Cell culture
Lung adenocarcinoma cell lines (PC-9, H1373, HCC827 and A549) and normal human lung epithelial cell line (BEAS2B) were collected from Cell Resource of CAMS (Beijing, China), cultured in RPMI-1640 medium with 10% FBS (Gibco, CA) and preserved at 37°C with 5% CO2.
shRNAs and anti-miRNA inhibitors
Genechem (Shanghai, China) synthetized shRNAs targeting LINC00337 (shRNA#1, 2) and YTHDF1 (sh-YTHDF1) and negative control shRNA (sh-NC) without exact target. RiboBio (Guangzhou, China) provided inhibitors against miR-1285-3p (anti-miR-1285-3p) and miR-NC (anti-miR-NC). Subsequent to seeding of PC-9 or A549 cells in six-well plates for twenty-four hours, they received transfection with Lipofectamine 2000 offered by Invitrogen (Shanghai, China) in the case of 40-60% confluence as per manufacturer’s guidance. Forty-eight hours later, cells were collected, and stable cells were chosen for four-week therapy with neomycin (500 µg/mL).
RNA isolation andquantitative real-time PCR (qRT-PCR)
As per the manufacturer's guidance, total RNA segregation was implemented using TRIzol from Invitrogen, and it was then synthesized into cDNA using stochastic primers via a PrimeScript RT reagent Kit from Takara (Dalian, China) or a miRNA reverse transcription PCR kit commercially offered by RiboBio. QRT-PCR analysis was implemented using the SYBR Premix Ex Taq kit from Takara. The utilized primers are listed below: LNC00337: 5'-CCAGACTGGAGAACCACAGC-3' (forward) and 5' CTGTGTCTATGTGCAGCCCT-3' (reverse), miR-1285-3p: 5'- TCTGGGCAACAAAGTGAG-3' (forward) and 5'-CTCAACTGGTGTCGTGGA-3' (reverse), YTHDF1: 5'-ACCTGTCCAGCTATTACCCG-3' (forward) and 5'-TGGTGAGGTATGGAATCGGAG-3' (reverse). Bulge-Loop miRNAs qPCR Primers were offered by RiboBio, and data were processed via StepOnePlus Real-Time PCR System offered by Applied Biosystems (Shanghai, China), whose results were evaluated with GAPDH or U6 expression as standard.
Western blot evaluation
RIPA extraction liquid from Beyotime (Jiangsu, China) in the presence of Protease Inhibitor Cocktail and PMSF (Roche, Shanghai, China) was utilized for lysis of assembled cells. Subsequent to examination of protein sample concentration via BCA Protein Assay Kit from Beyotime, the harvested proteins in commensurable amounts were set apart by SDS-PAGE (10% gel) and transferred to PVDF membranes, which underwent 1-h sealing by Tris-buffered saline (5% defatted milk) and 12-h primary antibody incubation at 4°C. Next, optical density method was adopted to quantitate autoradiographs using Quantity One software (Bio-Rad) with GAPDH (#2118; CST, Shanghai, China) as a reference. Anti-YTHDF1 (#86463), anti-E-cadherin (#3195), anti-Vimentin (#5741) antibodies were provided by CST.
Immunohistochemistry
Nude mouse tumor tissues implanted by paraffin received immunostaining, and expression level and position of target proteins were ascertained using avidin-biotin-peroxidase method. Next, primary antibodies against E-cadherin and Vimentin were diluted at 1:200 for later application. Tumor apoptosis and proliferation were appraised by independently probing Ki-67 and Bax. Lastly, slice visualization was achieved using a microscope from Olympus (Japan).
5-ethynyl-20-deoxyuridine assay (EdU) Assay
Cell proliferation was also proved by Ethynyl-2-deoxyuridine incorporation assay using an EdU Apollo DNA in vitro kit (RIBOBIO, Guangzhou, China) as per the manufacturer's guidance. Briefly, subsequent to transfection with the corresponding vector, cells received respective 2-h incubation at 37 °C, with 100 μl of 50 μM EdU/well. Via a fluorescence microscopy, the cells were identified. We carried out each experiment for three times.
Cell Counting Kit-8 assay
Cell Counting Kit-8 (Beyotime Inst Biotech, China) was used to determine cell proliferation. In a word, 5×103 cells/well underwent 1-day raising in a 96-well broad-bottomed plate at 37°C, followed by transfecting them with corresponding vectors. Finally, with a microplate reader from Bio-Rad (Shanghai, China), the absorbance was finally evaluated at 450 nm, and we carried out each experiment for three times.
Apoptosis and cell cycle experiments
As per the manufacturer's guidance, apoptosis determination was implemented via FACS using PE-Annexin V apoptosis detection kit from BD Pharmingen (Shanghai, China) subsequent to 48-h transfection, and cell cycle was assessed utilizing PI cell cycle assessment kits (BD Pharmingen). Each assay was implemented thrice.
Transwell assay
Transwell chambers were used to observe the invasion of lung adenocarcinoma cells. We seeded cancer cells in the upper chamber coated by Matrigel (Corning, USA, dilution ratio: 1:6) at a density of 105 cells per well and supplemented DMEM with 1% FBS. Later we filled 600μL DMEM with 10% FBS into the lower chamber, fixed cells by 4% methanol and stained them with crystal violet. Then we counted them in 5 random 200 microscopic fields, after cells being invaded to the lower surface of membrane and incubated at 37°C for 24 hours. We carried out each experiment for three times.
Dual-Luciferase Reporter Assay
Genechem designed and synthesized YTHDF1 full-length promoter reporter vector. Human YTHDF1 3'-untranslated region (UTR) fragment with supposed binding sites for miR-1285-3p reporter vector was offered by RiboBio. 48-h transfection later, Dual-Luciferase Reporter Assay System from Promega was adopted for luciferase activity determination as per the manufacturer's guidance, and luciferase activity ratio (Firefly/Renilla) was ascertained. Each assay was implemented thrice.
RNA pull-down assay
The DNA fragment with the full length LINC00337 or NC sequence received PCR amplification using a primer with T7 and was cloned into GV394 from Genechem, Shanghai, China). Restriction enzyme XhoI was utilized for linearization of DNAs. Next, T7 RNA polymerase (Takara) and Biotin RNA Labeling Mix from Roche (China) were employed for reverse transcription of biotin-labeled RNAs underwent reverse transcription. Thereafter, the products received DNase I (RNase-free, Roche) treatment and purification using the RNeasy Mini Kit (Qiagen, USA), and the extracted RNAs were employed for qRT-PCR assessment.
Tumor xenograft implantation in nude mice
We divided six-week-old nude mice into two groups (4 mice/group) randomly, raised them with unceasing food and water in sterile conditions without pathogens. To establish the lung adenocarcinoma xenograft model, we subcutaneously injected A549 cells into nude mice. We monitored tumor growth every week and calculated it as equation: Volume = (Length) × (Width)2/2. Animal assays took place in SPF Animal Laboratory at Xinxiang Medical University, and experiments were performed following the NIH guidelines on animal welfare.
Statistical analysis
Differences of data in normal distribution and equal variance were processed by 2-tailed Student t test (2‐group comparisons) or ANOVA, and the post hoc Bonferroni test (multigroup comparisons) was implemented as appropriate. Differences of data in non-normal distribution or unequal variance were processed by a nonparametric Mann-Whitney U test (2‐group comparisons) or the Kruskal-Wallis test followed by the post hoc Bonferroni test (multigroup comparisons). The standard we used to determine statistical significance was that P<0.05. We carried out all tests via SPSS 22.0 (SPSS, Chicago, IL, USA).