Special role of JUN in papillary thyroid carcinoma based on bioinformatics analysis

The thyroid cancer incidence has been increasing worldwide in recent years, and more cases of thyroid cancer are diagnosed every year [1]. Papillary thyroid carcinoma (PTC) is the most common malignancy in the thyroid and accounts for almost 80% of all thyroid cancers [2]. It is characterized by distinctive nuclear alterations including pseudoinclusions, grooves, and chromatin clearing [3]. Most patients with PTC have an excellent prognosis, but a small number of patients remain suffering with aggressive PTC which can develop invasive tumors and/or distant metastases [4]. Undoubtedly, PTC places an enormous economic burden on society and personality and greatly lowers the quality of one’s life. It is of great significance to study the mechanism of PTC genesis and explore new avenues to prevent PTC formation.

Previous study indicated that a number of different genetic changes were related to PTC, particularly the chimeric oncogenes formed by a fusion of a membrane receptor protein tyrosine kinase domain with another gene’s 5-prime terminal region. Oncogenic gene rearrangements involving the RET and NTRK1 have been found in PTC tissues [5, 6]. BRAF and RAS mutations are also observed in PTC cases, and the constitutive activation of effectors along the RET/PTC-RAS-BRAF signaling pathway contributed to the transformation of the thyroid cell to PTC [7‐9]. For the diagnosis and prognosis of PTC, several methods and markers are used. Immunohistochemical markers have been evaluated and tested in PTC tissues, such as CK19, HBME-1, RET, galectin-3, and CITED1. However, they are helpful only in some cases, for all of them have their limits and may bring some error diagnostics [10‐13]. Molecular studies also conferred some useful information for the diagnosis and therapy of PTC. Liu et al. have reported that CXCR7 gene involves in regulating proliferation and metastasis of PTC cell and provides a potential target for therapeutic interventions in PTC [14]. Minna et al. found that miR-199a-3p could act as a tumor suppressor in PTC [15]. Despite those researches on PCT, there are still so many mechanisms underlining PTC genesis and progression that needed further investigation, especially in the gene expression profile level.

In this study, we aimed to identify the differentially expressed genes (DEGs) in PTC tissues compared with normal thyroid tissue adjacent to PTC tumors. A series of bioinformatics analyses including DEGs identification, function enrichment, and interaction network construction were conducted to gain more insights into the molecular mechanisms of PTC genesis and progression. Our aim is to explore the pathogenesis of PTC and find potential diagnostic biomarkers/therapeutically targets of PTC by bioinformatic methods.

The incidence of PTC has increased worldwide over the past 15 to 20 years, especially in developed countries [31, 32]. To manage the increasing PTC patients effectively, a better understanding of the molecular mechanism involved in PTC is necessary. We applied bioinformatics techniques to investigate the DEGs in PTC and deeper explore the molecular mechanism underlying PTC genesis in this study. A total of 1800 overlapped DEGs were detected in two datasets. Enrichment analysis found the top five enriched GO terms in three ontologies and four significant enriched KEGG pathways were mainly concerned with regulation of pigmentation, cellular homeostasis, extracellular matrix, and intercellular junction. By constructing interactive network of the DEGs, we found that transcription factor HNF4A and DEG JUN had higher connection degrees in the network. By employing plugin ClusterOne, we got six subnetworks and DEG JUN appeared in two subnetworks.

In the constructed interaction network, we observed that the transcription factor HNF4A was connected with many other DEGs. However, its own expression has not changed in PTC. HNF4A regulates expression of genes involved in glucose metabolism and homeostasis [33]. It may play important roles in the occurrence and progression of PTC by interfering other genes’ normal expression and further disturbing cellular homeostasis. Nevertheless, further studies are still needed to illustrate its specific role in PTC genesis. We also discovered that the DEG gene JUN could interact with many other target proteins, implying its important role in PTC genesis. JUN is a proto-oncogene, and its encoding product is the first discovered oncogenic transcription factor [34]. Previous study demonstrated that it could promote tumor formation and maintain tumor cell survival between the initiation and progression stages [35]. The activation of JUN was also involved in the progress of breast cancer, gastric cancer, and colorectal carcinomas [36‐38]. But the role of JUN in PTC has not been studied, and here, we observed its significant different expression in PTC tissues and noticed its interaction with so many other proteins in the created interaction network. We deduced that the JUN may also play an important role in PTC genesis and progression and can be a possible potential diagnostic biomarker/therapeutical target gene of PTC.

Our deduction get further conferred by the cluster analysis, as the JUN appeared again in cluster 1 and cluster 5 (Fig. 4a, e). GO enrichment analysis manifested that cluster 1 was mainly concerned with “protein dimerization activity.” There were only two DEGs in cluster 1, namely JUN and HLF. The rest were all unchanged target proteins, and most of them were transcription factors, such as ATF2, ATF3, ATF4, ATF7, DDIT3, and FOSL1. HLF is a proto-oncogene whose expression product is a subset of the bZIP transcription factors and can cause abnormal transcriptional regulations of target genes which is related to leukemia development [39]. ATF2 binds with JUN to form a heterodimer and participates in reducing the amount of tumor necrosis factor (TNF) transcription through competitive binding [40]. The co-expressing JUN and ATF3 in two neuronal-like cell lines significantly enhanced JUN-mediated neurite sprouting [41]. The interactions of JUN with the aforementioned other transcription factors in regulation certain gene expression have also been reported [41, 42]. We presumed that the activated JUN might interact with some transcription factors and further induce abnormal gene expressions which lead to carcinogenesis.

The top significant GO term of function cluster 5 was “sequence-specific DNA binding.” Many transcription factors, such as ATF2, ATF3, ATF4, and ATF7, were also clustered to this module. What is noteworthy is the gene FOS, another proto-oncogene playing an important role in tumourigenesis and carcinogenesis [43], which changed its expression either in this module. The expression product of FOS can dimerize with JUN and form Activator Protein-1 (AP-1) complex. AP-1 binds to target genes at AP-1-specific sites at the promoter/enhancer regions and converts extracellular signals into changes of gene expression [44]. Some studies found that the JUN/FOS dimer, namely AP-1 complex, was involved in certain cancer genesis, and it can be the potential targeted therapeutic genes for certain cancer therapy. Magrisso et al. state that the expression of JUN and/or FOS are important events in colorectal tumorigenesis [45]. Wong et al. reported that the cyclooxygenase-2 inhibitor (SC-236) functioned the antitumor effects via inhibiting JNK-c-Jun/AP-1 activation, and the inhibition of JNK activation may have a therapeutic benefit against gastric cancer [37]. Zhang demonstrated that geldanamycin is a highly potent inhibitor of the AP-1 transcription factor and affects the activation of JNK in hypoxic HT29 human colon adenocarcinoma cells [36]. We supposed that the JUN/FOS dimer might also act as a promoter in PTC genesis. Design drugs targeting at this complex is potentially effective in PTC therapy. Even though, further immunohistochemical studies are still needed to confirm our results.