The High Expression of Minichromosome Maintenance Complex Component 5 Is an Adverse Prognostic Factor in Lung Adenocarcinoma

Background Minichromosome maintenance (MCM) genes are crucial for genomic DNA replication and are important biomarkers in tumor biology. In this study, we aimed to identify the diagnostic, therapeutic, and prognostic value of the MCM2–10 genes in patients with lung cancer. Methods We examined the expression levels, gene networks, and protein networks of lung cancer using data from the ONCOMINE, GeneMANIA, and STRING databases. We conducted a functional enrichment analysis of MCM2–10 using the clusterProfiler package using TCGA data. The correlation between the MCM2–10 expression and lung cancer prognosis was evaluated using Cox regression analysis. The influence of clinical variables on overall survival (OS) was evaluated using univariate and multivariate analyses. The TIMER database was used to evaluate the correlation between tumor infiltrating levels and lung cancer. Kaplan–Meier Plotter pan-cancer RNA sequencing was used to estimate the correlation between the MCM5 expression and OS in different immune cell subgroups in patients with lung adenocarcinoma (LUAD). Finally, the 1-, 3-, and 5-year predictions of LUAD were performed using nomogram and calibration analysis. Results The expression of MCM2, 3, 4, 5, 6, 7, 8, and 10 in lung cancer was higher than that for normal samples. The MCM5 expression was associated with poor OS in patients with LUAD, and prognosis was related to TNM stage, smoking status, and pathological stage. The MCM5 expression is correlated with immune invasion in LUAD and may affect prognosis due to immune infiltration. Conclusion MCM5 may serve as a molecular biomarker for LUAD prognosis.


Introduction
Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide [1]. Numerous studies have evaluated therapeutic approaches for reducing mortality rates in patients with lung adenocarcinoma (LUAD) [2]; however, the 5-year survival rate of patients with lung cancer from 2009 to 2015 was only 19% [3]. Further studies are required to identify accurate and promising prognostic biomarkers and efficient therapeutic targets to enhance survival rates in patients with lung cancer and to guide customized treatments [4].
The minichromosome maintenance (MCM) gene family plays key roles in DNA replication and cell cycle progression [5]. DNA replication errors can lead to tumorigenesis [6]. MCM family proteins are involved in the occurrence and development of cancer [7]. Indeed, several studies have shown that MCM proteins are highly expressed in various cancers, including pancreatic ductal adenocarcinoma [8], hepatocellular carcinoma [9], and colorectal cancer [10] and can be used as molecular markers for diagnosis and prognosis. Teresita et al. suggested that the progression of precancerous lung disease to carcinoma in situ is enhanced in MCM2-overexpressing cells [11]. MCM3 is involved in the carcinogenesis of multiple cancers [12] and is associated with the development of LUAD [13]. Yi et al. identified MCM4 as a potential lung cancer driver gene and demonstrated that MCM4 upregulation is associated with poorer survival in patients with lung cancer [14]. MCM6 levels are higher in primary lung tumors with both FHIT and p53 inactivation [15]. MCM7 is involved in tumor formation, progression, malignant transformation, and prognosis [16] and can be used as a potential biomarker for the poor prognosis of non-small-cell lung cancer [17]. MCM9 is an outlier within the MCM family, containing a long C-terminal extension comprising 42% of the total length, but with no known functional components and high predicted disorder [18]. MCM10 acts as an oncogene that promotes the progression of hepatocellular carcinoma [19]. However, a correlation between the MCM2-10 gene expression and immune infiltration in lung cancer has rarely been reported.
Accordingly, in this study, bioinformatic methods were used to analyze online public databases to assess the expression of MCM2-10 genes in patients with lung cancer and the relationship between this expression and tumor prognosis. Our findings may contribute to the screening, diagnosis, treatment, and prognosis of patients with lung cancer.

The Overexpression of Different MCM2-10 Genes in
Lung Cancer. The transcriptional expression of MCM2-10 genes in lung cancer and normal samples was investigated using the ONCOMINE database (http://www.oncomine .org/); Figure 1, Table 1 Figure 2).

