Clinical sample

Collected tumor tissues and matched adjacent tissues from 53 NPC patients admitted to West China Hospital of Sichuan University from January 2018 to December 2019. All cases were diagnosed by histopathological examination. None of them were treated with chemotherapy or radiotherapy. This study was approved by the Institutional Ethical Review Board of the Sichuan University West China Hospital, and written informed consent was obtained from all patients. The clinicopathological features of participants were listed in Additional file 5: Table S1. The 53 cases of nasopharyngeal carcinoma tissue and para-cancerous tissue specimens were collected strictly in accordance with the specimen collection specifications during the operation, some were frozen and stored at − 80 °C, some were fixed with 10% formalin, and dehydrated by an automatic dehydrator. Embed in paraffin for preservation.

Cell culture

We used ten NPC cell lines including HONE-1, CNE-1, CNE-2, HNE-1, C666-1, HK-1, S26, S18, SUNE-1 and 6-10B. The human immortalized nasopharyngeal epithelial cell lines (NP69 and N2Tert) served as a control. They were collected from the Cellbank of the Chinese Academy of science (Shanghai, China). Cells were grown routinely in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and cultured in a 37 °C humidified atmosphere with 5% CO2.

Microarray analysis

MiRNA expression was analyzed using the Arraystar Human miRNA Microarray v3.0 by KangChen Bio-tech as previously described. Strict screening criteria were used to identify differentially expressed miRNAs(log2FC > 2, FDR < 0.05). Heat map and volcano plots were generated with the “pheatmap” and “ggplot2” packages in R (version 3.4.4).

GEO database analysis

The data set used is from GEO database (https://www.ncbi.nlm.nih.gov/geo/), and the download data format is MINIML (NO: GSE12452, GSE64634). The m6A-related genes are derived from Juan Xu’s research on the molecular characterization and clinical significance of m6A modulators across 33 cancer types. PCA graphs are drawn by R software package ggord; The box plot is implemented by the R software package ggplot2; the heat map is displayed by the R software package pheatmap. All the above analysis methods and R package were implemented by R foundation for statistical computing (2020) version 4.0.3.

The extracted data were normalized and processed by log2 transformation. the microarray data were normalized using the preprocessCore package in R software (version 3.4.1). Probes were converted to gene symbols according to the platform annotation information of the normalized data. Probes with more than one gene were eliminated and the average value was calculated for genes corresponding to more than one probe. As an initial quality control step using variance stabilized counts with individual horse effect removed using the removeBatchEffect function of limma R package.

The Cancer Genome Atlas (TCGA) database analysis

Raw counts of RNA-sequencing data (level 3) and corresponding clinical information from 62 NPC samples were obtained from The Cancer Genome Atlas (TCGA) dataset (https://portal.gdc.cancer.gov/) in January 2020, in which the method of acquisition and application complied with the guidelines and policies. The m6A-related genes are derived from Juan Xu’s research on the molecular characterization and clinical significance of m6A modulators across 33 cancer types.

All the above analysis methods and R package were implemented by R foundation for statistical computing (2020) version 4.0.3 and software packages ggplot2 and pheatmap.

Plasmid construction and cell transfection

On the GenePharma (Shanghai, China), the transfected materials miR-100-3p mimics and inhibitor, si-METTL3-1,2, oe-ZFAS1 and sh-ZFAS1 were purchased. Twenty-four hours before transfection, HK-1 and HONE-1 cells in the exponential phase were digested by pancreatin and made into cell suspension. miR-100-3p mimics and inhibitor, si-METTL3-1,2, oe-ZFAS1 were used for cell experiment and the in vitro experiment. After trypsinization from flasks, cells were cultured in six-pore plates, incubated at 37 °C with 5% CO2 for 18–24 h. Three hours before transfection, cells at about 80–90% confluency were changed to the serum and antibiotic-free media. Then, cells were transfected using lipofectamin 2000 reagent (Life Technologies, Gaithersburg, MD, USA) referring to manufacturer’s instructions and incubated at the same conditions as above for 48 h. In addition to the in vivo experiment, based on the manual, concentrated lentiviral solutions of sh-ZFAS1 was mixed with two wells of nutrient solution contains HONE-1 cell, respectively. Finally, the cells were digested by pancreatin and injected into mice after incubation for 48 h.

