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Introduction

Lung cancer is one of the most common primary malignant tumors. According to the 2018 Global Cancer Statistics, lung cancer was the leading cause of cancer-associated mortality worldwide, with a 5-year survival rate of patients with lung cancer of 18% (1). The most common causes of lung cancer include smoking, air pollution, heredity factors and activation of cancer genes, such as Ras and Raf (2). Despite the fact that the majority of patients at early disease stages can be treated with surgery (3), the choice of treatment for patients at advanced stages are limited to radiotherapy and chemotherapy (4). Furthermore, in some patients, prolonged exposure to a single chemotherapeutic agent such as cisplatin may lead to the development of resistance (5). Thus, the development of novel targeted therapeutic strategies remains critical for improved prognosis and survival outcomes of patients with lung cancer.

Histone-lysine N-methyltransferase EZH2 (EZH2) is the core catalytic component of polycomb repressive complex 2 (PRC2), which is involved in transcriptional repression (6). EZH2 is considered an oncogene, as it is frequently overexpressed in several types of human cancer, including prostate (7), gastric (8) and breast cancer (9,10). Furthermore, RNA interference of EZH2 has been reported to significantly decrease cell viability in breast cancer (11,12). However, the molecular mechanisms by which EZH2 knockdown inhibits disease progression of lung cancer remain unclear.

The aim of the present study was to identify whether EZH2 was associated with cell viability, migration and invasion, as well as cell cycle progression and apoptosis, in the lung cancer A549 cell line. By identifying whether EZH2 acts as an oncogene in lung cancer, the present study may provide a novel diagnostic biomarker and a therapeutic target for the treatment of lung cancer.

Materials and methods

Bioinformatics analysis of human lung cancer

The GSE19804 dataset (13) was downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo) using the keywords ‘lung cancer’, ‘Homo sapiens’ and ‘Expression profiling by array or sequencing’, which includes 60 adjacent non-tumor (within 2 cm around tumors) and 60 tumor samples. GEO2R is an interactive online platform, which identifies differentially expressed genes (DEGs) by comparing samples in the GEO series (14). The cut-off criteria for the DEGs were an adjusted P<0.05 and a |log2 fold change (FC)| value of >2. The genes were aligned using their expression levels, and the data are presented as a volcano plot and a heat map. PROGgeneV2 (http://watson.compbio.iupui.edu/chirayu/proggene/database/?url=progge) is a web-based platform used to study prognostic implications of genes in different types of cancer. PROGgeneV2 was used to perform the survival analysis based on EZH2 expression in the GSE30219 dataset (15) downloaded from the GEO database.

Materials and reagents

The cell cycle kit (cat. no. 558662) and apoptosis detection kit (cat. no. 559763) were purchased from BD Biosciences, while the MTT reagent was purchased from Sigma-Aldrich; Merck KGaA. The primary antibodies (all diluted 1:1,000) against EZH2 (cat. no. 4905), cyclin D1 (cat. no. 2922), p21 (cat. no. 2947), cleaved(C)-caspase-3 (cat. no. 9661), C-caspase-9 (cat. no. 52873), N-cadherin (cat. no. 4061), E-cadherin (cat. no. 3195), vimentin (cat. no. 5741) and GAPDH (cat. no. 5174) were purchased from Cell Signaling Technology, Inc., and the EZH2 inhibitor (EPZ-6438) was obtained from Selleck Chemicals.

Cell culture

A total of five human lung cancer cell lines (H1650, PC9, A549, H1299 and H460), and a normal lung epithelium cell line, (BEAS-2B) were purchased from the Bank of Type Culture Collection of the Chinese Academy of Sciences and cultured in RPMI-1640 medium supplemented with 10% FBS and 100 U/ml penicillin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO2.

Reverse transcription-quantitative (RT-q)PCR

Total RNA was extracted from lung cancer A549 cells using TRIzol® regent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, and cDNA was generated using the PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd.) at 37°C for 30 min, according to the manufacturer's protocol. qPCR was subsequently performed using SYBR® Premix ExTaq™ (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol and detected using the LightCycler™ 480 system (Roche Diagnostics). The reaction conditions were as follows: Initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 40 sec and extension at 72°C for 40 sec. The following primer sequences were used for qPCR: EZH2 forward, 5′-GAAAGCCGCCCACCTC-3′ and reverse, 5′-AAACATCGCCTACAGAAAAGC-3′; and GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. Relative expression levels were calculated using the 2−ΔΔCq method and normalized to the internal reference gene GAPDH (16).

