Lung cancer cell lines A549 and H661 were obtained from the American Type Culture Collection and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml of streptomycin, and maintained in 5% CO₂ atmosphere at 37°C. Small interfering RNA (siRNA) sense, 5′-GCACAAACTTGATCATCCA-3′ targeting the human MCM10 (siMCM10) and negative control siRNA (NC; cat. no. NControl_05815) synthesized by Guangzhou RiboBio Co., Ltd. 100 nM siRNA and NC were transiently transfected into lung cancer cells using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The cells were used for subsequent experimentation after 48-h transfection.

A total of 38 pairs of primary lung cancer and adjacent normal tissue samples were obtained from patients who underwent surgical operations at the Renmin Hospital of Wuhan University between May 2017 and November 2017 (age range, 41-75 years; 23 males and 15 females). All patients provided informed consent. Samples were immediately frozen in liquid nitrogen for MCM10 mRNA expression analysis. Human lung tissue microarrays including 56 pairs of primary lung cancer and corresponding adjacent normal lung/bronchiole tissues were purchased from Shanghai Outdo Biotech Company Co., Ltd. The tissue microarrays contained 24 adenocarcinoma (ADC), 13 squamous cell carcinoma (SCC), 7 adenosquamous carcinoma (ASC), 5 large cell lung carcinoma (LCLC) and 7 of small-cell lungs carcinoma (SCLC) samples.

MCM10 transcript level in lung cancer was determined from the Oncomine database (https://www.oncomine.org). A total of 7 datasets were selected using the following filter settings: MCM10, analysis type: Cancer vs. Normal analysis; Cancer type: Lung cancer. Comparison of MCM10 across 9 analyses was performed based on the screening criteria. The details of the analyses are listed in Table I. Gene expression omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) was used as a source to downloaded GSE30219 (21), GSE19188 (22) and GSE31210 (23) microarrays datasets. A total of 22 cases in GSE31210 were excluded from univariate and multivariate Cox proportional hazards regression analysis due to incomplete resection or adjuvant therapy. Tumor recurrence was classified as either early or late using 2 years as the cutoff. The expression pattern of MCM10 and the corresponding clinical data were extracted and analyzed. Kaplan-Meier (KM) Plotter (http://kmplot.com/analysis/index.php?p=service&cancer=lung) was used to determine the prognostic significance of MCM10. The following settings were used to obtain the KM survival plots: Gene symbol, MCM10; split patients by, median; survival, overall survival (OS), follow up threshold, all; histology, all/ADC/SCC; all other settings, all. The categorization of MCM10 expression (low vs. high) was based on the median expression level. Log rank P-values and hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated and extracted from the KM Plotter webpage. The expression level of MCM10 in the Cancer Genome Atlas (TCGA) dataset was also analysed by UALCAN (http://ualcan.path.uab.edu). The role of MCM10 in biological pathways was investigated using gene set enrichment analysis (GSEA; version 3.0; Broad Institute, Inc.) and the GSE3141 (24) dataset with functional gene set files (GSEA file, c2.cp.v4.0.symbols.gmt) to access enriched gene sets.

Paraffin was removed from the microarray tissue samples by treating with xylene twice for 10 min followed by rehydration with gradient alcohol for 5 min. Sodium citrate buffer (Wuhan Servicebio Technology Co., Ltd.; 10 mM; pH 6) was used for antigen retrieval and the samples were heated at 100°C in a microwave oven. Endogenous peroxidase activity was inhibited by treating the slides with 3% hydrogen peroxide. The tissue microarray was incubated with primary antibodies for MCM10 at 4°C (1:100; cat. no. DF12162; Affinity Biosciences). After overnight incubation, the microarray slides were rinsed with PBS and incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Wuhan Servicebio Technology Co., Ltd.; 1:200; cat. no. GB23303) at 37°C for 30 min and the reaction was visualized by using a fresh solution of 3,3′-diaminoben-zidine. The same criteria were used for evaluate the staining intensity and proportion of positive stained cells in each sample. The staining intensity was scored as 0 (no staining), 1 (pale yellow), 2 (yellow) and 3 (brown). The proportion of stained cells was defined as 0 (0-5% positive staining), 1 (6-25% positive staining), 2 (26-50% positive staining), 3 (51-75% positive staining) and 4 (>75% positive staining). The multiplication of staining intensity score and proportion of positive cells score was used as the IHC index. The IHC index ≥5 was defined as high expression of MCM10, while IHC index ≤4 was defined as MCM10 low expression.

