Colon cancer was the fourth most commonly diagnosed malignant tumour worldwide in 2018, with increasing incidence in countries undergoing major developmental transition(30). Due to a lack of specific symptoms for early detection, patients are usually diagnosed at an advanced stage which leads to a poor prognosis(31). Therefore, it is crucial to uncover the underlying molecular mechanism and to explore key biomarkers for early colon cancer diagnosis.
In this study, we analysed three microarray datasets that included 127 tumours and 117 normal samples. A total of 436 DEGs were screened. Functional annotation showed that the DEGs were mainly enriched in biological processes associated with cell cycle phase transition, nuclear division, positive regulation of transferase activity, meiotic nuclear division, and DNA replication. These results suggest that these genes are closely related to the cell cycle. Many studies indicate that dysregulation of cell cycle progression is closely related to cancer progression(32, 33). Finetti et al. (34) found that several genes participate in regulating the cell cycle, like CDK1 and AURKA. Moreover, their expression was correlated with breast cancer prognosis. In our colon cancer study, we obtained many DEGs involved in cell cycle progression, including CCND1, BLM, BUB1, BUB1B, CCNA2, CCNB1, CDK1, and CDC20. Some genes are closely related to the transformation of cancer. For example, CCND1 belongs to the cyclin family whose members are characterised by dramatic periodicity in protein abundance throughout the cell cycle. Deregulation of CCND1 is observed frequently in numerous human cancers, including pancreatic cancer, head and neck squamous cell carcinoma, breast cancer, and colorectal carcinoma(35, 36). Accumulation of CCND1 in the nucleus causes uncontrolled cell cycle progression and acts as a tumour-initiating event.(37) Overexpression of cyclin D1 (T286A), an oncogenic mutant allele of CCND1, promotes stabilisation and overexpression of the DNA replication licensing factor, Cdt1, by inhibiting its proteolysis. This causes DNA re-replication and damage, and results in cellular aneuploidy, genomic instability, and further neoplastic growth(38). Cyclin dependent kinases (CDKs) are necessary functional partner kinases with cyclin D1. Thus, CDK inhibitors would be an effective drug for targeting malignant tumours(39). However, given the development of resistance and side effects of CDK inhibitors, further research is warranted(36).
Pathway analysis also revealed that DEGs were mainly enriched for terms associated with the cell cycle pathway. Cyclin A/B1/B2-associated events in the “G2/M transition” and “Regulation of TP53 Activity through Phosphorylation” pathways were closely related to tumorigenesis. Like the cyclin D1 mentioned above, cyclins A/B1/B2 are also cyclin members that bind to CDKs and regulate the cell cycle. Abundant evidence shows that G2/M phase arrest is closely related to the inhibition of tumour cell proliferation(40, 41). Additional studies focusing on cyclins aimed to identify novel therapeutic strategies for cancer treatment. Qiang(42) revealed that the microRNA MiR-219-5p downregulates CCNA2 expression and induces G2/M phase arrest to inhibit tumour formation in oesophageal cancer. Xuezi et al.(43) found CCNA2 is downregulated by the small molecule FH535 in colorectal cancer, which causes G2/M phase arrest and inhibits tumour proliferation. Downregulating CCNA2 and CCNB1 is a key mechanism for the anti-cancer activity of gossypin,a flavone extracted from Hibiscus vitifolius(44). Thus, inhibiting CCNA2 and CCNB1 may contribute to the development of novel anti-cancer drugs. The p53 signalling pathway significantly contributes to cell cycle regulation, suppression of tumour expression, metabolism, aging, development, and reproduction(45). Phosphorylation of p53 protein stabilizes the protein and extends its half-life, thus causing cell cycle arrest, apoptosis, and inhibited tumour cell proliferation(46). For instance, a study of natural polyphenols as anti-cancer agents revealed that polyphenols could induce apoptosis, which was achieved by stabilizing p53 protein through phosphorylation and showed remarkable effects in human gastric carcinoma cells(47). We also identified some pathways associated with metabolism, including triglyceride metabolism, carnitine metabolism, regulation of lipolysis in adipocytes, and Phase I - Functionalization of compounds. Among these pathways, we found that FABP4, which encodes fatty acid binding protein, was involved in fatty acid uptake, transport, metabolism, and is related to tumour metastasis. Kshipra et al.(48) observed that overexpression of FABP4 plays a key role in aggressive metastasis of ovarian cancer via various metabolites and protein pathways. Likewise, FABP4 has crucial effects on adipocyte-induced cholangiocarcinoma metastasis(49). Collectively, metabolic disorder is among the leading causes of tumour development. Thus, the study of tumour metabolism may provide new targets for tumour treatment.
The PPI network was built using STRING. Twenty hub genes were screened, which include CDK1, CCNB1, CCNA2, AURKA, CDC20, AURKB, TPX2, BUB1, CDC45, MAD2L1, KIF2C, NCAPG, DLGAP5, FOXM1, CENPF, CENPE, BUB1B, TTK, ASPM, and KIF20A. The functional annotation of these genes was most closely related to the cell cycle.
Survival analysis showed that higher mRNA expression of six hub genes was significantly related to longer OS in colon cancer patients, including CCNB1, CCNA2, AURKA, NCAPG, DLGAP5, and CENPE. Moreover, AURKA and CENPE exhibited favourable effects on both OS and DFS. Thus, the 6 hub genes showed clinical prognostic value and might provide potential therapeutic targets for colon cancer.
For early COAD diagnosis, we identified CDK1, CCNB1, CCNA2, MAD2L1, and DLGAP5, which were closely related to clinicopathological parameters. Our results showed that both the mRNA and protein expression of these five hub genes are higher in tumour tissue than in normal tissue, which indicates that the hub genes may be closely related to COAD progression. Previous studies observed that the expression of these genes was correlated with tumour size and stage(50–52). In our study, we found that mRNA expression of the five hub genes was significantly related to mild clinical pathological parameters, so these genes may play an important role in the early diagnosis of colon cancer. The relationship between these genes and COAD has not yet been fully determined, but our data indicate that the increased expression in early COAD stages may provide an indicator for early diagnosis.
We finally performed alteration analysis of eight hub genes which showed significant effects on survival analysis, including CDK1, CCNB1, CCNA2, AURKA, MAD2L1, NCAPG, DLGAP5, and CENPE. These result show that more than 40% of the patient tumours analysed had at least one hub gene alteration. AURKA was the most frequently altered (28%) of the 8 hub genes. The protein encoded by this gene is a cell cycle-regulated kinase that appears to be involved in spindle assembly, cytokinesis, centrosome maturation, and separation(53). Previous studies showed that AURKA is frequently upregulated and correlated with prognosis in several types of cancers, which may reveal an important role in human cancer(54, 55).
There were some limitations in this study. First, all the data analysed in our study was retrieved from online databases. Thus, further studies with larger sample sizes and biological experiments are required to validate our findings. Our future research will focus on experimental verification of these results. Second, we did not explore the underlying mechanisms of hub genes in COAD. Future studies should investigate the detailed mechanism between hub genes and COAD.