A total of 77 NSCLC tissue samples and paired adjacent normal tissue samples (distance, ≥3 cm) were obtained from patients undergoing surgery from May 2018 to January 2020 at the First Affiliated Hospital of Xinxiang Medical University (Xinxiang, China). Of these patients, 44 were male (mean age, 60.22 years) and 33 were female (mean age, 58.6 years). The histological type of the tumors was assessed using the World Health Organization Classification (20) by two independent pathologists. Ethics approval was granted by the Ethics Committee of the First Affiliated Hospital of Xinxiang Medical University (approval no. KN201808002). Oral consent was obtained from patients. All experiments were performed in accordance with approved guidelines and regulations (21).

Human NSCLC cell lines (A549, PC9, H1299, H1975 and HCC827) and normal pulmonary epithelial cells (BEAS-2B) were obtained from the American Type Culture Collection. A549, PC9, H1299, H1975 and HCC827 cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.). BEAS-2B cells were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.). All media were supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and all cells were incubated at 37°C with 5% CO_2.

Stable cell lines were constructed using lentivirus system. The plasmids used to make stably overexpressed/knockdown cell lines were based on the pcDNA3/Flag-METTL3 plasmid (Addgene; cat. no. 53739) or short hairpin (sh)RNA-METTL3 (GenePharma Co., Ltd.). To generate lentivirus particles, H1299 or H1975 cells were co-transfected with plasmids together with Trans-lentiviral packaging plasmids using Trans-lentiviral Packaging kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Lentivirus particles were harvested at 48 h post transfection and concentrated using Lenti-X concentrator according to manufactory instruction (Clontech Laboratories, Inc.). Stable cell lines was transduced with lentivirus particles in serum-free medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 8 µg/ml polybrene (Sigma-Aldrich; Merck KGaA) followed by selection with 2 µg/ml puromycin for ≥10 days. The cells with stable overexpression or knockdown of METTL3 were s identified by RT-qPCR and immunoblot analysis. The target sequences of shRNAs were as follows: shMETTL3#1, 5′-GCCAAGGAACAATCCATTGTT-3′; shMETTL3#2, 5′-GCTGCACTTCAGACGAATTAT-3′; and shMETTL3#, 5′GCTTACTATCTAGCATCACAT-3′.

Total RNA was isolated from cells using TRIzol^® Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Then, 1 µg total RNA was reverse-transcribed to complementary DNA using PrimeScript™ RT Master Mix (Takara Bio, Inc.). The reaction conditions were 37°C for 15 min and 85°C for 15 sec. mRNA expression was measured using a TB Green™ kit (Takara Bio, Inc.), according to the manufacturer's instructions. Quantification of the target and reference (GAPDH) genes was performed in triplicate using a LightCycler^® 480 II (Roche Diagnostics GmbH) with an initial denaturing step at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The primers used were as follows: GAPDH forward, 5′-CTGGGCTACACTGAGCACC-3′ and reverse, 5′-GGAACGCTTCACGAATTTG-3′; and METTL3 forward, 5′-AAGTGGTCGTTGAGGGCAATG-3′ and reverse, 5′-GCTTGGCGTGTGGTCTTT-3′. All primers were synthesized by Shanghai Shenggong Biology Engineering Technology Service, Ltd.. The analysis was conducted with the 2^−ΔΔCq quantification method (22).

Cells were seeded in 96-well plates at a density of 2,000 cells/well. Cells were incubated at 37°C in an incubator with 5% CO₂ for 0, 24, 48 and 72 h. Cell Counting Kit-8 (CCK-8; Sigma-Aldrich; Merck KGaA) was used to measure cell viability according to the manufacturer's instructions. The absorbance was measured at a wavelength of 450 nm using a plate reader (Bio-Rad Laboratories, Inc.).

Apoptosis was measured using flow cytometry with a FITC-Annexin V/PI detection kit (Wanleibio Co., Ltd.). A total of 1×10⁶ cells per well was seeded in 6-well plates and cells were then harvested after culturing for 48 h at 37°C. Afterwards, the cells were stained in the dark with FITC-Annexin V and PI at room temperature for 15 min. Subsequently, the percentages of apoptotic cells were measured using FACS flow cytometry (BD Biosciences) and analyzed using FlowJo 8.6.3 (Tree Star, Inc.).