After washing with PBS, total protein was extracted from transfected cells using RIPA (Beyotime Institute of Biotechnology) supplemented with a protease inhibitor cocktail PMSF (Kangchen BioTech Co., Ltd.) on ice for 30 min followed by quantification of the protein in the lysate using a BCA kit purchased from Thermo Fisher Scientific, Inc. Protein samples were separated using SDS-PAGE (10% gel) and transferred onto a polyvinylidene difluoride membrane at 200 mA for 2 h. Following an overnight incubation with an anti-MCM10 antibody (1:3,000; Abcam; cat. no. ab3733) or an anti-cyclin D1 (CCND1) antibody (1:3,000; ABclonal Biotech Co., Ltd.; cat. no. A10757) at 4°C, the membranes were incubated with a goat anti-rabbit IgG (1:10,000; ProteinTech Group, Inc., cat. no. 10285-1-AP) at 37°C for 1 h after washing with TBS with Tween-20. The protein signal was detected using an Novex™ ECL Chemiluminescent Substrate Reagent kit (Thermo Fisher Scientific, Inc.).

Total RNA from the cell lines and tissues were isolated using the TRIzol^® reagent (Takara Bio, Inc.) and quantified with NanoDrop™ 2000 (Thermo Fisher Scientific, Inc.). Using the PrimeScript RT-PCR kit (Takara Bio, Inc.), 2 µg RNA was converted to cDNA following the manufacturer's protocol. The quantification of gene expression was performed by qPCR using SYBR Premix Ex Taq™ (Takara Bio, Inc.). The Cq value of target gene was normalized according to the reference gene (β-actin) using the 2^−ΔΔCq method (25). The following thermocycling conditions were used: 50°C for 30 min, 94.5°C for 15 min; 40 cycles of 96°C for 30 sec and 59.7°C for 1 min. The primer sequences used for the qPCR analysis were as follows: MCM10 forward, 5′-CCCCTACAGACGATTTCT CGG-3′ and reverse, 5′-CAGATGGGTTGAGTCGTTTCC-3′; CCND1 forward, 5′-GCTGCGAAGTGGAAACCATC-3′ and reverse, 5′-CCTCCTTCTGCACACATTTGAA-3′; β-actin forward, 5′-GAAGAGCTACGAGCTGCCTGA-3′ and reverse, 5′-CAGACAGCACTGTGTTGGCG-3′; CDK4 forward, 5′-AT GGCTACAAGCAGATATGAG-3′ and reverse, 5′-TCACTC GGGGTTGCCCTC-3′. CCNB1 forward; 5′-AATAAGGCG AAGATCAACATGGC-3′ and reverse, 5′-TTTGTTACCAAT GTCCCCAAGAG-3′; P27 forward, 5′-AGGAGATGTAAC TATCGGACCTC-3′, and reverse, 5′-GTTCCCTTCGCCAAT CACTATT-3′; p21 forward, 5′-TCCTGGAGCAGACCACCC CG-3′ and reverse, 5′-GGGGTGGGACAGGCACCTCA-3′; P19 forward, 5′-GGGTTTTCGTGGTTCACATCC-3′ and reverse, 5′-CTAGACGCTGGCTCCTCAGTA-3′; CCNA2 forward, 5′-GGATGGTAGTTTTGAGTCACCAC-3′ and reverse, 5′-CACGAGGATAGCTCTCATACTGT-3′; CCNE1 forward, 5′-GCCAGCCTTGGGACAATAATG-3′ and reverse, 5′-CTTGCACGTTGAGTTTGGGT-3′; CDK6 forward, 5′-TC TTCATTCACACCGAGTAGTGC-3′ and reverse, 5′-TGAGGT TAGAGCCATCTGGAAA-3′; CDK2 forward, 5′-CCAGGA GTTACTTCTATGCCTGA-3′ and reverse, 5′-TTCATCCAG GGGAGGTACAAC-3′; CDK1 forward, 5′-GGATGTGCTTAT GCAGGATTCC-3′ and reverse, 5′-CATGTACTGACCAGG AGGGATAG-3′.

CCK-8 (Dojindo, Molecular Technologies, Inc.) and colony formation assays were used to determine the proliferation ability of lung cancer cells. For the CCK-8 assay, 3,000 cells were seeded in 96-well cell culture plates and allowed to grow overnight. The cells were then transfected with siRNA as aforementioned for 48 h. At indicated culture points (0, 24 and 48 h), 10 µl CCK-8 was added to each well and incubated for another 2 h at 37°C. The measurement of absorbance was done at a wavelength of 450 nm. For the colony formation assay, after 48 h of transfection, the cells were seeded in six-well cell culture plates at a density of 500 cells/well for 14 days. The number of colonies was counted after fixing and staining with 1% crystal violet (containing with 4% methanol) for 10 min at 37°C.

