Introduction
Worldwide, there were ~18.1 million new cancer cases and 9.6 million cancer-associated mortalities in 2018 (1). Among all types of cancer, lung cancer has the highest incidence and mortality rates, with 2.1 million new lung cancer cases and 1.8 million mortalities predicted in 2018, which accounts for ~18.4% of all cancer-associated mortalities (1). Non-small cell lung carcinoma (NSCLC) is the most common histological type of lung malignancy. Lung adenocarcinoma (LUAD) accounts for ~50% of all lung cancer cases (2). In the past few decades, clinical trials for the treatment and examination of lung adenocarcinoma have increased, including studies involving chemotherapy, molecular targeted therapy, stereotactic radiotherapy and immunotherapy (3-6). Although NSCLC has been investigated in numerous molecular studies aimed at developing new treatment strategies, the 5-year overall survival rate remains at 4-17%, and 65% of patients with NSCLC are classified as stage III or IV at diagnosis (4,7). Therefore, improved understanding regarding the regulatory mechanisms of NSCLC development and progression is urgently required for the improvement of cancer treatment.
Centrosome protein E (CENPE) is a kinesin-like motor protein that accumulates at the G2 phase of the cell cycle (8). Unlike other centrosome-associated proteins, it is not present during interphase and first appears at the centromere region of chromosomes during prometaphase (9). Therefore, cells with rapid proliferation have higher levels of CENPE expression and CENPE is upregulated in numerous types of solid cancer (10). Certain studies have identified that reducing the expression of CENPE can inhibit cancer cell proliferation. In prostate cancer, genetic deletion or pharmacological inhibition of CENPE was demonstrated to significantly decrease tumor growth (11). In high-grade glioma cells, knockdown of CENPE, kinesin family member 14 or non-SMC condensin I complex subunit G combined with temozolomide-treatment resulted in a combined suppressive effect on cell proliferation (12). In addition, small interfering RNA (siRNA)-induced knockdown of CENPE in human neuroblastoma cell lines can inhibit cellular proliferation (13). Targeting CENPE with the small molecular inhibitor GSK923295 inhibited the in vitro proliferation of 19 neuroblastoma cell lines and delayed tumor growth in three xenograft models (13). Furthermore, one study identified that CENPE can interact with FA complementation group A, which may serve an important role in cell cycle control and the pathogenesis of tumor (14).
In summary, previous studies have demonstrated that CENPE is associated with the proliferation of certain cancer cells. However, to the best of our knowledge, the role and regulatory mechanisms of CENPE in NSCLC have not been studied. The present study identified that the expression level of CENPE is higher in LUAD tissues compared with normal tissues. Cell proliferation in the A549 and PC9 cell lines was significantly inhibited following knockdown of CENPE. In addition, it was identified that the expression of CENPE in LUAD is directly regulated by FOXM1.
Materials and methods
Sample collection and cell culture
Between March 2018 and June 2018, seven LUAD tumor samples and seven paired normal lung tissue samples were collected from patients at The First Affiliated Hospital of Jinzhou Medical University (Jinzhou, China). The tumor and normal tissue samples were obtained by lobectomy, and the distance between the tumor tissue and normal lung tissue was ~6 mm. The patients included five males and two females, with a mean age of 56 years (range, 41-78 years). The present study was approved by the Ethics Committee of the First Affiliated Hospital of Jinzhou Medical University and written informed consent was obtained from each individual. The A549 and PC9 human LUAD cell lines were purchased from the American Type Culture Collection (Manassas) and cultured in DMEM high glucose (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO2.
siRNA and plasmid transfection
A549 or PC9 cells (5×105/well) were seeded in 6-well plates, cultured overnight and then transfected with 100 nmol/l CENPE siRNA, 100 nmol/l FOXM1 siRNA or 100 nmol/l negative control siRNA using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The sequences of the siRNAs were as follows: CENPE, 5′-GGCUGUAAUAUAA AUCGAA-3′ (11); FOXM1 5′-GCUCAUACCUGGUACC UAU-3 (15); and negative control, 5′-UUCUCCGAACGUG UCACGU-3′ (Shanghai GenePharma, Co., Ltd.).