Cell migration assays were performed using a cell migration assay kit (Corning, Inc.) and Transwell inserts. Briefly, 2×10⁴ cells were seeded in the upper chamber of a Boyden chamber (without serum in the RPMI-1640 medium, Invitrogen; Thermo Fisher Scientific, Inc.). The lower portion of the chamber was filled with 600 µl RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 20% FBS (Gibco; Thermo Fisher Scientific, Inc.). Following incubation for 24 h at 37°C, migrated cells in the lower chambers were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with 0.05% crystal violet (Sigma-Aldrich; Merck KGaA) for 10 min at room temperature. Cells were counted in five randomly selected fields of view under a light microscope (Leica GmbH DMI4000B) at 200× magnification.

m6A qPCR was performed as reported previously (23) to assess the relative abundance of the selected mRNA in m6A antibody immunoprecipitation (IP) and input samples. Isolated m6A-RIP RNA was quantified by qPCR. The primers used were as follows: Bcl-2 forward, 5′-ATCGCCCTGTGGATGACTGAGT-3′ and reverse: 5′-GCCAGGAGAAATCAAACAGAGGC-3′.

Total RNA was isolated and purified using PolyATtract mRNA Isolation Systems kit (cat. no. Z5310; Promega Corporation). m6A-seq was performed as previously described (24). The NEBNext Ultra Directional RNA Library Prep kit (cat. no. E7420L; Illumina, Inc.) was used for library construction. Clustered libraries were sequenced with an Illumina HiSeq 4000 (Illumina, Inc.) by Novogene Co., Ltd.

Total protein was extracted from cells using RIPA buffer containing protease and phosphatase inhibitor cocktail (both from Sigma-Aldrich; Merck KGaA). The protein concentration was determined with a BCA Protein Assay kit (Beyotime Institute of Biotechnology). Cellular proteins were separated by 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk and 0.1% Tween-20 in Tris-buffered saline at room temperature for 2 h. Membranes were probed at 4°C overnight with primary antibodies against METTL3 (1:2,000; cat. no. 86132S; Cell Signaling Technology, Inc.), Bcl-2 (1:1,000; cat. no. ab32124; Abcam) and GAPDH (1:5,000; cat. no. 5174S; Cell Signaling Technology, Inc.). After washing in PBST (0.5% Tween-20). Three times, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,000; cat. no. BA1070; Wuhan Boster Biological Technology, Ltd.) at room temperature for 1 h. The signal was visualized using ECL western blotting detection reagents (Tanon Science and Technology Co., Ltd). Protein expression levels were quantified using ImageJ software (version 1.50; National Institutes of Health).

A total of 15 female BALB/c nude mice (age, 6 weeks; weight, 18–20 g) were purchased from the Model Animal Center at Nanjing University (Nanjing, China) and raised under pathogen-free conditions in the Xinxiang Medical University Animal Center (Xinxiang, China). The mice were housed with filtered air, 12 h light/dark cycle, constant temperature (25°C), relative humidity (50±5%) and free access to food and water. In order to establish a human NSCLC xenograft model, 5×10⁶ H1299, sh-METTL3-H1299 and METTL3 stably overexpressed H1299 cells (2×10⁶ per mouse) were subcutaneously injected into mice. Tumor growth was observed daily. Tumor volume was calculated as follows: 0.5× (length × width²). At 24 days post-inoculation, the maximum diameter exhibited by a single subcutaneous tumor was 15 mm and mice were anesthetized by intraperitoneal administration of sodium pentobarbital (50 mg/kg), then sacrificed by cervical dislocation. Tumors were harvested and weighed for histological analysis. Lungs were extracted and fixed for 24 h at room temperature using 10% (v/v) neutral-buffered formalin and transferred to 70% ethanol until embedding in paraffin then sectioned at 3 µm. For hematoxylin-eosin staining, sections were incubated with hematoxylin for 2 min and eosin for 1 min. The experimental procedures were approved by the Laboratory Animal Care Committee at Xinxiang Medical College (approval no. XXMU-2020-146367).

