Data resource and comprehensive analysis

Gene expression profiles were obtained from The Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov/). Protein expression profiles were obtained from Clinical Proteomic Tumor Analysis Consortium (CPTAC) database (https://proteomic.datacommons.cancer.gov/pdc/). A summary of clinical data is shown in Supplementary Table S1-2.

Cell culture

Human bronchial epithelial (HBE) cells and lung cancer cell lines (A549, PC9, H1299, and H1650) were obtained from ATCC. All cells were cultured in RPMI-1640 medium (Hyclone, Logan, USA) supplemented with 10% FBS (Gibco, Grand Island, USA) and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) at 37°C in a humidified atmosphere (95% air, 5% CO2).

Cell transfections

The specific siRNA of RBM14 and YY1 overexpressed vector were ordered from Sangon Biotech (Shanghai, China). The sequence of si-RBM14 is 5′-GCATTCTGGCCATAGAGCTCGTATT-3′. The sequence of the scrambled siRNA is 5′-TGCTGACTCCAAAGCTCTG-3′. After being incubated overnight, the siRNAs or plasmids were transfected by using lipofectamine 2000 reagent (ThermoFisher Scientific, Waltham, USA). Forty-eight hours after transfection, cells were harvested for further analysis.

Reverse transcription-quantitative PCR

Total RNA was extracted using TRIzol® (Thermo Fisher Scientific). PrimeScript RT kit was used to reverse the mRNA into cDNA, and the SYBR-Green PCR Master One-Mix kit (TransGen, Biotech, Co., Ltd.) was used for quantitative real-time PCR. β-actin was used as an internal control. The sense and anti-sense primers were listed in Table 1.

Table 1 The primers used in this studyChromatin immunoprecipitation assays

A Simple ChIP Enzymatic Chromatin IP Kit (#9002, Cell Signaling Technology, Danvers, USA) was used to evaluate the accumulation of YY1, EP300, H3K9ac, and H3K27ac in RBM14 promoter according to the manufacturer’s instructions. Briefly, after being crosslinked with EBM-2 containing 1% formaldehyde, the crosslinked cells were collected in a lysis buffer containing 1% PMSF. Chromatin was digested by micrococcal nuclease, and 2% of aliquots of lysate were used as input control. Lysates were incubated with 3 μg primary antibody or normal rabbit IgG, followed by immunoprecipitation with protein G agarose beads and incubation at 4 °C overnight with gentle shaking. DNA crosslink was reversed by the addition of 5 mol/L NaCl and Proteinase K at 65 °C for 2 h. Immunoprecipitated DNA was purified and amplified by PCR using specific primers. The IgG (ab172730, Abcam), anti-EP300 (ab275378, Abcam), anti-YY1 (ab109228, Abcam), anti-H3K9ac antibody (ab32129, Abcam), and anti-H3K27ac antibody (ab4729, Abcam) were used. Immunoprecipitated DNAs were analyzed by qPCR. Primer sequences targeting RBM14 promoter (−91~+7; ch11: 66616629-66616727, named RBM14-promoter) were as follows: sense, 5′-CATTCCTGAGGAGGACTGCC-3′ and anti-sense, 5′-TCTTCATTTTGTCGCCGCAG-3′.

Western blotting

Cells were lysed by RIPA buffer (P0013C, Beyotime, Jiangsu, China), and the protein concentration was measured using a BCA kit (P0010, Beyotime). The protein was separated using SDS-PAGE and transferred onto a PVDF membrane (ISEQ00010, EMD Millipore). After blocking with 5% skimmed milk, the membrane was incubated with primary antibodies against RBM14 (ab70636, Abcam), YY1 (#63227, Cell Signaling Technology), p300 (#54062, Cell Signaling Technology), and β-actin (ab179467, Abcam) over-night at 4°C. After washing, the secondary antibody (Cell Signaling Technology) was added for 1 h of incubation. Protein signals were analyzed using an enhanced chemiluminescence substrate reagent kit (P0018M, Beyotime).

Co-immunoprecipitation

Cells were lysed with IP buffer (1 mM EDTA, 20 mM HEPES, 150 mM NaCl, 0.05% sodium deoxycholate, and 0.05% NP-40) added protease inhibitors and phosphatase inhibitor cocktails. After centrifugation at 12,000 rpm for 10 min, the cell lysates were incubated with indicated antibody and protein A/G PLUS agarose beads (#9863, #37478, Cell Signaling Technology) overnight at 4 °C. After washing, the beads were lysed with loading buffer and boiled at 100 °C for 10 min. Then, the protein was detected by Western blotting.

Cell Counting Kit-8

The transfected cells (1 × 104 per well) were cultured in 96 well plates for 72 h of incubation. Then, 10 μl Cell Counting Kit (CCK)-8 solution (C0038, Beyotime) was added to each well and incubated at 37°C for 2 h. The absorbance was detected at 450 nm using a microplate reader.

Apoptosis

Apoptotic cells were detected using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (#556547, BD Biosciences) according to the manufacturer’s instructions. Briefly, cells were re-suspended in 600 μl binding buffer. Then, 5 μl Annexin V/FITC and 5 μl propidium iodide (PI) were added. After being stained in dark for 15 min at room temperature, cells were analyzed by using a FACS Calibur (BD Biosciences).

Glycolysis

Forty-eight hours after transfection, PC9 and A549 cells were cultured in the phenol red-free medium for 24 h. The level of glucose and lactate in the culture supernatant was detected by Glucose Assay Kit (#361510, Rsbio, Shanghai, China) and Lactate Assay Kit (#A019–2, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. A Seahorse XF Glycolysis Stress Test kit (#103020-100, Agilent Technologies, Santa Clara, USA) was used to evaluate ECAR according to the manufacturer’s instructions. The basal ECAR was measured under the basal condition, followed by the sequential addition to each well of glucose (10 mM), oligomycin (2 mM), and 2-deoxyglucose (100 mM).

Statistical analysis

All data analyses were performed using GraphPad Prism software (v6.02). Quantitative results are displayed as the mean ± standard error. Two-tailed Student’s t test and one-way analysis of variance (ANOVA) followed by Tukey’s test were used to compare the differences in two groups and multiple groups. Kaplan–Meier survival curve was analyzed with the log-rank test. Each experiment was conducted three times at a minimum. P < 0.05 was considered statistically significant.