Introduction
Thyroid cancer is the most common type of cancer of the endocrine system, with cases increasing worldwide (1,2). Thyroid cancer may be classified into numerous types according to the histopathological characteristics. Medullary thyroid carcinoma (MTC) is a form of thyroid cancer which originates from the parafollicular cells of the thyroid (3). It is the third most common type of thyroid cancer and accounts for ~3% of all thyroid cancer cases. Approximately 1 in 4 of MTC cases are caused by mutations in the rearranged during transfection (RET) proto-oncogene (4). The majority of MTC cases are sporadic, presenting with metastatic disease at diagnosis (5). Nearly all patients with distant metastases succumb to this disease (6).
Presently, RET mutation have been suggested to be an indicator of the poor prognosis of MTC; however, this is not sufficient for understanding the underlying molecular mechanisms of MTC tumourigenesis. Soh et al (7) demonstrated that vascular endothelial growth factor receptor 2 was involved in the pathogenesis of MTC via promotion of pro-invasive and pro-angiogenic phenotypes. Additionally, dysregulation of the Dickkopf/Wnt signaling pathway inhibitor 4 has been identified in MTC (8). A previous study revealed that aberrant expression levels of microRNAs (miRNAs) have a potential role in tumourigenesis (9), which may provide novel insight in MTC research. Notably, increasing evidence has supported the important role of miRNAs in cancers including thyroid cancer (10,11). For example, He et al (12) reported that three miRNAs, including miR-221, −222, and −146, are overexpressed in papillary thyroid cancer. Furthermore, miR-197 and −346 are significantly overexpressed in follicular thyroid cancers (13). Although great advances have been made in understanding the functions of miRNAs in thyroid cancers, the underlying molecular mechanisms of this disease remain to be elucidated.
The present study aimed to use GSE40807 miRNA microarray data provided by Lassalle et al (14) to identify differentially expressed miRNAs (DEMs) between human MTC and healthy control tissues. Subsequently, transcription factor (TF)-miRNA and miRNA-target gene regulatory networks were constructed. Finally, the target genes of DEMs were performed functional enrichment analyses to predict their potential functions that may be associated with MTC. To the best of our knowledge, this is the first time that the dataset of GSE40807 was analyzed.
Discussion
Patients with progressive MTC have limited treatment options (28). Thus, understanding the underlying molecular mechanism of carcinogenesis may facilitate diagnosis and therapy options of this disease. In the present study, 15 upregulated and 17 downregulated DEMs were identified. In the constructed TF-miRNA regulatory network, hsa-miR-9-5p was regulated by 9 TFs and hsa-miR-1 was regulated by 8 TFs. The TFs of NF-κB1 and MYC regulated 4 and 3 DEMs, respectively. Additionally, the above two miRNAs served key roles in the miRNA-target gene regulatory network. Their target genes were primarily enriched in the MAPK signaling pathway and during focal adhesion. These miRNAs and signaling pathways may be important biomarkers for MTC diagnosis and treatment.
In the miRNA-target gene regulatory network, hsa-miR-1 was upregulated, and its target genes, including MAPK1, were enriched in numerous signaling pathways associated with cancer, including MAPK. MAPKs are a family of protein kinases whose functions are conserved during evolution from unicellular organisms (29). The MAPK signaling pathway consists of numerous key signaling components and phosphorylation events which control multiple fundamental cell processes including proliferation, differentiation and apoptosis (30). This signaling pathway has been frequently identified in activated in human cancers, which leads to malignant phenotypes including autonomous cell proliferation (31). Notably, Zatelli et al (32) demonstrated that the growth of the TT MTC cell line depends on activation of the MAPK signaling pathway, which suggests its role in MTC. In addition, MAPK signaling is important in regulating cytokine signaling pathways (33). Cytokines are released in response to inflammation and immunity, and have important roles in cancer development and progression (34). It has been reported that undifferentiated thyroid cancer cells secrete cytokines (35). Taken together, the MAPK signaling pathway may serve important roles in MTC via hsa-miR-1 and its target gene MAPK1.
In addition to hsa-miR-1, hsa-miR-9-5p upregulated in the miRNA-target gene regulatory network, and was regulated by 9 TFs in the TF-miRNA regulatory network, including NF-κB1 and IL-1ß. NF-κB is a transcription regulator activated by various intra- and extracellular stimuli. A previous study demonstrated that NF-κB1 regulates the expression of genes involved in numerous processes, including proliferation and apoptosis (36). Inappropriate activation of NF-κB has been associated with numerous inflammatory diseases, whereas persistent inhibition of NF-κB may lead to delayed cell growth (37). NF-κB has been demonstrated to be associated with the development of colorectal (38), breast (39), bladder (40), prostate (41) and advanced thyroid (36) cancers. On the other hand, IL-1β, a member of the IL-1 cytokine family, is an important mediator of the inflammatory response. A previous study reported that inflammation is a critical component of tumor progression (42). Zeki et al (43) suggested that IL-1 regulates G1 cell cycle progression and arrest in papillary thyroid carcinoma cells. Therefore, NF-κB1, IL-1ß and their regulated DEM hsa-miR-9-5p may serve important roles in MTC progression.
Additionally, TFs of MYC were demonstrated to regulate 3 DEMs including hsa-miR-195-3p, hsa-let-7b-3p and hsa-let-7f-1-3p. MYC is a multifunctional, nuclear phosphoprotein which serves roles in cell cycle progression, apoptosis and cellular transformation (44). The MYC gene has been widely implicated in numerous human cancers (45,46). Khosla et al (47) revealed that MYC mRNA expression levels increased in apoptotic TT cells, suggesting its role in MTC. Its regulated miRNA hsa-miR-195-3p has been identified to be abnormally expressed in a variety of cancers. For example, levels were upregulated in breast cancer and downregulated in gastric, hepatocellular and bladder cancers (48–50). The roles of hsa-let-7b-3p and hsa-let-7f-1-3p in cancer remain to be elucidated; thus, it was hypothesized that these miRNAs may be involved in MTC via regulation from the MYC gene. Taken together, MYC and its regulated DEMs, hsa-miR-195-3p, hsa-let-7b-3p and hsa-let-7f-1-3p, may serve important roles in the development of MTC.
The present study identified numerous key miRNAs and TFs that may be associated with MTC using comprehensive bioinformatics methods. However, no experiments with tissues or cells were performed to validate the expression levels of these miRNAs and TFs; a key limitation of this study. Additionally, there were only 14 pairs of miRNA microarrays in the dataset. Further studies with experimental validations and more samples are required to validate these observations.
In conclusion, the results of the present study indicated that the DEMs hsa-miR-1, hsa-miR-9-5p and hsa-miR-195-3p may have the potential to be used as diagnostic and therapeutic targets of MTC. Additionally, hsa-miR-1 and its target gene MAPK1 may serve a role in MTC, involving in MAPK signaling pathway. Additionally, TFs of IL-1ß and MYC may be implicated in the development of MTC.