Cell lines K562, H1299, A549 and H460 were purchased from the American Type Culture Collection. In the three lung cancer cell lines, H1299 lacks the expression of p53 protein. A549 and H460 cells are detectable for p53 (19). A549/DDP (DDP-resistant A549 cell line) was purchased from Shanghai ExCell Biology, Inc. Cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS; Gemini Company, http://www.gembio.com/products) at 37°C and in a humidified atmosphere containing 5% CO₂ for 1 week.

Lipofectamine^® 3000 reagent (Thermo Fisher Scientific, Inc.) was used to transfect pBORIS plasmids purchased from OriGene Technologies Inc.. The empty vector control was constructed in our previous study (7). According to the requirement of Lipofectamine^® 3000, 70% confluent cells were prepared for the transfection. Plasmid DNA (1 µg) was transfected per well into6-well plates. Plasmid (50 ng) was transfected per well into 96-well plates. Lipofectamine RNAiMAX (Thermo Fisher Scientific, Inc.) was used to knockdown genes according to the manufacturer's instructions. Two siRNAs (siRNA-1 and siRNA-2) that targeted BORIS mRNA were used to knockdown BORIS. All small interfering RNAs (siRNA) were chemically synthesized by Xiang Yin Biotechnology Co., Ltd. (Table SI). The DDP treatment on transfected cells were performed one day after transfection.

Cells (5×10⁵) were harvested from cell culture plates. RNA was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and ethanol precipitation. Samples were quantified using the NanoDrop 2000 system (Thermo Fisher Scientific, Inc.) and proceeded to reverse transcription. The kit (cat. no. CW0741) used for reverse transcription was purchased from cwbiotech Beijing (https://www.cwbiotech.com/home.html). Subsequently, gene expression levels were quantified using SYBR Green based qPCR (cwbiotech Beijing). The thermocycling conditions were as follow: Initial denaturation at 95°C for 3 min; 45 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec, elongation at 72°C for 30 sec; and afinal extension at 72°C for 30 sec. Amplification and signal capture were performed by Bio-Rad CFX connect real-time system (Bio-Rad Laboratories). GAPDH and β-actin were used as the internal control genes to evaluate the expression levels of the candidate genes. The primer sequences used for qPCR are presented in Table SII.

Total protein was extracted from collected samples using RIPA buffer (Beyotime Institute of Biotechnology) supplemented with phenylmethylsulfonyl fluoride (cat. no. CAS 329-98-6; Sigma-Aldrich; Merck KGaA) and protease inhibitor cocktail (cat. no. 04693159001; Roche Applied Science). Total protein was quantified using the bicinchoninic acid kit (cat. no. P0012S; Beyotime Institute of Biotechnology) and 30 µg protein/lane was separated via 10% SDS-PAGE gel. The separated proteins were subsequently transferred onto nitrocellulose membranes and blocked with 5% skimmed milk for 1 h at room temperature. The membranes were incubated with BORIS polyclone primary antibody (cat. no. Ab126778; Abcam) overnight at 4°C. The BORIS antibody was diluted by 0.1% in 2% skimmed milk. Following the primary incubation, membranes were incubated with secondary antibody conjugated with HRP (cat. no. DW-0990; Dawen Biotechnology Co., Ltd., www.dawenbio.com) for 2 h at room temperature, which was diluted by 0.05% in 2% skimmed milk and used for subsequent experimentation. Protein blots were detected using the Advansta ECL system (cat. no. K-12043-D10; Advansta Inc., http://advansta.com) and visualized using the Bio-Rad chemidoc XRS+ system (Bio-Rad Laboratories, Inc.).

For the cell viability assays, 2,000 cells were seeded onto 96 well-plates/well. Cells were incubated with MTT (500 µg/ml, cat. no. M2128, Sigma-Aldrich; Merck KGaA) at 37°C for 4 h in the dark. Following the MTT incubation, the purple formazan crystals were dissolved using 100 µl dimethyl sulfoxide for 15 min at room temperature, and viability was subsequently analyzed at a wavelength of 490 nm, using a BioTeck Synergy 2 plate reader system (http://www.mtxlsi.com/bio-tek-synergy-2.htm).

For the colony formation assays, treated cells were seeded onto 6 well plates and fixed with 4% formaldehyde for 20 min at 37°C, and subsequently rinsed twice with PBS. Cells were stained with 0.5% crystal violet for 30 min at room temperature. Images were capturedusing white light channel in Bio-Rad chemidoc XRS+ system (Bio-Rad Laboratories, Inc.).

The Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit (cat. no. V13241) was obtained from Thermo Fisher Scientific, Inc. Following treatment with DDP, ~10⁶ cells were harvested and washed twice in cold PBS. According to the manual, 1X annexin binding buffer was used to dilute the Annexin V and propidium iodide (PI). Cells were then resuspended and incubated in Annexin V/PI working buffer at room temperature for 15 min in the dark. Subsequently, cells were stored in the dark and on ice. FACS was performed using a FACSCalibur (BD Biosciences) to detect apoptotic cells. Data analysis was performed using FlowJo software (version no. 7.6.1; FlowJo LLC).

Cells mounted on coverslips were prepared for treatments. After treatments cells were fixed with 4% formaldehyde for 20 min at 37°C and subsequently permeabilized with 0.3% Triton X-100 for 10 min at room temperature. Cells were subsequently blocked with 1% bovine serum albumin (BSA; Sangon Biotech Co., Ltd.) for 30 min at room temperature. Cells were incubated with primary antibody (1:200 in 1% BSA) overnight at 4°C. Cells were washed three times with PBS and subsequently incubated with secondary antibodies conjugated with tetramethyl rhodamine isothiocyanate (TRITC) or fluorescein-5-isothiocyanate (FITC; 1:200 in 1% BSA) for 1 h at room temperature. Cell nuclei were stained using DAPI (0.5 µg/ml) at room temperature for 5 min. Coverslips were re-washed four times and visualized using a Leica fluorescence microscope (magnification, ×20). BORIS primary antibody (cat. no. Ab126778) was purchased from Abcam and γH2AX antibody (cat. no. 05-636) was purchased from EMD Millipore. Primary antibodies were diluted 1:200 in 1% BSA. Flag antibody (cat. no. R1180B) was purchased from OriGene Technologies, Inc. FITC conjugated rabbit antibody (cat. no. DW-GAR001, www.dawenbio.com) and TRITC conjugated mouse antibody (cat. no. DW-A0521, www.dawenbio.com) were purchased from Dawen Biotechnology Co., Ltd..

TUNEL apoptosis detection kit (FITC) was bought from Shanghai Yeasen Biotechnology Co., Ltd.. The TUNEL reaction system was incubated at 37°C for 2 h in the dark. The cells were washed twice with PBS for 5 min. DAPI (1:6,000) was used to stain the nucleus in a dark room for 5 min in room temperature. After 2 washes with PBS, coverslips were sealed with glycerol and mounted onto slides. A Leica fluorescence microscope was used to visualize the cells and more than three scopes of images were captured by 20× objective lens for further analysis.

RNA-seq data and the clinical information for patients with NSCLC treated with DDP were downloaded from The Cancer Genome Atlas (TGCA) data portal and manually curated (11) (Table SIII). Clinical characteristics for the lung cancer cases (n=156) include age, sex, ethnicity, BORIS expression [fragments per kilobase of exon model per million reads mapped (FPKM)], clinical-stage (Tumor-Node-Metastasis; TNM) (3) and survival span. Treatment outcomes were recorded for 23 patients (Table SIV). Cancer tissues from 16 of the 23 patients were collected and analyzed before DDP treatment, which were shown to be ‘prospective’ in the present study. Cancer tissues from 7 of the 23 patients were collected and analyzed after DDP treatment, which were shown to be ‘retrospective’ in the present study (Table I). Survival probabilities of 156 NSCLC patients were evaluated when the patients were divided into two groups using a cut-off value of 0.0035 (FPKM).

Lung cancer classification for stagingwas referenced to worldwide standard (20). BORIS expression differences between patient groups were analyzed using the χ² test (Table I). The survival probabilities of investigated patients were analyzed via the Kaplan-Meier method and compared using the log-rank test. BORIS expression differences between NSCLC cell lines were analyzed by one-way ANOVA followed Tukey post-hoc test. All experiments were performed in triplicate. Statistical analysis was performed using SPSS 24.0 software (IBM Corp.) or GraphPad Prism software (version 5.0; GraphPad Software, Inc.). Statistical differences were analyzed by paired Student's t-test and presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

A total of 156 patients with NSCLC who received DDP treatment were extracted from TCGA (http://www.tcga.org/) (Tables I, II and SIII). The median of BORIS expression among the patients was 0.0035 (FPKM), which was used as a cut-off to study the association between BORIS and the overall survival rate in the present study. A higher BORIS expression level was significantly associated with a short survival span (P=0.019; Fig. 1A). In addition, BORIS expression levels in female patients were significantly lower than male patients. It is suggested that BORIS may be induced at high levels in male patients (Table I). BORIS expression in 23 cases with records of treatment outcomes was analyzed (Table SIV). BORIS expression data were extracted from The Cancer Genome Atlas database (http://www.tcga.org). Though no paired tissues were collected from the same patient before and after DDP therapy, BORIS levels declined from an average of 0.0071 FPKM in the prospective tissues to an average of 0.0027 FPKM in retrospective tissues (Fig. 1B). These data suggested that BORIS expression was associated with the mortality rate of NSCLC upon DDP chemotherapy.