Cell cycle progression was assessed using cell cycle detection kit (KeyGEN BioTech Co., Ltd.; cat. no. KGA511) 48 h post transfection. Cells were resuspended in 70% ethanol at −20°C overnight. The cells were washed with cold PBS, subsequently treatment with 100 µl RNase for 30 min at 37°C and 400 µl PI for 30 min at 4°C before flow cytometry analysis. The DNA content was analyzed with ModFit software (version 3.2; Verity Software House). Cell apoptosis was detected by flow cytometry (BD FACSARia™ III; BD Biosciences) after staining with Annexin V-allophycocyanin and PI (BD Biosciences; cat. no. 556547) according to the manufacturer's protocol. The apoptosis rate was calculated as the sum of early and late apoptosis.

GraphPad Prism (version 5.0; GraphPad Software, Inc.) and SPSS (version 17.0; SPSS, Inc.) were used for statistical analysis. Differences between paired samples were analyzed using a paired Student's t-test. Independent groups were analyzed using an unpaired Student's t-test. Multiple groups were compared using one-way ANOVA followed by Dunnett's test. The association between the expression level of MCM10 and the clinicopathological factors was explored using the Chi-square test. Univariate and multivariate survival analyses were performed using the Cox proportional hazards regression model. Factors with prognostic significance in the univariate analysis were included in the subsequent multivariate analysis. The diagnostic value of MCM10 was evaluated by receiver operating characteristic (ROC) analysis and area under the curve (AUC). Data are presented as the mean ± SEM of three independent experiments, each performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

Quantification of the MCM10 expression level in 38 paired lung cancer and adjacent normal tissue samples was performed (Fig. 1A). The results revealed a significantly elevated MCM10 mRNA expression level in lung cancer tissues compared with the normal tissues (Fig. 1B). Subsequently, the GSE30219 dataset was selected to further assess the differences in MCM10 expression between cancerous and normal lung tissue samples. The results were consistent with the data derived from patients recruited in the present study (Fig. 1C). The expression analysis of MCM10 in different histological subtypes was carried out using the Oncomine database and GSE19188 dataset. The Oncomine results demonstrated the overexpression of MCM10 in lung tissues across 9 analyses. The median rank of MCM10 was 1,013 among upregulated genes by cluster analysis in lung cancer (Fig. 1D and Table I). MCM10 expression was significantly higher in ADC, SCC and SCLC compared with normal lung tissues (Fig. 1E). The expression level of MCM10 was also significantly upregulated in lung cancer compared with normal samples included in TCGA database (Fig. S1). Moreover, stage IA of lung cancer exhibited a higher MCM10 expression than normal lung tissue (Fig. 1F). In order to evaluate the diagnostic value of MCM10, the GSE30219 dataset was used to perform ROC analysis. The result showed that MCM10 discriminated a lung cancer patient from a healthy individual with a ROC area under the curve of 0.927 (95% CI, 0.0.887-0.966) (Fig. S2).

Furthermore, upon investigation of the MCM10 expression level in 56 pairs of cancer and adjacent normal tissue samples from the lung tissue microarray by IHC, the positive staining of MCM10 was mainly found in the nucleus and partly in the cytoplasm of the lung cancer cells (Fig. 2A). Among the 56 cases of lung cancer, 33 (58.9%) lung cancer tissues showed high expression (IHC index ≥5) of MCM10, whereas the adjacent normal lung/bronchiole tissues had a low level or negative MCM10 expression (IHC index ≤4). In addition, MCM10 expression was upregulated in all histopathological types of lung cancer compared with the adjacent normal tissue (Fig. 2B). These findings suggested an oncogenic potential of MCM10 in the pathogenesis of lung cancer.

GSE31210 dataset was used to investigate whether MCM10 expression was associated with clinical pathological features of lung cancer. An increased expression level of MCM10 was significantly associated with the pathological properties of lung cancer, such as pathological stage, early recurrence and late recurrence, without having a significant association with age and gender (Table II).

The KM plotter database was used to analyze the prognostic potential of MCM10 in lung cancer. The categorization of MCM10 expression (low vs. high) was based on the median expression level. High MCM10 expression level contributed to the shorter OS time of patients with lung cancer [n=1,926; HR, 1.82; 95% CI, 1.6-2.07, log rank P<1×10⁻¹⁶; Fig. 3A]. The current study also analyzed the association between MCM10 expression and the prognosis of patients with ADC and SCC. The results revealed that increased MCM10 expression had a negative effect on the survival of ADC patients [n=720; HR 2.25; 95% CI, 1.76-2.87, log rank P=2.6×10⁻¹¹; Fig. 3B]. However, no difference was observed among SCC patients [n=524, HR 1.05; 95% CI, 0.83-1.33), log rank P=0.69, Fig. 3C]. In addition, analysis of the GSE30219 dataset (n=293), revealed a shorter OS time for patient with high MCM10 expression compared with patients with low MCM10 expression (Fig. 4A). The prognostic value of MCM10 was also evaluated with respect to clinicopathological features of lung cancer patients. A positive association between MCM10 expression and decreased survival probability was observed in lung cancer patients with less malignant characteristics such as age ≤60 years, early T-stage, negative lymph node infiltration and no distant metastasis. The KM analysis indicated a longer median OS for patients with reduced MCM10 expression in the stratified subgroups (all P≤0.0149; Fig. 4B-E).

