The occurrence and development of tumors is a complex process involving multiple genes and metabolic pathways. The self-renewal ability of cancer stem cells is considered to be the main reason promoting cancer progression and resistance to drug therapy[26]. Numerous patients with hepatitis B exist in China, where liver cancer is a common malignant tumor. Due to discrete early clinical symptoms, lack of sensitive and specific markers, and limited diagnostic methods patients are often treated during the middle and late stages of disease. The survival rate has not improved much in recent years [27, 28]. Therefore, determining the key molecules causing liver cancer, exploring its internal mechanisms and finding potential therapeutic targets are future research directions. The rise of high-throughput sequencing technology has made the search for tumor markers very convenient. This technique can measure the expression level of thousands of genes simultaneously, which is a powerful tool to study the gene expression profiles between cancer and normal samples. Furthermore, this method provides a theoretical basis for our experimental follow-up cell study[29].
Since research pertaining to the key molecules and pathogenesis of liver cancer is not very clear, three microarray datasets were downloaded from GEO, which encompassed 158 pairs of samples. GSE64041 and GSE54236 were used to identify the differentially expressed genes. After they were identified, the function and pathways of these DEGs were studied.
Accordingly, 317 DEGs were identified in GSE64041, where 87 differentially expressed genes were found to be upregulated while 230 differentially expressed genes were found to be downregulated. The GO analysis showed that the differentially expressed genes were mainly enriched in the oxidation-reduction process,mitotic nuclear division,tryptophan catabolic process to kynurenine,cellular response to tumor necrosis factor,tryptophan catabolic process,monooxygenase activity,iron ion binding,heme binding,oxidoreductase activity, acting on paired donors,and oxygen binding. In the KEGG pathway analysis, these DEGs were mainly observed to be related to retinol metabolism, oocyte meiosis, metabolic pathways, cell cycle, PI3K-and Akt signaling pathway. However, 342 DEGs were identified in GSE54236, in which the number of differentially expressed genes that were upregulated and downregulated were 146 and 196, respectively. The GO functional enrichment analysis demonstrated that the differentially expressed genes were mainly enriched in mitotic nuclear division, cell division, sister chromatid cohesion, chromosome segregation, G2/M transition of mitotic cell cycle,microtubule binding, ATP-dependent microtubule motor activity, microtubule motor activity, protein kinase binding, and protein binding. According to the KEGG analysis, the differentially expressed genes were mainly involved in the cell cycle, oocyte meiosis, progesterone-mediated oocyte maturation, p53 signaling pathway, and histidine metabolism. Tumors are the result of unregulated cell division and proliferation, a process that consumes much energy. Many different types of tumors, including breast cancer, kidney cancer, lung cancer, prostate cancer and colorectal cancer, have differences in metabolism[30-33]. The process of glucose metabolism in tumor cells is significantly different from that in normal cells[34, 35]. Studies have pointed out that in tumor cells, the mitochondria that produce energy are damaged, hence, most tumor cells use glucose and amino acids as substrates. Moreover, under aerobic conditions, energy is obtained only through anaerobic glycolysis outside the mitochondria, termed the "Warburg" effect[36, 37]. This type of metabolism is inefficient and consumes more energy while producing the same amount of ATP as aerobic oxidation, resulting in loss of weight in patients with advanced cancers. Monooxygenase is also called liver microsomal enzyme in the liver. The main component of monooxygenase is cytochrome P450 (CYPs), which is mainly involved in detoxification and drug metabolism (including retinol metabolism). Studies have pointed out that CYP450 family genes are significantly associated with liver disease and liver cancer. For example, the downregulation of CYP2A6 and CYP2C8 is related to the overall survival and recurrence rates of liver cancer[38]. Additionally, the high expression of CYP4A11 is associated with a better prognosis in patients with liver cancer[39], and CYP activity is affected in varying degrees in patients with liver fibrosis or cirrhosis. The PI3K-Akt pathway is a common pathway in cells[40],which is considered to play a role in accelerating the cell cycle, promoting cell proliferation and inhibiting cell apoptosis. Currently, certain targeted therapeutic drugs designed for this pathway have made some progress[41, 42],and inhibitors targeting PI3K and AKT have also entered clinical trials [43]. Chromosome separation and sister chromatid binding are important processes in cell division. Chih-JuiChang et al. found that DNA topoisomerase Ⅱ (TOPO Ⅱ) is essential for the later stages of sister chromatid separation[44]. TP53 encodes the p53 protein, which is an important regulatory factor of the cell cycle, that induces cell cycle arrest and regulates cell apoptosis[45]. The inactivation of the p53 protein is a late event of liver cancer, increasing the malignant degree of liver cancer and contributing to resistance to treatment[46]. Another study suggested that the TP53 pathway is related to immunity. In this regard, TP53 mutation can promote the expression of PD-L1 and increase the infiltration of T lymphocytes in lung adenocarcinoma[47].