Western blot analysis

Total protein was extracted from A549 cells using RIPA lysis buffer supplemented with protease inhibitors cocktail and PMSF (1:100) (all Beyotime Institute of Biotechnology). Protein concentration was determined using the Pierce Micro BCA protein assay system (Pierce; Thermo Fisher Scientific, Inc.). A total of 50 µg protein/lane was separated using SDS-PAGE on a 10% gel and subsequently transferred onto polyvinylidene difluoride membranes (Sigma-Aldrich; Merck KGaA). The membranes were blocked with 5% skimmed milk in TBS-Tween (1% Tween-20) for 1 h at room temperature. After washing with TBS-Tween, the membranes were incubated with primary antibodies against EZH2, cyclin D1, p21, C-caspase-3, C-caspase-9, N-cadherin, E-cadherin, vimentin and GAPDH overnight at 4°C. After washing, membranes were incubated with goat anti-rabbit secondary antibody (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence kit (cat. no. NCI4106; Thermo Fisher Scientific, Inc.) and scanned using the lmageQuant 4000 system (Cytiva). The optical density of the target bands were quantified using the Quantity One system v4.6.2 (Bio-Rad Laboratories, Inc.).

RNA interference

A549 cells were transfected with 50 nM EZH2 small interfering (si)RNA (Shanghai Genechem Co., Ltd.) using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h before subsequent experimentation, according to the manufacturer's protocol. The following siRNA sequences were used: EZH2 forward, 5′-GTGCCCTTGTGTGATAGCACAA-3′ and reverse, 5′-GGCACTTTCATTGAAGAACTAA-3′; and non-targeted negative control (NC) forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′.

MTT assay

The viability of cells was detected using the MTT assay, according to the manufacturer's protocol. Briefly, A549 cells were trypsinized and seeded into 96-well plates at a density of 1×104, following siRNA transfection for 48 h or treatment with EPZ-6438 (40 µM) for 24 h, and treatment with different concentrations (0, 2, 4, 8 or 16 µM) of cisplatin (CDDP; Sigma-Aldrich; Merck KGaA) for 24 h. Cells were subsequently incubated with 20 µl MTT solution (in 5 mg/ml PBS) at 37°C for 4 h. Following which, the purple formazan crystals were dissolved using 150 µl dimethyl sulfoxide for 20 min at room temperature, and cell viability was subsequently analyzed at a wavelength of 570 nm. All experiments were performed in triplicate.

Colony formation assay

A total of 500 A549 cells were plated into 35 mm dishes prior to transfection with siRNA or treatment with EPZ-6438 (40 µM) for 24 h, followed by treatment with 4 µM CDDP for 24 h, and maintained at 37°C for 2 weeks. Cell colonies were subsequently washed three times with PBS, fixed with ethanol for 15 min at room temperature and stained with 1% crystal violet for 20 min at room temperature. Cell colonies were observed using a Canon camera (Canon, Inc.).

Cell cycle assay

Cell cycle analysis was performed using propidium iodide (PI) staining (BD Biosciences), according to the manufacturer's protocol. Briefly, cells were digested with EDTA-free trypsin and centrifuged at 500 × g for 5 min at 4°C. A total of 1×106 cells were harvested and fixed with 70% ethanol overnight at 4°C. Subsequently, PI supplemented with RNase A was added and the DNA content was sorted using flow cytometric analysis (BD Biosciences). Total cell quantity at each phase was analyzed using ModFit LT v5.0 software (Verity Software House, Inc.).

Flow cytometric analysis of apoptosis

The extent of apoptosis was determined using the PE-Annexin V Apoptosis assay kit (BD Biosciences), according to the manufacturer's protocol. Briefly, cells were digested with EDTA-free trypsin and centrifuged at 500 × g for 5 min at 4°C. Cells were harvested and washed three times with PBS, 48 h post-transfection, prior to resuspension in 500 µl binding buffer. Subsequently, cells were incubated with 5 µl PI and 5 µl Annexin V-FITC in the dark at room temperature for 10 min and apoptotic cells were analyzed using the FACScan flow cytometer (BD Biosciences).