Isolation of cytoplasmic and nuclear RNA

Follow the manufacturer’s instructions to isolate and purify cytoplasmic and nuclear RNA using the cytoplasmic and nuclear RNA purification kit (Sigma-Aldrich). U6 is regarded as an internal reference for nuclear RNA.

RNA isolation and qRT-PCR

The RNA in the two cell lines was extracted with TRIzol reagent (Invitrogen) following the instruction of manufacturer. The concentration and purity of extracted RNA were detected by a NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Then cDNA was synthesized using the Reverse Transcription System Kit (Applied Biosystems, Foster City, CA, USA). The detection of ZFAS1, GAPDH and miR-100-3p expression was performed by RT-PCR using SYBR Green PCR Master Mix (Takara, Dalian, China) and TaqMan MicroRNA Assay Kit (Applied Biosystems) on Bio-Rad iQ5 Multicolor Real-Time qRT-PCR Detection system (Bio-Rad, Hercules, CA, USA). GAPDH was a reference gene for ZFAS1 and METTL3, while U6 was the reference gene for miR-100-3p. The primers were synthesized by Sangon (Additional file 5: Table S2) (Shanghai, China). All statistics were analyzed based on 2−ΔΔCt method.

m6A RNA methylation quantification

According to the manufacturer’s instructions, the EpiQuik m6A Methylation Quantitative Kit (colorimetric; Epigentek) measured the total m6A level of the extracted RNA. Poly-apurified RNA (200 ng) was used for each sample analysis.

RNA immunoprecipitation (RIP)

RIP was performed using the EZ-Magna RIP kit (Millipore) according to the instructions. Briefly, 24 h after transfection, cells were collected and RIP assay was performed using m6A antibody (2 mg/sample; Synaptic system) or AGO2 antibody (5 mg/ sample; Abcam). IgG was used as a negative control. As described earlier, quantitative RT-PCR was used to detect the co-precipitated RNA. In the analysis of mature RNA, comparative CT (DDCT) methods were used. The input fractional CT values were used to normalize the differences in sample preparation, and negative control (IgG) CT was used to adjust the background scores.

CCK-8 and Wound-healing assays

For CCK-8 detection, cells were seeded into 96-well plates at the density of 1000 cells per well, and 10 mL of CCK-8 (Dojindo) was added to each well on days 0–5. Then incubated with the cells in 37℃ 2 h, the density of light measured at 450 nm.

In the wound healing assays, the cells were inoculated in a 6-well plate and cultured to the fusion state. After starvation in serum-free medium for 24 h, monolayers were linearly scraped to introduce artificial wounds and captured at 0 h and 24/48 h.

Luciferase reporter assays

293 T cells were transferred to 24-well plates in each well and incubated before transfection. Then the cells were co-transfected using miR-100-3p, the wild type or mutant ZFAS1 and ATG10. After 48 h of transfection, the cells were washed by PBS before incubated with shake for 15 min at room temperature. Then cell lysis buffer was collected at 4 °C and centrifuged for 2 min. The supernatant was collected and refrigerated at -80 °C refrigerator. Cell lysis buffer was added to 96-well plate with 10 μl per well. During the detection, 30 μl luciferase reagents II (Promega, Madison, WI, USA) were added. Then 30 μl stop buffer was added to terminate the activities. The luciferase activities were detected by the Dual-Glo luciferase reporter assay kit (Promega) for 48 h post transfection.

Flow cytometric analysis of apoptosis

After transfection, 2 × 105 cells from the transfection group and the NC group were collected, washed and resuspended with precooled PBS according to the Annexin V Apoptosis Detection Kit (Life Technologies). Then the cells were incubated for 15 min before washed and resuspended in 500 μl of binding buffer. Ten microliter of Annexin-V–FITC and propidium iodide respectively were added and the mixture was analyzed using the FACS Calibur. The data was analyzed by FACS Diva software.