Tissue sections were incubated with primary antibodies against METTL3 (1:50; cat. no. 86132S; Cell Signaling Technology, Inc.), Bcl-2 (1:50; cat. no. ab32124; Abcam) and Ki67 (1:100; cat. no. ab16667; Abcam) at 4°C overnight. Following washing with PBST containing 0.05% Tween-20, the sections were incubated with HRP-conjugated anti-rabbit secondary antibody (1:5,000; cat. no. BM3894; Wuhan Boster Biological Technology, Ltd.) at room temperature for 1 h. The sections were developed with 0.05% 3-diaminobenzidine tetrahydrochloride at room temperature for 10 sec, which was followed by counterstaining with 10% Mayer's hematoxylin for 4 min at room temperature. IHC results were analyzed by two experienced pathologists. A total of five random fields under a light microscope (Leica GmbH DMI4000B) at 200× magnification were chosen to calculate the percentage of positively stained cells vs. the total number of tumor cells. The staining proportion of the positive cells was divided into four groups as follows: -, 0 positive cells observed; +, <30% vs. positive tumor cells observed; ++, 30–60% vs. positive tumor cells observed; and +++: >60% vs. positive tumor cells observed. The - and + groups were classed as METTL3-low expression group, while the ++ and +++ groups were classed as METTL3-high expression group for subsequent analysis. Low- and high-expression groups were combined following scoring and analyzed by Fisher's exact t-test.

The Gene Expression Profiling Interactive Analysis (GEPIA) database (gepia.cancer-pku.cn/) was used to evaluate the mRNA expression levels of METTL3 in NSCLC and normal tissue. Log rank test was used for Kaplan-Meier analysis. The Kaplan-Meier plotting tool (kmplot.com/analysis/) was used to evaluate the relapse-free survival of patients with NSCLC expressing low or high levels of METTL3. The association between METTL3 and Bcl-2 mRNA expression levels in NSCLC was analyzed using GEPIA database (gepia.cancer-pku.cn/) and Pearson's correlation coefficient.

A total of three independent repeats of experiments were performed. All data are shown as the mean ± SD. Data were analyzed using SPSS software 22.0 (IBM Corp.). Associations between two groups were evaluated using χ² test and Pearson's correlation analysis. Comparisons between two groups were performed using Student's unpaired two-tailed or paired t-test. Multiple groups were analyzed using one-way ANOVA followed by Bonferroni's post hoc test for pairwise comparisons. P<0.05 was considered to indicate a statistically significant difference.

First, it was determined that the expression of METTL3 was upregulated in the NSCLC cohort using bioinformatics tools (gepia.cancer-pku.cn/; Fig. 1A). In addition, RT-qPCR analysis of METTL3 mRNA expression in samples from patients with NSCLC showed that the expression of METTL3 was significantly higher in NSCLC tumor samples than in normal paracancerous tissue (Fig. 1B). m6A quantitative analysis showed that the m6A content level was significantly higher in tumor than in normal paracancerous tissue (Fig. 1C). Western blot and IHC analysis showed that the protein expression of METTL3 was increased in NSCLC compared with normal paracancerous tissue (Fig. 1D and E). Clinical and pathological data are shown in Fig. 1F. A significant positive association between high METTL3 expression and tumor differentiation, tumor stage and metastasis was observed. These results indicated that METTL3 served a significant role in NSCLC development and progression. Consistently, METTL3 expression was increased in the NSCLC cell lines compared with the normal human lung epithelial cell line BEAS-2B (Fig. 1G). Kaplan-Meier survival analysis indicated that patients with tumors with high METTL3 expression had a poor survival rate and outcome (Fig. 1H).