Wound healing assay

A total of 1×105 cells/well were seeded into 24-well plates and cultured in RPMI-1640 supplemented with 1% penicillin, 1% streptomycin and 10% FBS at 37°C in 5% CO2 until they reached 90% confluence. Subsequently, the cell monolayers were scratched using a 200 µl pipette tip to produce a consistent width. Cells were washed twice with PBS to remove non-adherent cells, while the adherent cells were serum-starved in serum-free medium overnight. Migratory cells were observed in five randomly selected fields under a light microscope (magnification, ×40) at 0 and 24 h. The width of the wounds was measured using ImageJ software 1.52 (National Institutes of Health).

Transwell and Matrigel assays

The migration and invasion assays were performed as previously described (17). Briefly, 4×104 cells were plated in the upper chambers of 8-µM-pore sized Transwell plates (BD Biosciences) in serum-free medium and cultured for 37°C for both assays. A Matrigel assay was performed to detect cell invasion. The Transwell membranes were precoated with 50 µl Matrigel (1 mg/ml; BD Biosciences) at 37°C for 4 h. A total of 500 µl RPMI-1640 supplemented with 10% FBS was added to the lower chambers. Following incubation at 37°C in 5% CO2 for 24 h, both the migratory and invasive cells in the lower chambers were washed twice with PBS and fixed with ethanol for 10 min at room temperature. Non-invasive cells in the upper chambers were removed using a cotton swab. Invasive cells, and the migratory cells were stained with 1% crystal violet for 20 min at room temperature. Stained cells were observed under a light microscope (magnification, ×100).

Statistical analysis

Statistical analysis was performed using GraphPad Prism software version 7.0 (GraphPad Software, Inc.). The survival curve was calculated using the Kaplan-Meier estimator method and the log-rank test was used to compare the two survival curves. Data are presented as the mean ± standard deviation of three independent experiments. Unpaired Student's t-test was used to compare differences between two groups, while one-way ANOVA followed by the least significant difference post hoc test was used to compare difference between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Results

EZH2 expression is upregulated in human lung cancer

Analysis of the GSE19804 dataset identified 265 DEGs, including 53 upregulated and 212 downregulated DEGs in lung cancer compared with normal tissues (P<0.05 and |Log2FC| >2; Fig. 1A). The DEGs are shown in Table SI, while the top 20 DEGs in lung cancer are presented in Fig. 1B.

Figure 1. EZH2 expression is upregulated in human lung cancer tissues and cell lines. (A) Differentially expressed genes were selected using volcano plot filtering (P<0.05; FC >2). The red dots represent upregulated genes, while the green dots represent downregulated genes and the grey dots represent genes with |FC|<2. (B) Heatmap of the top 20 dysregulated genes. Red indicates upregulation and green indicates downregulation. The values between −3 and 3 indicate the FC degree in gene expression. The 60 GSM on the left side were collected from tumor tissues of patients with lung cancer, while the 60 GSM on the right side were collected from normal tissues of the same patients. (C) Altered EZH2 expression was demonstrated to be associated with overall survival time. (D) EZH2 expression in lung cancer cell lines (H1650, PC9, A549, H1299 and H460) and normal human lung epithelial cell line (BEAS-2B) was detected using reverse transcription-quantitative PCR and western blot analyses. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. BEAS-2B. EZH2, histone-lysine N-methyltransferase EZH2; FC, fold change; GSM, Gene Expression Omnibus samples.

Survival analysis was performed for patients with lung cancer, using the GSE30219 dataset. The results demonstrated that high EZH2 expression was associated with a shorter overall survival time in patients with lung cancer, thus an unfavorable prognosis, compared with those with low EZH2 expression. The 3- and 5-year survival rates of patients with high EZH2 expression were lower than those of patients with low EZH2 expression (Fig. 1C).

EZH2 mRNA and protein expression levels were also determined in the six lung cancer cell lines using RT-qPCR and western blot analyses. The results show that EZH2 was upregulated in all the lung cancer cell lines (H1650, PC9, A549, H1299 and H460) compared with that in the BEAS-2B cell line (Fig. 1D). As the expression level of EZH2 was highest in the A549 cell line, this was used for further experimentations. Taken together, these results suggest that EZH2 may be associated with tumorigenesis and progression of lung cancer.