In order to determine the final hub gene, the third microarray dataset was used to overlap the differentially expressed genes of the three microarray datasets, resulting in 43 hub genes. By establishing the PPI network and using Cytoscape plugins, four hub genes were finally identified, of which two were found to be related to prognosis: ECT2 and FCN3.
ECT2 is epithelial transformation sequence 2[48] and a guanosine nucleotide exchange factor (GEFs), which catalyzes the transformation between GDT and GTP, activating Rho enzyme[49],and regulating cell division[50]. Many studies have found that ECT2 is abnormally expressed in many tumors. For example, Zhang et al found that ECT2 is overexpressed in pancreatic cancer and is related to methylation [51]. Moreover, Sano et al. confirmed the high expression of ECT2 in gliomas, which predicted poor prognosis [52]. Xu et al also confirmed that ECT2 and miR-223 form an axis of action and regulate osteosarcoma development [53]. An increasing number of studies have found that ECT2 regulates tumor progression through a variety of ways, plays a carcinogenic role in mistakenly activating Rho [54, 55], overactivates the ras/mapk pathway, which leads to tumor formation[56],and promotes tumor cell invasion by regulating the EMT process [57]. The mechanism of ECT2 in liver cancer has also been studied, which is consistent with the results of the present study in regard to ECT2. Chen et al believed that the expression of ECT2 is upregulated in liver cancer, and ECT2 can promote the expression of the related gene RACGAP1, which mediates the activation of Rho enzyme, leading to the early recurrence of liver cancer [58].
FCN3 is a member of the FCN gene family, which varies greatly between races [59]. The results of this study demonstrated that the expression of FCN3 was found to be decreased in liver cancer, where FCN3 was mainly observed to be involved in complement activation, consistent with the results of other studies. Accordingly, it is suggested that FCN3 is highly expressed in normal liver tissues but stably low in liver cancer. Compared to FCN1 and FCN2, FCN3 has a higher ability with respect to complement activation. [60, 61]. In addition, the expression of FCN3 was found to be closely related to the disease. For example, Chen et al. found that the decrease in serum FCN3 was associated with insulin resistance, while lower serum FCN3 predicted the development of type 2 diabetes [62]. Zheng et al. discovered that the expression of FCN3 increased in the vitreous effusion of patients with proliferative diabetic retinopathy, hence, FCN3 may serve as a new therapeutic target for the treatment of proliferative diabetic retinopathy [63]. Szala et al. found that the expression of FCN3 in patients with ovarian cancer was significantly lower than that in benign ovarian tumors and normal ovarian tissues [64]. Shi et al. put forward that the expression of FCN3 was low in lung squamous cell carcinoma [65]. The mechanism of FCN3 in liver cancer has yet to be reported, which warrants further elucidation.
At present, AFP is still used as a diagnostic marker for liver cancer. Due to the lack of good sensitivity and specificity, missed diagnoses and misdiagnosis may often occur. Although liver cancer markers have repeatedly emerged over time, no unified conclusion currently exists[66-69]. In this study, differentially expressed genes were found from a large dataset (316 samples in total). In order to improve accuracy, the identified genes were verified in TCGA, in which a difference in their expression was observed (Figure.8). Afterward, their function, prognosis and targeted miRNA were analyzed. Overall, this study provided novel insights in understanding the pathogenesis of liver cancer as well as the search for tumor markers. The mechanism of FCN3 in diabetes, ovarian cancer and lung squamous cell carcinoma has previously been studied, but its role in liver cancer is not clear. In the future, we intend to verify the expression of FCN3 in tissues, identify its downstream target genes, and explore the effects of its expression changes on the proliferation and invasion of liver cancer.