The FOXM1 expression plasmid and control plasmid were purchased from Guangzhou FulenGen Co., Ltd. (Guangzhou, China) and transfected into A549 cells due to the lower expression of FOXM1 in these cells compared with PC9 cells (16). Transfections were performed in 6-well plates using Lipofectamine® 3000 transfection reagent (Gibco; Thermo Fisher Scientific, Inc.,), according to the manufacturer's protocol. In total, the cells were transfection with 2 µg plasmid/well. Cells were harvested and subjected to analysis 48 h after transfection.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from tissue samples and cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reverse transcribed into cDNA using the RT reagent kit with gDNA Eraser (Takara Bio, Inc.). The reaction conditions were as follows: 42°C for 2 min, 37°C for 15 min and 87°C for 5 sec. qPCR was performed in a reaction mix with SYBR Green (Takara Bio, Inc.) and was performed in triplicate with an ABI 7500 Prism Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following conditions were used: 95°C for 30 sec for initial denaturation followed by 40 cycles of 95°C for 5 sec and 60 °C for 34 sec. All qPCR primers were purchased from Invitrogen (Invitrogen; Thermo Fisher Scientific, Inc.) and the sequences were as follows: CENPE forward, 5′-GATTCTGCCATACAAGGCTACAA-3′ and reverse, 5′-TGCCCTGGGTATAACTCCCAA-3′; FOXM1 forward, 5′-GGAGCAGCGACAGGTTAAGG-3′ and reverse, 5′-GTTGATGGCGAATTGTATCATGG-3′; and GAPDH forward, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-GCCATCACGCCACAGTTTC-3′. Relative expression levels were calculated using the 2−∆∆Cq method (17).
Western blot analysis
Total protein was extracted from the cells or tissue samples using RIPA lysis buffer (Beyotime Institute of Biotechnology) with 1% protease inhibitor phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology). The lysates were centrifuged at 1,3000 × g for 15 min at 4°C and protein concentration was measured using a BCA kit (Beyotime Institute of Biotechnology). Subsequently, 40 µg protein/lane was separated by 10% SDS-PAGE and the proteins were then transferred to a PVDF membrane (Roche Applied Science). The membrane was blocked with 5% BSA (Wuhan Boster Biological Technology, Ltd.) for 1 h at room temperature and then incubated at 4°C overnight with primary antibodies. Following incubation with appropriate HRP-conjugated secondary antibodies, including goat anti-mouse IgG (cat. no. BA1050; 1:5,000; Wuhan Boster Biological Technology, Ltd.) and goat anti-rabbit IgG (cat. no. BA1054; 1:5,000; Wuhan Boster Biological Technology, Ltd.), for 1 h at room temperature, the membranes were visualized with an ECL kit (Beyotime Institute of Biotechnology) The primary antibodies for western blot analysis included mouse monoclonal anti-GAPDH (1:2,000; cat. no. BM1985; Wuhan Boster Biological Technology, Ltd.), rabbit anti-FOXM1 (1:1,000; cat. no. ab184637; Abcam) and rabbit anti-CENPE (1:1,000; cat. no. ab133583; Abcam).
Cell proliferation assay
Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) and 5-ethynyl-2'-deoxyuridine (EdU; GeneCopoeia, Inc.) assays were used to detect proliferation. In the CCK-8 assay, 24 h post-transfection, cells were seeded in a 96-well plate at a density of 3,000 cells/well and cultured overnight at 37°C in a humidified atmosphere containing 5% CO2. CCK-8 reagent was then added to the wells and the absorbance at 450 nm was detected following culture for 24, 48 and 72 h at 37°C.
The iClick™ EdU Andy Fluor™ 647 Flow Cytometry assay kit (GeneCopoeia, Inc.) was used to evaluate cell proliferation in the EdU assay. A total of 24 h post-transfection, A549 and PC9 cells, cultured in DMEM high glucose supplemented with 10% FBS, were treated with EdU (10 µM) for 6 h and the assays were performed according to manufacturer's protocol. The cells were then analyzed using a flow cytometer and analysis was performed with FlowJo v10 software (FlowJo LLC).
Chromatin immunoprecipitation (ChIP) assay
The FOXM1 binding site on the CENPE promoter region was analyzed using the Cistrome Data Browser (http://cistrome.org/db/#/) (18), with the following parameters: Biological sources; Neuroblastoma cell, Brain; and Factors; FOXM1, SK-N-SH, CENPE (19). The CENPE promoter sequence was obtained from the Eukaryotic Promoter Database (https://epd.epfl.ch/) (20). Subsequently, the binding site was analyzed using the AnimalTFDB3.0 database (http://bioinfo.life.hust.edu.cn/AnimalTFDB/) (21). The following two pairs of primers were used in the present study: Primer 1 (114 bp) forward, 5′-GACGGCAATTCTGTTTGGGT-3′ and reverse, 5′-CTGCCAAAAGCTAGCGAACG-3′; and primer 2 (218 bp) forward, 5′-CCCTCTCCTGTTTAGCAGTG-3′ and reverse, 5′-CCGCCATCCTATCAGGCTG-3′.