In order to investigate the role of MCM10 expression as an independent predictor of a shorter OS and recurrence-free survival in early stages of lung cancer, the GSE31210 dataset was used for prognosis analysis. This dataset contained 204 cases of I-II stage lung cancer; however, 22 cases were excluded due to incomplete resection or adjuvant therapy. Univariate analysis showed that high MCM10 expression (P=0.001) and pathological stage II (P<0.0001) were associated with poor OS in lung cancer patients (Fig. 5A). The incorporation of the significant characteristics into the multivariate analysis revealed that pathological stage (HR, 2.027; 95% CI, 1.291-3.184; P=0.002) and MCM10 expression (HR, 3.251; 95% CI, 1.289-8.199; P=0.012) were independent prognostic factors of OS. Furthermore, the current results also indicated that MCM10 expression was an independent prognostic factor for recurrence-free survival both in univariate analysis (HR, 4.475; 95% CI, 2.353-8.510; P<0.0001) and multivariate analysis (HR, 3.423; 95% CI, 1.768-6.628; P<0.0001; Fig. 5B).

GSEA was performed using the GSE3141 dataset to understand the cellular processes in the pathogenesis of lung cancer mediated by increased expression of MCM10. The samples were divided into two groups (high vs. low) using the median expression level of MCM10, and the top 20 related biological processes that satisfied P<0.05 and false discovery rate <0.25 are shown in Table III. The results suggested that the significant gene set regulated by MCM10 was mainly associated with cell cycle progression and proliferation (Fig. 6A and Table III). To confirm the GSEA analysis of MCM10, MCM10 expression was transiently knocked down in A549 and H661 cells followed by the assessment of the effect of MCM10 expression on cell proliferation using the CCK-8 assay. The results showed that the downregulation of MCM10 significantly suppressed cell proliferation (Fig. 6B and C). Consistent with the findings of the CCK-8 assay, colony formation assay also showed that lung cancer cell growth was significantly inhibited when MCM10 was knocked down (Fig. 6D).

Several previous studies demonstrated the function of MCM10 in DNA replication (9,12,26); however, the effect of MCM10 on cell cycle regulation in lung cancer remains to be elucidated. Flow cytometry analysis was used to determine the effect of MCM10 knockdown on lung cancer cell cycle progression. As shown in Fig. 7A, the depletion of MCM10 significantly increased and decreased the proportion of G₀/G₁ and G₂/M phase cells, respectively compared with the NC group in A549 cells, while knockdown MCM10 only caused G₀/G₁ arrest in H661 cells. An apoptosis assay was performed after 48 h of transfection; however, no significant differences were observed between the two groups (Fig. S3). To explore the mechanism of proliferation inhibition induced by MCM10 knockdown, RT-qPCR was performed to screen a spectrum of genes related to cell cycle regulation between NC and MCM10 knockdown groups. CCND1, a gene responsible for G₁/S transition, was decreased to the greatest extent at the mRNA level following siMCM10 transfection (Fig. 7B and C). To confirm the effect of MCM10 on CCND1 expression at the protein level, proteins were extracted from A549 and H661 cells transfected with siMCM10 or NC. Western blotting results revealed that CCND1 was expressed at a low level in siMCM10 cells compared with the NC group (Fig. 7D).

The potential value of CCND1 expression in predicting the survival of patients with different pathological subtypes of lung cancer was determined in the current study (Fig. S4). The results showed that high CCND1 expression only predicted a worse outcome in patients with stage I-II and negative regional lymph node infiltration compared with low CCND1 expression. Due to the aforementioned association between MCM10 and CCND1, the current study explored the combination of MCM10 and CCND1 in the prognosis prediction of patients with lung cancer. Patients were divided into an MCM10 and CCND1 low expression group and an MCM10 and CCND1 high expression group to obtain KM plots based on the GSE30219 dataset. Combination of low MCM10 and CCND1 expression was associated with a longer median OS (Fig. 8A). In addition, low MCM10 and CCND1 expression was associated with better outcomes compared with high MCM10 and CCND1 expression in patients with less aggressive characteristics such as age ≤60 years, early T-phase, negative lymph node infiltration and no distant metastasis (Fig. 8B-E), demonstrating that the combination of MCM10 and CCND1 expression may be a prognostic indicator for early-stage lung cancer patients.