Functional Enrichment of MCM2-10 in Patients with
Lung Cancer. Gene-gene interaction (Figure 3(a)) and protein-protein networks (Figure 3(b)) of MCM2-10 were constructed. The functional enrichment of 30 molecules obtained from the protein-protein network was predicted using the clusterProfiler package. GO terms were analyzed according to BP, MF, and CC (Figure 3(c) and Supplemental Table 1). The BP associated with MCM2-10 included DNAdependent DNA replication, DNA replication, and DNA replication initiation. The MF were associated with DNA replication origin binding, DNA helicase activity, 3′-5′-DNA helicase activity, catalytic activity, acting on DNA, and helicase activity. The CC were associated with MCM complex, nuclear chromosome, telomeric region, chromosome, telomeric region, chromosomal region, and nuclear replication fork. In the KEGG analysis, five pathways were associated with MCM2-10, and the cell cycle pathway accounted for the highest proportion. The cBioPortal online tool was then used to evaluate the frequency of MCM2-10 alteration in lung cancer. In total, 1144 samples from TCGA were analyzed, and the percentage of genetic alterations in MCM2-10 for lung cancer varied from 1.1% to 5% (Figure 3(d)).

Clinical
Value of MCM5 in Lung Cancer. We explored the prognostic value of MCM genes in the OS of patients with lung cancer. The mRNA expression of MCM5 (p = 0:008) was closely linked to worse OS in patients with lung cancer (Figure 4(d), Table 2). MCM5 was highly expressed in patients with lung cancer and was closely related to TNM stage, pathological stage, sex, age, smoking status, primary therapy outcomes, and OS, PFI, and DSS events ( Figure 5 and Supplemental Table 2). Furthermore,    7 BioMed Research International we explored the correlation between MCM5 expression and clinicopathological parameters on OS in patients with lung cancer, and poor OS was associated with LUAD, TNM stage, smoking status, and pathological stage (Figures 6(a) and 6(c)-6(g)).

Correlation between the MCM5 Expression and Immune
Infiltration Level. The TIMER online tool was used to investigate the correlation between the MCM5 expression and immune cell infiltration in lung cancer. The somatic copy number alteration module showed that the arm-level gain of MCM5 was significantly associated with immune cell infiltration levels in LUAD and LUSC (Figure 7(a)). MCM5 was positively correlated with the infiltration levels of Th2, NK CD56dim, Tgd, and Treg cells in lung cancer (Figure 7(b)). The MCM5 expression was positively correlated with the infiltration levels of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and DCs in LUAD (Figure 7(c)). MCM5 was positively correlated with infiltration levels of CD4+ T cells and DCs in LUSC (Figure 7(c)).

Prognostic Analysis of the MCM5 Expression Based on Immune Cells in LUAD Patients and Prognostic Predictive
Model. The high MCM5 expression was closely related to LUAD prognosis and immune cell infiltration. We further explored whether the high MCM5 expression affected the prognosis because of immune infiltration. The Kaplan-Meier Plotter pan-cancer RNA-seq LUAD (n = 513) data were analyzed for the prognosis of enriched and decreased immune cells. (Figure 8(a)). These findings reveal that MCM5 may affect the prognosis of patients with LUAD, in part due to immune infiltration. Finally, we used nomogram and calibration analysis to predict the 1-, 3-, and 5-year OS of patients with LUAD using clinically related factors such as age, smoking status, pathological stage, and primary therapy outcome (Figures 8(b) and 8(c)).