The ChIP assay was performed using the SimpleChIP™ Enzymatic Chromatin IP kit (cat. no. 9003; Cell Signaling Technology, Inc.), according to the manufacturer's protocol. In brief, 4×106 A549 cells were fixed with formaldehyde at room temperature for 15 min and lysed on ice for 5 min using the lysate in the kit, and then chromatin was harvested and fragmented using sonication. Antibodies specific to FOXM1 (1:100; cat. no. ab184637; Abcam) were used to recruit the target DNA overnight at 4°C and the complex was precipitated by Protein G magnetic beads for 2 h at 4°C. Normal rabbit IgG (1:300; cat. no. 2729; Cell Signaling Technology, Inc.) was used as the negative control and incubated with the sample overnight at 4°C, followed by precipitation of the complex with Protein G magnetic beads for 2 h at 4°C. Following immunoprecipitation, the protein-DNA complex was reversed and the DNA was purified. The enriched DNA was subjected to PCR analysis. PCR was performed with PCR Master mix (cat. no. KT201; Tiangen Biotech, Co., Ltd.), 2 µl DNA, 0.5 µl of each forward and reverse primers, and 7 µl ddH2O. The reaction conditions were as follows: 95°C for 4 min, 35 cycles of 95°C for 1 min, 60°C for 1 min and 72°C for 1 min, and a final step of 72°C for 5 min. The PCR products were screened on 2% agarose gel and signals were detected using enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology), followed by scanning using a FluorChem HD imaging system (ProteinSimple). The following primers were used to detect the co-immunoprecipitated CENPE promoter region by PCR: P r imer 1 (114 bp) for wa rd, 5′-GACGGCAATTCTGTTTGGGT-3′ and reverse, 5′-CTGCCAAAAGCTAGCGAACG-3′; and primer 2 (218bp) forward, 5′-CCCTCTCCTGTTTAGCAGTG-3′ and reverse, 5′-CCGCCATCCTATCAGGCTG-3′.
The Cancer Genome Atlas (TCGA) data analyses
To analyze the differential CENPE/FOXM1 expression levels in normal and LUAD tissues, and the survival of patients with NSCLC the Gene Expression Profiling Interactive Analysis server (http://gepia.cancer-pku.cn/) (22) was used to performed Kaplan-Meier analysis followed by a log-rank test. The CENPE and FOXM1 RNA-sequencing data were downloaded from the cBioPortal (http://www.cbioportal.org/index.do) (23). The expression difference analysis was performed with an unpaired Student's t-test using UALCAN (http://ualcan.path.uab.edu/index.html) (24) and Pearson's correlation analysis was performed using GraphPad Prism 6 software (GraphPad Software, Inc.).
Statistical analysis
Each experiment was independently repeated three times. Statistical analysis was performed with GraphPad Prism 6 software (GraphPad Software, Inc.). All data are presented as the mean ± standard deviation. Data were analyzed using Student's t-test for two groups. P<0.05 was considered to indicate a statistically significant difference.
Results
CENPE enhances the proliferation rate of LUAD cells
CCK-8 and EdU assays were used to examine the effect of CENPE on LUAD cell proliferation. The siRNA was designed to knockdown CENPE in A549 and PC9 cells. CENPE mRNA and protein levels were significantly reduced following RNA interference (P<0.001; Fig. 1A and B). The EdU assay demonstrated that the proliferation of LUAD cells was inhibited following CENPE-knockdown (P<0.001; Fig. 1C and D). The CCK-8 assay demonstrated the same phenomenon (P<0.01; Fig. 1E and F).
Expression of CENPE in human LUAD tissues
The expression of CENPE in lung adenocarcinoma and adjacent normal tissues was evaluated by RT-qPCR and western blot analysis. The results demonstrated that the mRNA levels of CENPE in the LUAD tissues were significantly upregulated compared with those in the paired normal tissues (P<0.01, Fig. 2A), and similar results were observed following western blot analysis of protein levels (Fig. 2B). Subsequently, the expression of CENPE in TCGA data was analyzed, and the expression level of CENPE was significantly higher in the LUAD samples compared with the normal tissue samples (P<0.0001; Fig. 2C). In addition, all tumor grades expressed higher levels of CENPE compared with normal lung tissue and the expression of CENPE was significantly higher in stage II tumors compared with stage I tumors (Fig. 2D). Furthermore, Kaplan-Meier analysis of TCGA data revealed that a lower CENPE expression was associated with improved patient outcomes (P<0.0001; Fig. 2E).