In order to investigate the functional role of METTL3 in NSCLC, METTL3 was knocked down using three independent shRNAs (shRNA-1, shRNA-2 and shRNA-3) in an NSCLC cell line (H1299). Efficient knockdown of METTL3 was confirmed by RT-qPCR (Fig. 2A) and western blotting (Fig. 2B). The CCK-8 assay results showed significantly decreased viability of NSCLC cell lines (H1299, H1975) after silencing METTL3 (Fig. 2C). Moreover, flow cytometry assay revealed that METTL3 silencing accelerated apoptosis in NSCLC cell lines (H1299, H1975; Fig. 2D). Migration assay revealed that METTL3 silencing repressed migration (Fig. 2E). m6A-qPCR validation showed that the percentage of m6A content was significantly decreased when METTL3 was knocked down in H1299 and H1975 cell lines (Fig. 2F). Collectively, these results indicated that silencing METTL3 suppressed viability and migration and promoted apoptosis in NSCLC cell lines.

Next, stable METTL3-overexpressing NSCLC cell lines (H1299 and H1975) were constructed by lentiviral infection. Upregulation of METTL3 expression was confirmed by RT-qPCR (Fig. 3A) and western blotting (Fig. 3B). METTL3 overexpression significantly promoted NSCLC cell line (H1299 and H1975) viability, as determined by CCK-8 assay (Fig. 3C). Moreover, flow cytometry revealed that overexpression of METTL3 inhibited the apoptosis of NSCLC cell lines (H1299 and H1975; Fig. 3D). Furthermore, METTL3 overexpression significantly increased migration of NSCLC cell lines in the Transwell assay (Fig. 3E). Quantitative analysis of m6A showed that METTL3 overexpression increased m6A content (Fig. 3F). Based on these results, it was concluded that METTL3 overexpression promoted NSCLC cell viability and migration.

A previous study reported that Bcl-2 may be mediated by METTL3 in breast cancer (21). In order to identify the underlying molecular mechanisms by which METTL3 promotes NSCLC progression, m6A-seq was performed. The results showed that the GGAC motif was highly enriched within m6A sites in the immunopurified RNA (Fig. 4A). In order to verify that METTL3 targets Bcl-2 mRNA for m6A modification, ethylated RNA immunoprecipitation qPCR was performed to validate the m6A-seq data. The results showed that METTL3 knockdown significantly decreased m6A levels of Bcl-2 mRNA (Fig. 4B). RT-qPCR analysis showed that Bcl-2 expression was significantly decreased at the mRNA level following METTL3 knockdown in H1299 and H1975 cells (Fig. 4C). Bcl-2 protein expression was significantly downregulated in METTL3-knockdown H1299 and H1975 cells (Fig. 4D). Then, the online bioinformatics tool GEPIA database (gepia.cancer-pku.cn/) was used for correlation analysis and our results showed that the METTL3 expression level also showed a positive correlation with Bcl-2 in tumors from individuals with NSCLC (Fig. 4E). Thus, it was concluded that Bcl-2 was a target of METTL3.

In order to confirm the potential contribution of METTL3 to tumor progression in vivo, BALB/c nude mice were subcutaneously injected with H1299 cells with METTL3 overexpression or knockdown to develop an NSCLC xenograft model. After 24 days, the tumors were removed, photographed and weighed (Fig. 5A and B). Tumors arising from cells with METTL3 overexpression were significantly larger and heavier than those arising from control cells (Fig. 5C). By contrast, knockdown of METTL3 significantly repressed tumor growth (volume) compared with that of tumors arising from control cells (Fig. 5C). Hematoxylin-eosin staining indicated that knockdown of METTL3 decreased the number of metastatic nodules, while overexpression of METTL3 increased the number of lung metastatic nodules compared with control (Fig. 5D). In order to confirm whether METTL3 promoted tumor growth in vivo by regulating the expression of Bcl-2, IHC staining and western blot analysis were performed. The results indicated that METTL3 overexpression increased Bcl-2 expression and METTL3 knockdown inhibited Bcl-2 expression (Fig. 5E-F). Collectively, these results suggested that METTL3-mediated m6A modification promoted NSCLC progression by enhancing expression of Bcl-2 in vivo.