Identification of mini‐chromosome maintenance 8 as a potential prognostic marker and its effects on proliferation and apoptosis in gastric cancer

Abstract Mini‐chromosome maintenance (MCM) proteins play important roles in initiating eukaryotic genome replication. The MCM family of proteins includes several members associated with the development and progression of certain cancers. We performed online data mining to assess the expression of MCMs in gastric cancer (GC) and the correlation between their expression and survival in patients with GC. Notably, MCM8 expression was undoubtedly up‐regulated in GC, and higher expression correlated with shorter overall survival (OS) and progression‐free survival (PFS) in patients with GC. However, the role of MCM8 in GC has not been previously explored. Our in vitro experiments revealed that MCM8 knockdown inhibited cell growth and metastasis. Moreover, MCM8 knockdown induced apoptosis. Mechanistically, the expression levels of Bax and cleaved caspase‐3 were increased, whereas Bcl‐2 expression decreased. Additionally, we demonstrated that MCM8 knockdown suppressed tumorigenesis in vivo. Overall, these results suggest that MCM8 plays a significant role in GC progression.

helicase activity in vitro. 9,10 Subsequently, MCM8 and MCM9 were discovered, similar to other members of the MCM2, MCM3, MCM4, MCM5, MCM6, MCM7 proteins 3 and are crucial for DNA pre-replication and initiation of the S phase. 11,12 In addition, they facilitate homologous recombination repair as a heterohexameric MCM8 and MCM9 complex at DNA damage sites. 11,13,14 MCM10 is an additional essential protein for the initiation of DNA synthesis. 15,16 Recently, increasing evidence has suggested that MCMs are up-regulated in multiple malignancies including cervical cancer, 17 breast cancer, 18 oesophageal squamous cell cancer, 19 chronic myelogenous leukaemia, 20 human gliomas and non-small-cell lung cancer. [21][22][23][24] However, there is less evidence demonstrating the relationship between MCM family proteins and GC. Therefore, we assessed the mRNA expression of MCMs in GC when compared with normal adjacent parental tissues by GEPIA (Gene Expression Profiling Interactive Analysis). Additionally, we analysed the relationship between MCM expression and the progression and prognosis of GC using the Kaplan-Meier plotter analysis. We observed that MCM2, MCM5 and MCM8 expression was up-regulated in GC samples when compared with adjacent normal parental samples and correlated with a poor prognosis.
Meanwhile, there have been several articles reveal that MCM2 and MCM5 may serve as prognostic indicators of patients with gastric cancer, but there is no such article on MCM8. Moreover, there is no in vitro or in vivo study on the function of MCM8 in gastric cancer. [25][26][27] We performed further analyses on MCM8. We detected the mRNA expression of MCM8 and its association with overall survival (OS) and progression-free survival (PFS) in different GC databases. Furthermore, functional assays indicated that MCM8 knockdown significantly inhibited cell growth and metastasis, but induced cell apoptosis. Intrinsic and extrinsic pathways are two major apoptotic pathways. The intrinsic pathway, also called the mitochondrial pathway, is attributed to the essential involvement of mitochondria. 28 The intrinsic pathway of apoptosis is regulated by a family of proteins called the Bcl-2 family. Some of these proteins (such as Bad, Bax or Bid) are pro-apoptotic, while others (such as Bcl-2 and Bcl-XL) are anti-apoptotic. The balance between pro-and anti-apoptotic Bcl-2 proteins determines the sensitivity of cells to apoptotic stimuli. 29 Cleaved caspase-3 is an important indicator of apoptosis. 28 Thus, we further determined the expression levels of Bcl-2, Bax and cleaved caspase-3 to uncover the underlying mechanism. In addition, our study showed that MCM8 knockdown suppressed the development and progression of cancer in vivo. Therefore, MCM8 may be a potential target for GC treatment.

| Bioinformatics and survival analysis
MCM mRNA expression in GC cancers vs normal tissues was analysed using GEPIA (http://gepia.cancer-pku.cn/) and Oncomine (http://www.oncom ine.org). For the survival analysis, Kaplan-Meier plotter (http://kmplot.com/analy sis/)30 was used to assess the OS and PFS in patients. Based on the median MCM8 expression level, patients with GC were classified into low and high expression groups, analysing the relationship using the log-rank test.

| Plasmid construction and lentiviral transduction
Three independent shRNAs targeting MCM8 and a control shRNA were designed by Shanghai HanBio Company (Shanghai, China). The shRNAs were cloned into the lentivirus-based vector pHBLV-U6-MCS-PGK-PURO. The targeting sequence of the shRNAs and primers for plasmid construction is presented in Table S1. The lentiviruses carrying MCM8 shRNA or control shRNA were purchased from Shanghai HanBio Biotechnology. Briefly, cells were seeded in 24-well plates and infected with shRNA or sh-Ctrl lentivirus at a multiplicity of infection

| Quantitative reverse transcription polymerase chain reaction (RT-qPCR)
Total RNA was extracted from cells using RNAiso Plus reagent (Takara; Dalian, Liaoning, China) and reverse-transcribed (oligo) into cDNA with the PrimeScript RT kit (Takara). Next, gene expression was detected as mRNA levels by qPCR with the ABI 7500 Fast Real- After incubation with the chemiluminescence detection reagent, the bands were visualized and analysed with the ImageLab software. The protein level of β-actin was used as a loading control.

| Colony formation assay
In brief, transduced cells were reseeded into 6-well plates (1000 cells/well) and incubated for 10-12 days at 37°C in a humidified atmosphere containing 5% CO 2 . RPMI-1640 medium was replaced every 2-3 days; then, colonies were washed with PBS, fixed them with 4% formaldehyde and then stained with crystal violet. Finally, the total number of colonies was counted, and images were obtained.