EZH2 knockdown restricts cell viability and induces apoptosis

To evaluate the function of EZH2 in lung cancer cells, A549 cells were transfected with siRNA-EZH2, and non-targeting siRNA was used as the NC. The results demonstrated that the mRNA and protein expression levels of EZH2 were significantly decreased in A549 cells transfected with siRNA-EZH2 compared with that in the NC cells, using RT-qPCR and western blot analyses, respectively (Fig. 2A).

Figure 2. EZH2 knockdown inhibits cell viability and induces apoptosis. (A) EZH2 mRNA and protein expression levels were significantly decreased in cells transfected with siRNA-EZH2 compared with that in the NC. (B) MTT assay demonstrated that the viability of A549 cells deceased following EZH2 knockdown. (C) The colony formation assay was performed using A549 cells, with or without EZH2 knockdown. (D) The cell cycle distribution was analyzed using flow cytometry, with or without EZH2 knockdown and analyzed quantitatively. (E) NC and siRNA-EZH2 transfected cells were stained with Annexin V/propidium iodide for flow cytometric analysis and analyzed quantitatively. (F) Western blot analysis of cyclin D1, p21, C-caspase-3 and −9 proteins in A549 cells, treated with NC and siRNA-EZH2 and analyzed quantitatively. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. NC. EZH2, histone-lysine N-methyltransferase EZH2; si, small interfering; NC, negative control; C, cleaved; FITC, fluorescein isothiocyanate.

To determine the biological function of EZH2 in lung cancer cells, the viability of A549 cells following EZH2 knockdown was analyzed using MTT and colony-formation assays. The MTT assay results demonstrated that the cell viability was significantly decreased in siRNA-EZH2 transfected cells compared with that in the NC cells (Fig. 2B). Similarly, colony formation ability was also decreased following EZH2 knockdown (Fig. 2C). In addition, EZH2 knockdown also induced G1 phase cell cycle arrest, while the cell percentage in the G2 phase was also decreased in A549 cells (Fig. 2D).

Flow cytometric analysis was performed to determine whether the inhibitory effects on cell viability were associated with apoptosis, following EZH2 knockdown (Fig. 2E). The number of cells in early stage apoptosis was significantly higher in the cells treated with EZH2 siRNA compared with that in the NC cells, 0.7±0.21 vs. 2.9±0.48% cells (P<0.05). In addition, the percentage of apoptotic cells in late stage apoptosis was also significantly increased in siRNA-EZH2 transfected cells compared with that in the NC cells. Furthermore, the protein expression levels of the cell cycle proteins, cyclin D1 and p21 significantly decreased, while the levels of the apoptosis-associated proteins, C-caspase-3 and −9 significantly increased in cells following EZH2 knockdown (Fig. 2F). These results indicate that EZH2 may play a role in regulating cell viability and apoptosis.

EZH2 knockdown inhibits cell migration and invasion

The migratory ability of A549 cells was assessed using wound healing and Transwell assays. Both assays revealed that the migratory rate was decreased in A549 cells transfected with EZH2 siRNA compared with that in the NC cells (Fig. 3A and B, respectively). In addition, the Matrigel assay found that A549 invasion was also decreased in cells transfected with EZH2 siRNA compared with that in the NC cells (Fig. 3B). Furthermore, decreased protein expression levels of epithelial-mesenchymal transition (EMT)-associated proteins (N-cadherin and vimentin), and increased levels of E-cadherin were detected in siRNA-EZH2 transfected cells compared with that in NC cells (Fig. 3C).

Figure 3. EZH2 knockdown inhibits cell migration and invasion in lung cancer cells. (A) Wound healing and (B) Transwell and Matrigel assays were used to detect the migration and invasion of lung cancer cells treated with NC and siRNA-EZH2 and analyzed quantitatively. Magnification, ×40. (C) The protein expression levels of N-cadherin, E-cadherin and vimentin were detected in cells transfected with NC and siRNA-EZH2 and analyzed quantitatively. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 and **P<0.01 vs. NC. EZH2, histone-lysine N-methyltransferase EZH2; NC, negative control; si, small interfering.