CENPE levels are associated with the expression of FOXM1
To further understand the regulatory mechanisms of CENPE, genes associated with CENPE expression were analyzed using TCGA database. It was identified that CENPE expression was highly correlated with transcription factor FOXM1 expression (r=0.8509; P<0.0001; Fig. 3A). Furthermore, in the tissue samples collected in the present study, the expression levels of FOXM1 and CENPE were significantly correlated (r=0.7057; P<0.0001; Fig. 3B). In addition, it was identified that FOXM1 expression was significantly higher in LUAD tissues compared with normal tissues (P<0.05; Fig. 3C). Analysis of the data from TCGA demonstrated the same result (P<0.001; Fig. 3D). All grades of tumors expressed higher levels of FOXM1 compared with normal lung tissue and the expression of CENPE was significantly higher in stage II tumors compared with stage I tumors (Fig. 3E). Additionally, Kaplan-Meier analysis of TCGA data demonstrated that lower FOXM1 expression was associated with an improved patient outcome (P=0.0015; Fig. 3F).
Silencing of FOXM1 inhibits proliferation of LUAD cells
To verify that FOXM1 regulates the expression of CENPE, siRNAs were designed to knockdown FOXM1 expression in A549 and PC9 cells. It was first demonstrated that FOXM1 was significantly knocked down in both cell lines and CENPE expression was also significantly reduced (P<0.01; Fig. 4A and B). The EdU assay revealed that silencing FOXM1 significantly inhibited the proliferation of LUAD cells (Fig. 4C and D; P<0.01). The CCK-8 assay further demonstrated that knockdown of FOXM1 significantly inhibited the proliferation of LUAD cells (P<0.05; Fig. 4E and F).
Overexpression of FOXM1 promotes proliferation of LUAD cells
To further confirm that FOXM1 regulates CENPE and promotes proliferation of LUAD cells, FOXM1 was overexpressed in A549 cells. RT-qPCR and western blot analysis demonstrated that FOXM1 was significantly overexpressed, and CENPE levels were significantly higher in cells overexpressing FOXM1 (P<0.001; Fig. 5A and B). After FOXM1 was overexpressed, the CCK-8 results revealed a significantly increased proliferation rate (P<0.05; Fig. 5C). Furthermore, the EdU assay demonstrated that overexpression of FOXM1 significantly promoted proliferation of A549 cells (P<0.001; Fig. 5D and E).
CENPE expression is directly regulated by FOXM1
From a previous study, it is understood that transcription factors can bind to the promoter region of genes and directly regulate their transcription (25). Therefore, the present study further analyzed the regulatory mechanism of FOXM1 on CENPE. First, the ChIP-sequencing data of FOXM1 was analyzed using the Cistrome database. The results demonstrated that FOXM1 has a binding peak in the promoter region of CENPE (Fig. 6A). The FOXM1 binding site on the CENPE promoter region (-499 to 100) was then analyzed. It was identified that there are two FOXM1 binding sites in the region of CENPE (Fig. 6B). Primers were then designed using DNA sequences that bind to the peak positions to further validate the conclusions of the binding. The ChIP assay of A549 cells confirmed that the CENPE promoter region contains a binding site for FOXM1 (Fig. 6C). To further confirm this conclusion, the band strengths obtained by CHIP from the control and FOXM1-interference groups were compared, and a lower intensity was observed for the FOXM1-interference group (Fig. 6D). Therefore, the current results indicate that FOXM1 can directly regulate the expression of CENPE in LUAD.
Discussion
Mitosis is a key event of the cell cycle. The ability to maintain proliferation is a hallmark of cancer and microtubules serve an important part in maintaining a sustained cell cycle (9). At the same time, numerous microtubule-associated proteins are highly expressed in tumor tissues (9), and targeting microtubule-associated proteins is one way to treat tumors (26). Therefore, further understanding of the regulatory mechanisms of microtubule-associated proteins in lung cancer may improve understanding regarding the cell cycle and also provide new targets for the treatment of tumors. CENPE is a microtubule-associated protein that is highly expressed in numerous types of cancer, including prostate cancer (11) and glioma (27). The present study identified a high expression of CENPE in LUAD tissues and CENPE-knockdown was demonstrated to inhibit the proliferation of LUAD cell lines.
