Although THCA generally has a good prognosis, some high-risk patients develop metastasis and recurrence and can thus die from the disease. Therefore, relevant diagnostic and prognostic indicators of THCA need to be identified. Our analysis of the relationship between mutations and CNVs in m6A regulatory genes and clinical pathology showed that advanced pathological stage and T stage were significantly associated with changes in m6A regulatory genes. We then evaluated the effect of changes in m6A regulatory genes on their expression profiles and found that the expression of six m6A regulatory genes (i.e., FTO, METTL3, YTHDF1, YTHDF2, ZC3H13, and WTAP) significantly correlated with their different copy number states. An increased copy number was associated with mRNA expression, while copy deletion was associated with reduced mRNA expression. We also found a significantly lower PFI in patients with abnormal copy numbers than in those with normal copy numbers, indicating that abnormal copy number is a factor indicative of a poorer prognosis in patients with THCA. We also analyzed the PFI in different states of each gene. Because the number of patients with a single-gene CNV change was too small, the results were not significant. Furthermore, pathologic stage was an independent prognostic factor for patients with THCA. Therefore, we speculated that CNVs are involved in the occurrence of thyroid cancer by affecting cancer-related signaling pathways in which m6A regulatory genes are involved by changing their mRNA expression.
The global incidence of THCA has continued to increase in recent decades. Data from the National Cancer Registry of China show that the annual incidence markedly increased by 4.9% during 2000–2003 to 20.1% during 2003–2011 [10]. A series of cancer-related pathways are dysregulated in THCA development. Several valuable THCA molecular markers such as BRAF, RAS point mutations, RET/PTC, TERT, and PAX8/PPAR-γ are increasingly being used in clinical practice [11, 12]. However, there are no concise prospective data to support the use of molecular markers alone to determine the degree of treatment or to predict the prognosis of patients with THCA.
New evidence indicates that m6A is involved in various aspects of RNA metabolism, including pre-splicing of mRNA, 3′-end processing, nuclear export, translation regulation, mRNA attenuation, and noncoding RNA processing [7, 13–15]. m6A RNA modification affects tumor proliferation [16], differentiation, tumorigenesis, invasion [17], and metastasis [18] by regulating proto-oncogenes and tumor suppressor genes. Similarly, CNVs play a crucial role in the occurrence, development, and outcome of several cancers, such as lung, endometrial, prostate, and gastric cancers [19–22]. However, the role of m6A methylation modification and CNVs in THCA is unknown. In this study, we analyzed mutation data and CNV data of m6A regulatory genes in 492 patients with THCA from the TCGA database and found that KIAA1429 and YTHDC1 with frameshift mutations, YTHDC2 with splice donor variant changes, and ZC3H13 with stop codon mutations had the greatest impact on gene function, indicating that “writers” and “readers” may play an important role in the occurrence of thyroid cancer. The “writers” METTL3 and METTL14 are more likely to be mutated or undergoing CNVs in other genes in clear cell renal cell carcinoma [23], while the changes of “erasers” FTO and ALKBH5 have been proven to be more important in breast cancer, glioblastoma, and hematological malignancies [24–26]. The differences in genes related to different tumor types suggest that the regulation of m6A at the cellular level is complicated, and further studies are needed to investigate the regulatory mechanism of m6A in thyroid cancer.
The “writer” ZC3H13 gene, which is a canonical CCCH zinc finger protein that harbors a somatic frameshift mutation in colorectal cancer, suppresses colorectal cancer proliferation and invasion by inactivating Ras-ERK signaling [27], indicating that ZC3H13 may serve as a tumor suppressor gene. However, Gewurz et al. found that ZC3H13 may be a key upstream factor of NF-κB responsible for its activation [28]. Activation of NF-κB signaling accelerates tumor proliferation and invasion [29], suggesting that ZC3H13 may be an oncogenic protein. ZC3H13 may bind with K-ras, which is frequently mutated in various cancers such as non-small cell lung cancer and colon carcinoma [30, 31] and is strongly associated with cancer progression [32]. Thus far, the expression and biological function of ZC3H13 in malignant tumors are still unclear. It is speculated that the ZC3H13 gene may also play a major part in the occurrence and development of THCA by regulating cancer-associated signaling pathways and biological processes. Further studies are needed to identify the specific impact of ZC3H13 on the regulation of the downstream genes. With improvements in CNV detection technology, its pathogenic mechanism and relationship with gene mutations are expected to be widely recognized. CNVs in m6A regulatory genes are expected to provide new directions for the diagnosis and prognosis of thyroid cancer.
This study has some limitations that need to be considered when interpreting the findings. First, the number of samples with increased copy numbers and samples with normal copy numbers (26 vs. 240) was not evenly distributed. Second, a relationship between the expression levels of ZC3H13 and the risk of THCA was not observed in this study; thus, further research is necessary to clarify this ambiguity. Despite these limitations, the study remains valuable because to the best of our knowledge, this is the first study to investigate the genetic changes of m6A regulatory genes in THCA and to analyze the relationship of mutations and CNVs in m6A regulatory genes with the clinical pathology of THCA. To further clarify the specific target mRNAs of m6A modification in the occurrence and development of THCA, we plan to conduct m6A-Seq and m6A MERIP studies in clinical samples to support our findings.