EZH2 knockdown enhances sensitivity of lung cancer cells to CDDP by inhibiting viability and enhancing apoptosis

To further assess the function of EZH2 during treatment with CDDP, the cell viability of NC and siRNA-EZH2 transfected cells following CDDP treatment was analyzed using the MTT and colony formation assays. The viability (Fig. 4A) and colony formation ability (Fig. 4B) of A549 cells transfected with siRNA-EZH2 were significantly reduced compared with that in NC cells, following CDDP treatment (0, 2, 4, 8 or 16 µM) and 4 µM CDDP treatment, respectively. To determine whether the chemo response effects of EZH2 on A549 cells was associated with apoptosis, transfected cells following CDDP treatment were detected using flow cytometry. The number of early stage apoptotic cells in the EZH2 knockdown cells (9.99±1.26%) was significantly increased compared with that in the NC cells (6.37±0.69%), with 4 µM CDDP for 24 h (Fig. 4C). In addition, the percentage of late-stage apoptotic cells was significantly increased in EZH2 knockdown cells (8.51±0.94%) compared with that in the NC cells (3.98±0.43%), with 4 µM CDDP for 24 h. Furthermore, the expression levels of the apoptosis-associated proteins, C-caspase-3 and −9, increased in siRNA-EZH2 cells treated with CDDP compared with those in NC cells (Fig. 4D).

Figure 4. Downregulation of EZH2 enhances sensitivity to CDDP by inhibiting cell viability and promoting apoptosis. (A) MTT and (B) colony formation assays were performed to detect the proliferative ability of cells transfected with siRNA-EZH2 or treated with the EZH2 inhibitor, following CDDP treatment, respectively. (C) A higher apoptosis rate was observed in cells transfected with siRNA-EZH2 or treated with the EZH2 inhibitor, following CDDP treatment, compared with that in the control groups. (D) The expression levels of C-caspase-3 and −9 were detected using western blot analysis in cells transfected with siRNA-EZH2 or treated with the EZH2 inhibitor, following CDDP treatment. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. NC+CDDP or Control+CDDP. EZH2, histone-lysine N-methyltransferase EZH2; si, small interfering; NC, negative control; CDDP, cisplatin.

Notably, an inhibitor of EZH2 (EPZ-6438) was used to simulate EZH2 silencing. A549 cells treated with CDDP (4 µM) alone or in combination with EPZ-6438 (40 µM) for 24 h were subsequently analyzed using the MTT, colony formation and apoptosis assays. The viability (Fig. 4A) and colony formation ability (Fig. 4B) of A549 cells treated with the EZH2 inhibitor were significantly decreased compared with those in control cells, following CDDP treatment (0, 2, 4, 8 or 16 µM) and 4 µM CDDP treatment, respectively. The number of early stage apoptotic cells in cells treated with the EZH2 inhibitor (11.30±1.35%) was significantly increased compared with that in the control cells (4.18±0.53%); additionally, the percentage of late-stage apoptotic cells was significantly increased in cells treated with the EZH2 inhibitor (12.64±1.64%) compared with that in the control cells (3.42±0.31%), with 4 µM CDDP for 24 h (Fig. 4C). Furthermore, the expression levels of the apoptosis-associated proteins, C-caspase-3 and −9, increased in cells treated with the EZH2 inhibitor and with CDDP compared with those in control cells (Fig. 4D). Therefore, the results were consistent to those observed with siRNA-EZH2. Overall, the results of the present study demonstrated that EZH2 played a vital role in CDDP response, and EZH2 inactivation increased chemosensitivity to chemotherapeutic agents of lung cancer cells. Thus, EZH2 inhibitor may be used to be enhance chemosensitivity of lung cancer cells.

Discussion

According to the 2018 Global Cancer Statistics Report, lung cancer is the world's leading malignant tumor in morbidity (11.6%) and mortality (18.4%) rates (18). Both surgical resection or chemotherapy have become less effective in the treatment of lung cancer, resulting in the recurrence (19). Thus, it remains critical to research molecular targets associated with lung cancer progression or chemoresistance, to develop novel therapeutic strategies for cancer treatment. Taken together, the results of the present study demonstrated that EZH2 silencing may sensitize lung cancer cells to the chemotherapeutic agent, CDDP. Thus, EZH2 may act as a novel target of lung cancer progression.