Firstly, the current study identified that CENPE is highly expressed in lung adenocarcinoma using TCGA database, and in grade I and II lung adenocarcinoma the expression was revealed to increase with increasing grade. Subsequently, it was demonstrated that the proliferation of A549 and PC9 cells was inhibited following CENPE-knockdown, indicating that CENPE is associated with the proliferation rate of lung adenocarcinoma. Therefore, CENPE may be a marker of the proliferation rate of a tumor. In other tumor types, there are reports that CENPE can be used as a marker for tumor proliferation. In prolactin pituitary tumors, it was identified that seven genes, including ADAM metallopeptidase with thrombospondin type 1 motif 6 (ADAMTS6), collapsing response mediator protein 1 (CRMP1), pituitary tumor transforming gene, aspartokinase (ASK), cyclin B1 (CCNB1), aurora kinase B and CENPE, were associated with tumor recurrence or progression, and five of these genes (ADAMTS6, CRMP1, ASK, CCNB1 and CENPE) were also associated with the pathological classification (28). In glioma, RNA levels of eight major mitotic spindle assembly checkpoint genes, including CENPE, have been reported to be significantly associated with glioma grade and survival time (27). In invasive ductal carcinoma, CENPE was identified to be one of the top ten potential crucial genes (29). In addition, studies have identified that the expression of CENPE is associated with drug tolerance (30-32). In paclitaxel-resistant ovarian cell lines, it was revealed that CENPE expression is lower (31).
Following bioinformatics analysis, the present study identified that CENPE has a significant correlation with FOXM1 expression, and FOXM1 is also highly expressed in lung adenocarcinoma. In addition, the overall survival rate of patients with high expression of FOXM1 was worse; however, there was a difference between the survival trend of FOXM1 and the survival trend of CENPE. FOXM1 is a transcription factor that participates in all stages of tumor development, predominantly through control of the cell cycle and proliferation, regulating the expression of genes involved in the G1/S and G2/M transitions and M phase progression (33). The forkhead box O3 and FOXM1 transcription factors, functioning downstream of the essential PI3K-Akt, Ras-ERK and JNK/p38MAPK signaling cascades, are crucial for cell proliferation, differentiation, cell survival, senescence, DNA damage repair and cell cycle control (34). Therefore, numerous studies have identified that FOXM1 can promote the proliferation of a number of types of tumor. It has been reported that DEP domain containing 1, negatively regulated by microRNA (miR)-26b, promotes cell proliferation and tumor growth by upregulating FOXM1 expression, indicating an important underlying mechanism of regulating the progression of triple negative breast cancer (35). G2 and S phase-expressed-1 contributes to cell proliferation, migration and invasion by regulating the p53/forkhead box 1 (FOXM1)/CCNB1 pathway and predicts poor prognosis in bladder cancer (36). In U2OS cells, enforced expression of FOXM1 suppressed diallyl disulfide-induced antiproliferation and anti-invasion (37). In glioblastoma cells, miR-876-5p may inhibit the development of glioblastoma by directly targeting FOXM1 (38). In ovarian cancer, hsa_circ_0061140 has been demonstrated to promote cell growth and metastasis through regulation of the miR-370/FOXM1 pathway, mediating the epithelial-mesenchymal transition (39). In ovarian cancer cells, FOXM1 transcriptionally activated PCNA-associated factor to drive cell proliferation (40). In meningioma, the FOXM1/Wnt signaling axis is associated with a mitotic gene expression program, poor clinical outcomes and proliferation (41). In colorectal cancer, one study reported that FOXM1 is critical for the proliferation and growth of colorectal cancer (42). In summary, the aforementioned studies suggest that the expression of FOXM1 is associated with proliferation and mainly regulates the G2/M phase. Therefore, the present study hypothesized that the expression of CENPE may be regulated by FOXM1. Through interference, overexpression and ChIP experiments, it was demonstrated that CENPE can be directly regulated by FOXM1.
In conclusion, the current study revealed that CENPE is highly expressed in LUAD tissues. In addition, FOXM1 is correlated with the expression of CENE and is associated with the proliferation of lung cancer cells. The ChIP assay demonstrated that FOXM1 binds directly to the promoter region of CENPE. Therefore, the current data indicate that CENPE can promote the proliferation of LUAD cells and is directly regulated by FOXM1. In the future it will be important to further understand the signaling pathways that regulate CENPE expression, and the mechanism by which CENPE regulates the cell cycle. In addition, more studies should investigate centromere-associated proteins that are abnormally expressed in tumors. The study of more centromere-associated proteins may provide new targets and a theoretical basis for targeted therapy of cell cycle-associated proteins.