Discussion
Recent studies have suggested that dysregulation of MCMs leads to tumor initiation, progression, and chemoresistance via modulation of the cell cycle and DNA replication stress

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BioMed Research International [41]. The MCM protein plays a key role in the proliferation and prognosis of lung cancer [16]. Bioinformatic analysis was used to detect mRNA expression, prognostic value, genetic mutations, functional enrichment, protein-protein network, and immune infiltration of MCMs in patients with lung cancer.
MCM2 plays a role in the proliferation, circulation, and migration of lung cancer cells [42]. MCM3 regulates cell proliferation by binding to cyclin D1 [43]. Mutations in MCM4 disrupt the functions of MCM2-7, resulting in genomic instability and cancer progression [44]. MCM5 is an important DNA replication initiation factor and is strongly downregulated following the overexpression of the long noncoding RNA CARMN [45]. MCM6, MCM7, and MCM8 collaborate with other MCM family members to promote cancer cell proliferation through cell cycle and DNA replication [46,47]. The MCM9 protein is involved in the unwinding activity [48]. MCM10 mediates DNA replication by collaborating with other cell-dividing cyclins [49]. The BP and associated pathways of MCM2-10 were elucidated by GO and KEGG enrichment analyses, which are useful for investigating the pathological mechanisms of lung cancer. The BP associated with MCM2-10 includes DNA-dependent DNA replication, DNA replication, DNA replication initiation, G1/S transition of mitotic cell cycle, and cell cycle G1/S phase transition. The cell cycle and DNA replication pathways are associated with MCM2-10. MCM2-8 and MCM10 were highly expressed in paired lung cancer samples and may be involved in the development of lung cancer through the cell cycle and DNA replication.
Correlation analysis between the MCM2-10 expression and OS revealed that only MCM5 was closely related to poor OS in patients with lung cancer. The MCM5 gene affects the prognosis of LUAD by regulating BP and pathways, such as cell cycle and DNA replication [50]. In this study, MCM5 was highly expressed in tumors, which is related to TNM stage, pathological stage, sex, age, smoking status, prognostic events, and primary therapy outcomes. MCM5 was positively correlated with poor OS in patients with LUAD and was influenced by TNM stage, smoking status, age, and pathological stage. These results suggest that MCM5 is involved in the development of lung cancer, may be used as a molecular target for diagnosis and treatment, and is an independent prognostic marker of lung cancer. Furthermore, we revealed that the armlevel gain of MCM5 was significantly associated with immune cell infiltration levels in lung cancer. MCM5 positively correlated with B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and DCs in LUAD. Enriched type 1 T-helper cells, decreased macrophages, and type 2 T-helper cells showed no significant correlation between the MCM5 expression and OS in patients with LUAD, whereas decreased type 1 T-helper cells, enriched macrophages, and type 2 T helper cells were related to the OS of patients with LUAD. MCM5 may partially influence the OS of patients with LUAD by immune cell infiltration. However, the exact role of MCM5 in the tumor immune microenvironment requires further investigation. Furthermore, we revealed that the arm-level gain of MCM5 was significantly associated with immune cell infiltration levels in lung cancer. MCM5 positively correlated with the infiltration levels of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, DCs, and partially influenced the OS of patients with LUAD by immune cell infiltration. Thus, MCM5 may serve as a molecular biomarker for immunotherapy. However, the exact role of MCM5 in the tumor immune microenvironment requires further exploration.
Our study had certain limitations. The data were collected online from open databases. In future studies, large clinical datasets are required to verify our findings, and the      14 BioMed Research International role of MCM2-10 in the pathogenesis of lung cancer should be further explored.

Conclusions
Our findings demonstrated that MCM2-8 and 10 were highly expressed in lung cancer, and only MCM5 affected the prognosis of patients with lung cancer. The influence of MCM5 on the prognosis of lung cancer patients was related to LUAD, smoking, pathologic stage, and TNM stages. We further confirmed that the abnormal expression of MCM5 in LUAD was related to immune cell infiltration, and immune cell infiltration may contribute to the prognosis of LUAD partly. The above findings suggested that MCM5 can be used as a molecular marker for the prognosis of LUAD.

Data Availability
The data in this paper were mined from online public databases.

Conflicts of Interest
The authors claim that the research was conducted without any commercial or financial relationships that could be interpreted as potential conflicts of interest.