EZH2 has been demonstrated to act as a transcriptional inhibitor and is a polycomb group protein (20). Previous studies have reported that EZH2 is overexpressed in several types of cancer (21,22). For example, EZH2 promotes viability and migration of breast cancer cells (23). The present study screened the GEO database using the key words ‘lung cancer’, ‘Homo sapiens’ and ‘Expression profiling by array or sequencing’, identifying the GSE19804 dataset with sufficient samples for data analysis, including 60 adjacent non-tumor and 60 tumor samples. Both bioinformatics and western blot analyses demonstrated that the expression level of EZH2 was increased in lung carcinoma tissues and cell lines, which could promote carcinogenesis in lung cancer. Furthermore, survival analysis indicated that increased levels of EZH2 in lung carcinoma was associated with poor prognosis, suggesting that higher levels of EZH2 may be used as a latent molecular biomarker for the prognosis of cancer. After a large number of clinical observations and data statistics (24,25), it was revealed that the recurrence and metastasis of most tumor patients (80%) occurred ~3 years after radical surgery, while those of 10% of patients occurred ~5 years after treatment. Therefore, the present study analyzed the 3- and 5-year survival rates of patients with high EZH2 expression and low EZH2 expression. Similar results have also been demonstrated in other types of human malignant cancer, including colon (26), breast (27), lung (28), bladder (29), ovarian (30) and prostate cancer (31). Taken together, the results of the present study highlight a novel potential role of EZH2 as a molecular target for drug development in lung cancer.

Wang et al (32) reported that siRNA-mediated suppression of EZH2 in bladder cancer induces apoptosis; however, Rao et al (33) demonstrated that siRNA-EZH2 has no effect on apoptosis in ovarian cancer. The results of the present study are consistent with the findings by Wee et al (34), suggesting that the function of EZH2 varies in different types of cancer. Furthermore, EZH2 knockdown in A549 cells suppressed viability, whilst inducing apoptosis and producing cell cycle arrest in the G1 phase. Taken together, these results confirm that EZH2 was associated with both tumor cell viability and apoptosis in lung cancer.

Metastasis is a complex process and can cause difficulties in the treatment of lung cancer (35), which has been defined as a multi-step process by which cell invasion contributes to metastasis (36). Previous studies have demonstrated that high expression levels of EZH2 was associated with cancer recurrence (37), distant metastasis (38), invasion (39) and angiogenesis (40) in numerous types of cancer, including melanoma and breast cancer. EZH2 is an adhesion protein expressed in breast and gastric cancer that promotes cancer metastasis by promoting ribosome synthesis; ribosomes are the cellular components that produce proteins, and their increased synthesis provides the conditions for cell metastasis (41). The results of the present study indicated that transfection with siRNA-EZH2 in lung cancer cells significantly inhibited invasion and migration. Furthermore, the expression levels of the EMT pathway proteins verified the molecular mechanisms of metastasis. Thus, EZH2 is a protein involved in the migration and invasion of lung cancer cells, whereby increased EZH2 expression may have a selective advantage on the migratory and invasive abilities of lung cancer cells. The detection of EZH2 expression can be performed as an additional tool to identify patients with lung cancer, with tumor progression and metastatic risk.

EPZ-6438 is a novel EZH2-specific inhibitor, which has demonstrated efficacy in different types of cancer, such as non-Hodgkin's lymphoma (42,43). In the present study, EPZ-6438 was used in combination with CDDP, which was demonstrated to have an additive effect on lung cancer cells. Treatment with the EZH2 inhibitor enhanced the CDDP-induced inhibition of cell viability and promoted apoptosis. However, further investigations into the molecular mechanism underlying EZH2 in the response of CDDP are required. Notably, the results of the present study highlight the potential applications of EZH2 inhibitors in future clinical treatment interventions and anti-chemotherapy resistance. Further studies should perform additional downstream experiments to reveal the underlying molecular mechanism and should verify the present results using animal models, which are the limitations of the present study.

In conclusion, the results of the present study demonstrated that EZH2 played a vital role in molecular diagnosis and in the CDDP response of lung cancer. Suppression of EZH2 inhibited tumorigenesis and enhanced chemosensitivity, thus suggesting that EZH2 may be used as a novel molecular therapeutic target for lung carcinoma.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was funded by the Shanghai Sailing Program (grant no. 17YF1415800) and the National Natural Science Foundation of China (grant no. 81802803).

Availability of data and materials

All data generated or analyzed during this study are included in this published article. The datasets generated and/or analyzed during the current study (GSE19804 and GSE30219) are available in the GEO repository (https://www.ncbi.nlm.nih.gov/geo/).

Authors' contributions

ZC, WW and ZD designed the study and analyzed the data. WW, HW, WZ and YH performed the experiments. ZC drafted the initial manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References