatant (100 μL) was added to the RIP immunoprecipitation buffer containing the magnetic bead-antibody complex to make the final total volume as 1 mL and labeled as Input that was incubated for 3 h at 4°C overnight. The supernatant was discarded on a magnetic separator after centrifugation with 0.5 mL of RIP elution buffer each tube, which was repeated 5 times. The magnetic bead-antibody complex was resuspended in 150 μL of proteinase K buffer, incubated at 5°C for 30 min; the antibody was detached from the magnetic beads, centrifuged briefly and placed on a magnetic separator. The supernatant was transferred to a new tube for RNA purification and RT-qPCR was used to detect the corresponding expression level.
Chromatin immunoprecipitation (ChIP) assay
Experiment was guided in accordance with the instructions of ChIP test kit (Millipore). The final concentration of 1% formaldehyde together with S18 and 6–10B cells was incubated on a plate. Then, glycine terminated the crosslinking and the cells were conjugated at 2000 rpm for 5 min and sonicated together with SDS Lysis Buffer. The cells were conjugated at 10,000 g to snip impurities. The ultrasonically disrupted product (100 μL) was supplemented with 900 μL of ChIP Dilution Buffer, 20 μL of 50 × PIC and 60 μL of ProteinA Agarose/SalmonSpermDNA, and then were mixed at 4°C for 1 h, precipitated for 10 min and centrifuged at 700 rpm for 1 min, of which 20 μL was set as the input. One tube with 1 μL of EZH2, H3K27me3 and IgG antibody and the other tube without any antibodies were incubated overnight at 4°C, rinsed, eluted and de-crosslinked, and DNA samples were recovered for RT-qPCR detection.
Statistical analysis
All data analyses were conducted using SPSS21.0 (IBM Co., Armonk, NY, USA). Data were expressed as mean ± standard deviation. Comparisons between two groups were conducted by t-test, while comparisons among multiple groups were assessed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. The connection between the expression of HOTAIR and the clinicopathological features of NPC patients was determined by chi-square test. Survival and prognosis of NPC patients was analyzed by Kaplan-Meier. p < 0.05 was indicative of statistically significant difference.