| Cell cycle analysis
FxCycle PI/RNase Staining Solution was applied to transfected cells at
After staining, the samples were immediately analysed using a flow cytometer (FACS Caliber, Becton-Dickinson).

| Tumour xenograft
Six-to-eight-week-old female nude mice were purchased from Charles River (Beijing, China). HGC27 cells (4 × 10 6 ), with or without MCM8 knockdown, were subcutaneously injected into the rightside dorsal flank of each mouse. Tumours were isolated on day 38, with the length (a) and width (b) of tumours recorded every 4 days.
The tumour volume was calculated using the following formula, V = ab 2 /2 (cm 3 ). Additionally, the tumours were imaged on day 38 after injection.

| Statistical analysis
Data were analysed using SPSS 20.0 (IBM Corp., Armonk, NY, USA). The qPCR results were evaluated with one-way ANOVA, and other results were analysed using Student's t test, presenting the means ± standard deviation (SD) obtained from three independent experiments. A P-value of <.05 was considered statistically significant.

| mRNA expression of MCMs in GC samples
The mRNA expression levels of MCMs in GC and normal tissues were compared based on data from GEPIA. As shown in Figure 1, and MCM9 did not significantly differ between GC and normal tissues.

| Relationship between MCM expression and prognosis in patients with GC
In patients with GC patients, the association of MCMs with OS and PFS was analysed by the Kaplan-Meier method. As shown in as a prognostic biomarker. However, no significant difference was observed between the low and high expression groups for PFS (logrank P = .082) ( Figure 3F).

| MCM8 knockdown inhibits the growth of GC cells
Three independent shRNAs targeting MCM8 were designed, and knockdown efficiencies were determined using RT-qPCR.
We selected sh-MCM8-1, presenting the highest efficiency  Figure 4G). The colony number reduced from 60 ± 5 to 30 ± 3 (P = .0014) and from 30 ± 3 to 14 ± 1 (P = .0014) in AGS and HGC27 cells, respectively ( Figure 4H). To further elucidate the possible mechanism underlying cellular growth inhibition induced by MCM8 knockdown, the cell cycle assay was evaluated by flow cytometry. As shown in Figure 4I, MCM8 knockdown significantly increased the percentage of cells in the G2/M phase. The percentages were increased from 8.9 ± 0.2% to 10.8 ± 0.4% (P = .0022) in AGS cells and from 13.0 ± 1.0% to 18.7 ± 1.6% (P = .0077) in HGC27 cells ( Figure 4J). These results suggested that MCM8 knockdown may inhibit proliferation by inducing G2/M phase arrest.

F I G U R E 2
The relationship between MCM expression and poor prognosis in patients with GC. Survival analysis was based on MCM expression in patients with GC from the public clinical microarray data set using the Kaplan-Meier plotter analysis (*P < .05). Survival was analysed using a log-rank test. MCM, mini-chromosome maintenance; GC, gastric cancer

| MCM8 knockdown induces apoptosis in GC cells
The effect of MCM8 on cell apoptosis was investigated. Annexin V-FITC and 7-AAD were used to stain apoptotic cells. As shown in Figure 5A,B, MCM8 knockdown significantly increased the percentage of apoptotic cells, from 7.5 ± 0.5% to 16.2 ± 2.2% for AGS cells and from 10.2 ± 1.5% to 18.2 ± 1.7% for HGC27.

| MCM8 knockdown suppresses cell migration and invasion
To determine whether MCM8 affects cell metastasis, Transwell migration and Matrigel invasion assays were performed. As shown in Figure 6A, MCM knockdown reduced the number of migrated cells. The number of migrated cells was reduced from 628 ± 15 to 372 ± 40 (P < .0001) and from 389 ± 17 to 199 ± 10 (P < .0001) for AGS and HGC27, respectively ( Figure 6B). Consistent with the results of the Transwell migration assay, the results of the Matrigel invasion assay showed that MCM8 knockdown significantly reduced the number of invasive cells ( Figure 6C,D). These findings indicated that MCM8 promotes cell metastasis.

| MCM8 knockdown suppresses cell growth in a mouse xenograft model
To study the effect of MCM8 on cancer progression in vivo, HGC27 cells, with or without stable MCM8 knockdown, were subcutaneously injected into nude mice. Our results showed that MCM8 knockdown suppressed tumorigenesis ( Figure 7A). The volume and weight of tumours were significantly reduced in the MCM8knockdown group when compared with those in the control group ( Figure 7B,C).

| D ISCUSS I ON
In this study, we observed that several MCM proteins were upregulated in GC samples when compared with normal tissue samples according to data obtained from online databases. We GAPDH was used as a reference. *P < .05, ***P < .001, ***P < .0001. D, Protein levels of Bcl-2, Bax and cleaved caspase-3 were determined using Western blotting. β-actin was used as the loading control. E, Semi-quantification of Western blotting. The integrated band density was determined using ImageLab Software, and β-actin was used as the reference. **P < .01, ***P < .001. RT-qPCR, quantitative reverse transcription polymerase chain reaction the balance of proliferation/apoptosis could be attributed to MCM8 overexpression in GC development. However, the precise underlying mechanism requires further investigation.
In summary, we observed MCM8 overexpression in GC tissues and demonstrated a correlation between MCM8 up-regulation and poor patient survival. MCM8 knockdown exerted anti-tumour activity both in vitro and in vivo. These findings indicate the biological function of MCM8 in GC and suggest that MCM8 could be used as a potential biomarker for this cancer.

ACK N OWLED G EM ENTS
This work was supported by grants of the Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.