In vivo nude mouse models

All of the experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Sichuan University. Female BALB/c nude mice (ages 4–5 weeks, 18–20 g) were purchased from the Charles River Laboratories. For the tumor growth model, 1 × 106 HONE-1 sh-NC or sh-ZFAS1 cells were injected into the axilla of the mice, and the tumor size was measured every 3 days (Fig. 8A). On day 30, the mice were killed, and the tumors were dissected and weighed.

Immunohistochemistry

Immunohistochemical staining was done on formalin-fixed and paraffin embedded tissue sections from tumors tissue. The tissue was placed in the incubator at 65 °C for 2 h before removing paraffin by xylene. After gradient elute the tissue by absolute ethyl alcohol, 95% ethyl alcohol, 85% ethyl alcohol, washed the tissue by redistilled water. In the third step, target retrieval was achieved with BOND Novocastra Epitope Retrieval Solution 1 (Leica Biosystems) at 100 °C for 20 min. Monoclonal Anti-ki67 antibody (2 µg/ml, Abcam) were used as primary antibody. After that, secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Abcam) were used. In this way, ki67 was stained in the tissue and the tissue was photographed by optical microscope.

Immunofluorescence assay

The cells were fixed and blocked with 3% bovine serum albumin, and then incubated with rabbit anti-LC3II (ab48394, 1:1000, USA) at 4 °C overnight. Then it was incubated with a red fluorescent secondary antibody (Intertek) for 2 h, stained with DAPI (1 μg/mL) for 5 min, sealed, and observed and photographed under a fluorescence microscope (Olympus Optical Co., Ltd).

Hematoxylin–eosin (HE) staining

The 5-μm tissue sections were deparaffinized in xylenol for 20 min, rehydrated with reduced ethanol concentrations (100%, 90%, 70%) for 5 min, and then washed with water. For HE staining, the slides were stained in Mayer’s Hematoxylin Solution for 5 min and then counterstained with Eosin for 5–10 min.

RNA stability assays

HON3-1 cells with or without METL3 knockout were treated with Actinomycin D at a final concentration of 5 μg/ml for 0, 2 and 4 h, and then collected. Total RNA was separated with TRIzol (Invitrogen) and analyzed by RT-PCR.

Western blot (WB)

First of all, proteins were isolated from extraction reagent (Thermo Fisher Scientific), followed with separated and transferred some proteins to Immobilon™-P membranes (Merck Millipore, Billerica, USA). Secondly, incubating ATG10 (ab124711,1:5000), LC3 (ab192890, 1:2000), P62 (ab109012, 1:10,000), Beclin1 (ab210498, 1:1000), p-PI3K (ab182651, 1:1000), PI3K (ab32089, 1:1000), p-AKT (ab38449, 1:1000), AKT (ab179463, 1:10,000), p-mTOR (ab109268, 1:5000), mTOR (ab134903, 1:10,000), E-cadherin (ab76055, 1:500), N-cadherin (ab76011, 1:10,000), vimentin (ab92547, 1:2000) and GAPDH (ab8245, 1:5000) (Abcam) primary antibodies against mouse in the membrane at 4 °C for a night time. Washed with TBST after incubation immediately, hybridized membranes with horseradish peroxidase (HRP)-linked antibody goat anti-rabbit IgG (1:2000, Abcam) for 1 h. At least, checking the antibody binding by an enhanced chemiluminescence kit.

Statistical analysis

SPSS 22.0 software (SPSS Software, USA) was used for statistical analysis and data was expressed with mean ± standard deviation. Data were compared by unpaired t-test (differences between two groups) or one-way ANOVA analysis (differences among groups). * p value < 0.05 was regarded statistically significant.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Disclaimer:

This article is autogenerated using RSS feeds and has not been created or edited by OA JF.

Click here for Source link (https://www.biomedcentral.com/)