Next, the proliferation rates of a panel of lung cancer cell lines was studied, which revealed a positive association between BORIS expression levels and cell proliferation (Fig. 1C and D). All cell lines tested demonstrated a statistically significant difference (Fig. 1C). Among the cell lines that were assessed, human pulmonary alveolar epithelial cell line (HPAEpiC) was a normal cell line. HPAEpiC did not express BORIS, suggesting that BORIS may just function in cancer cells. In order to further investigate its effect on cell proliferation, BORIS was silenced or overexpressed in NSCLC cell lines. A549/DDP cells express a relative higher BORIS compared with A549 (Fig. 1C). Next, two siRNAs targeting BORIS generated a similar knockdown efficiency with >50% decrease (Table SI and Fig. 2D). The knockdown efficiencies of siRNA-1 were all >70% in A549 and H460 cell lines (Fig. S1); therefore, in the present study, BORIS siRNA-1 was used for subsequent experimentation. BORIS knockdown suppressed the colony formation of NSCLC cells and induced the expression of p21, a cyclin-dependent kinase inhibitor (Fig. 2A and B). It has previously been demonstrated that elevated expression of p21 induces cell cycle arrest (21–23). Likewise, overexpression of BORIS promoted the proliferation of NSCLC cells and inhibited the expression of p21 in H1299 and A549 cells (Fig. 2B and C). Among the investigated NSCLC cell lines in the present study, BORIS was highly expressed in H1299 cells and expressed at low levels in A549 cells. Thus, the regulations of p21 by BORIS was investigated in these two cell lines, which express different BORIS levels (Fig. 1C). As BORIS knockdown inhibited cell cycles of NSCLC cells, the genome stability was investigated by assessing γH2AX, which is sensitive to DNA damage (24). BORIS was silenced by either siRNA-1 or siRNA-2 and the knockdown efficiencies were verified via western blotting and RT-qPCR analyses. The results demonstrated that BORIS knockdown induced γH2AX (Fig. 2D), suggesting that BORIS deficiency induces DNA damage in NSCLC cells. Collectively, these data demonstrated that BORIS expression was associated with NSCLC cell proliferation, indicating that BORIS may protect lung cancer cells from DNA damage.

In order to determine whether BORIS contributes to DDP resistance, BORIS was first silenced or overexpressed in H1299 cells, and subsequently treated by DDP at various concentrations from 1–4 µg/ml. BORIS knockdown inhibited H1299 cell proliferation. The supplement of 1 or 2 µg/ml DDP synergized BORIS knockdown for cell proliferation suppression (Fig. 3A). Conversely, BORIS overexpression promoted cell proliferation. BORIS overexpression resisted 1–4 µg/ml DDP treatment (Fig. 3B). Subsequently, the extent of apoptosis which was induced by 2 µg/ml DDP treatment was investigated in BORIS overexpressed H1299 cells compared with the negative control. The results demonstrated that BORIS overexpression inhibited DDP induced apoptosis from 13.41 to 8.68%, and necrosis from 5.60 to 3.46% (Fig. 3C). Taken together, these results suggest that BORIS protects NSCLC cells from injury by DDP treatment, thus, confirm the importance of BORIS in DDP resistance.

As BORIS resisted DDP-induced NSCLC suppression (Fig. 3), it was proposed that BORIS may inhibit DDP-induced DNA damage. pBORIS plasmid (BORIS-flag) was transiently transfected in A549 cells that were treated subsequently by DDP to induce DNA damage. Cells with overexpression of BORIS demonstrated a decreased γH2AX signal (Fig. 4A and B), indicating that BORIS attenuated DDP-induced DNA damage, thus promoting the proliferation of NSCLC. DNA damage was detected by the TUNEL assay in H1299 and A549/DDP when BORIS were silenced (Fig. 4C). BORIS may be beneficial for the DNA repair system of NSCLC cells to sustain genome stability under the treatment of DDP. The expression levels of few representative genes of DNA repair system were investigated, such as BRCA1, ERCC1, CMYC and MSH6 (Table SII). BORIS was overexpressed by pBORIS or silenced by siRNA-1 (siBORIS in Fig. 4D) in H1299 and H460 cells. Fold changes of the investigated genes are presented in Fig. 4D. MSH6 was regulated consistently and significantly by BORIS in H1299 and H460 cells (Fig. 4D). Collectively, the results of the present study suggest that BORIS may enhance the mismatch repair (MMR) system in NSCLC cells to resist DDP chemotherapy.