CCA, the second primary liver cancer, is usually diagnosed at an unresectable advanced stage. After disease progression, there are few treatment options for gemcitabine and cisplatin first-line chemotherapy, resulting in a poor prognosis[17]. Over the past four decades, the incidence of CCA has been increasing. The increase in CCA mortality was due to the anatomical location and growth pattern of CCA and the lack of clear diagnostic criteria, the diagnosis is difficult[18]. In recent years, studies on CCA have been reported, but its pathogenesis remains unclear and needs further study. Therefore, this study provides a promising target for the treatment of CCA, which is of great importance for its diagnosis, treatment and prognosis.
In the present study, we analyzed the gene expression profiles of 310 CCA samples and 50 normal samples from messenger RNA microarray datasets GSE132305, GSE89749, and GSE45001 in the GEO database. From these 3 datasets, 301 DEGs, 2,436 DEGs, and 3,445 DEGs were respectively identified, as well as a total of 492 mutual DEGs were screened.
GO analysis of abnormally expressed genes showed that upregulated genes were mainly associated with metabolic process, positive regulation of smooth muscle cell proliferation, cell surface receptor signaling pathway and extracellular exosome. Previous studies have shown that exosome mediated factors can promote tumor initiation, metastasis and drug resistance through intercellular communication[19]. Therefore, exosomes can be used as a promising diagnostic source of cancer[20]. The downregulated genes were mostly associated with extracellular matrix organization, cell adhesion, protein binding and structural molecule activity, which may be related to the fast multiplication of cancer cells. According to the results of KEGG analysis, the mutual DEGs were mainly involved in P53 signaling pathway, adipocytokine signaling pathway, chemical carcinogenesis, fat digestion and absorption and drug metabolism-cytochrome P450. The P53 signaling pathway is involved in apoptosis, growth inhibition, inhibition of cell cycle progression and acceleration of DNA repair[21]. Therefore, p53 is mutated in the vast majority of tumor cells and in more than 50% of all malignant tumors. Also, obesity could account for up to 20% of cancer-related deaths. The association is due to the metabolic and inflammatory changes of adipose tissue, which destroy the physiological homeostasis of local tissues and systems[22]. This suggests that obesity may increase the risk of CCA.
In order to obtain the hub genes among the identified DEGs, 492 mutual DEGs were analyzed with the PPI network base on the STRING database. A total of 21 genes were screened with degrees ≥ 20, particularly in ALB, MYC, APOB, IGF1 and KNG1. MYC, a transcription factor, plays an important role in regulating cellular processes, such as, survival, proliferation, metabolism, and signal transduction to control of DNA replication. It is also a oncogene. If MYC is overexpressed due to genetic factors or other mechanisms, it can lead to canceration in normal tissues, which is also the pathogenesis of many tumors, including various solid tumors and lymphoid malignancies[23]. In this study, we mainly explored its relationship with CCA. A previous research suggests that tumors with higher versus lower MYC protein expression should more aggressive, and hence that MYC protein expression should be associated with poorer prognosis[24]. ALB, a biomarker of nutritional and inflammatory conditions, is often used to evaluate the nutritional status of cancer patients. ALB is also associated with the prognosis of many cancers, such as oral cancer, head and neck cancer, and ovarian cancer[25, 26, 27]. Thus, higher serum ALB levels have a significant effect on the prognosis of patients with CCA after hepatectomy[28]. APOB, a key component of fat metabolism, has previously been reported that apolipoprotein levels are associated with overall cancer risk as well as breast, lung and colorectal cancer risk in men.These findings may contribute to future cancer prevention strategies[29]. IGF1, very similar to insulin in structure, is a peptide growth factor that promotes cell proliferation and inhibits apoptosis[30, 31]. Significantly increased levels of IGF1 in the biliary was reported to be related to malignant biliary obstructions, which implied IGF1 may have great effects on treatment of CCA[32]. KNG1, a cysteine proteinase, has identified as a serum biomarker for the early detection of advanced colorectal adenoma and colorectal cancer[33], as well as a potential prognostizc biomarker for oral cancer[34]. Therefore, KNG1 may exerts a enormous function on cancer.
In this study, the genes ALB, MYC, APOB, IGF1 and KNG1 were demonstrated to be involved in CCA, as well as these genes may be used as potential diagnosis biomarkers, treatment targets and prognosis markers for patients with CCA. Among them, we investigated MYC in depth as a potential therapeutic target. Targeted therapies for MYC have been proposed, including the inhibition of MYC transcription, partner protein dimerization, activating post-translational modifications, and turnover[35].Studies on inhibition of MYC transcription have focused on BET inhibitors[36] and complex DNA structures called G-quadruplexes. The dimer interference mainly included MAX Inhibitor MYCMI-6, Ki-MS2-008 and so on. The post-translation regulation of MYC mainly includes the PIN1 proline isomerase, kinases that phosphorylate S62-MYC and enzymes that affect MYC ubiquitin-dependent proteolysis[19]. However, these studies still have drawbacks. For example, although 15 different BET inhibitors are under clinical evaluation, clinical responses are limited, often leading to relapse, and inconsistent with their effect on MYC expression[37, 38].
In this study, we used LibDock, ADME/TOPKAT, CDOCKER and Molecular Dynamics Simulation, five sections of Discovery Studio for virtual screening and analysis. The compounds with higher libdock score had better energy optimization and stable conformation. According to the libdock score, the top 20 compounds were selected for further study. The pharmacological properties of these compounds were evaluated by ADME and toxicity prediction. The results showed that ZINC000008689961 and ZINC000027646625 are soluble in water and had good absorption level. In addition, they had no hepatotoxicity and were non-inhibitors of cytochrome P4502D6 (CYP2D6). Furthermore, compared with other compounds, these two compounds had lower mutagenicity, rodent carcinogenicity and developmental toxicity. To sum up, ZINC000008689961 and ZINC000027646625 were considered as safe drug candidates and further analysis were performed. Next, we studied the bonding mechanism and chemical bond of the selected candidate compounds. CDOCKER module computation indicated that the binding affinity of ZINC0000868961 and ZINC000027646625 with MYC was high. Eventually, their stabilities in natural environment was studied by molecular dynamics simulation. The results showed that the potential energy of these complexes gradually stabilizes with the passage of time. It is suggested that ZINC000008689961 and ZINC000027646625 may interact with MYC and their complexes were stable in natural environment. In opinion of all the results above, these two compounds could be developed and refined as drugs.
Afterwards, the anti-cholangiocarcinoma effects of Dhea and 2–14,15-Eg in vitro were detected by MTT assay, colony-forming assay and scratch assay. In MTT assay, the cellular viability in cell lines HuCCT1 decreased following the increasing drug doses of Dhea and 2–14,15-Eg. In colony-forming assay, the number and size of colony formation in Dhea and 2–14,15-Eg group were significantly lower than those in the control group, which was consistent with the results of Dhea and 2–14,15-Eg inhibiting the proliferation of CCA cells. In scratch assay, the wound width in control group decreasing as time goes by, and were smaller than in Dhea and 2–14,15-Eg group after 24h sharply which implied that Dhea and 2–14,15-Eg strongly inhibited migration of CCA cells.
This study not only provides new ideas for the study of MYC inhibitors, but also provides mind for the development of CCA drugs. But as we all know, without thousands of improvements and refinements, no single drug can be directly marketed. Consequently, the refinement and improvement of them is of great momentousness in the follow-up research.