Eva‐1 homolog A promotes papillary thyroid cancer progression and epithelial‐mesenchymal transition via the Hippo signalling pathway

Abstract Recently, the incidence of thyroid cancer is increasing worldwide. Papillary thyroid cancer (PTC) is the most common histological type of thyroid cancer. Whole‐transcriptome sequence analysis was performed to further understand the primary molecular mechanisms of the occurrence and progression of PTC. Results showed that Eva‐1 homolog A (EVA1A) may be a potential gene for the PTC‐associated gene in thyroid cancer. In this work, the role of EVA1A expression in thyroid cancer was investigated. Real‐time PCR was performed to detect the expression level of EVA1A in 43 pairs of PTC and four thyroid cancer cell lines. The Cancer Genome Atlas (TCGA) database was used to evaluate the relationship between the expression level of EVA1A and the pathological feature of PTC. The logistic regression analysis of the TCGA data set indicated that the expression of EVA1A was an independent risk factor for tumour, nde and metastasis (TNM) in PTC. This study shows the down‐regulation of EVA1A inhibited the colony formation, proliferation, migration and invasion of PTC cell lines. In the protein level, knockdown of EVA1A can regulate the expression of N‐cadherin, vimentin, Bcl‐xL, Bax, YAP and TAZ. This study indicated that EVA1A was an oncogene associated with PTC.

poorly differentiated thyroid cancer and parafollicular C cell-derived medullary thyroid cancer. 5 PTC is a well-differentiated thyroid cancer accounting for about 80% of all thyroid cancers. 6 Despite the good prognosis of thyroid cancer, up to 25% of patients with PTC has a significant risk for recurrence. 7 EVA1A is a novel endoplasmic reticulum and lysosome-associated protein-coding RNA located on chromosome 2p12. 8 In several cancers, such as hepatocellular carcinoma and non-small cell lung cancer, the expression of EVA1A is low in carcinoma tissues, and EVA1A can inhibit tumour cell growth through autophagy and apoptosis. 9,10 In contrast to its expression in hepatocellular carcinoma and non-small cell lung cancer, the EVA1A expression in thyroid cancer tissues is significantly higher than that in the normal thyroid tissue according to The Cancer Genome Atlas (TCGA) database (P < .0001).
This phenomenon is also observed in bladder urothelial carcinoma, colon adenocarcinoma and lymphoid neoplasm diffuse large B-cell lymphoma. (P < .05). However, the relationship between EVA1A and thyroid cancer remains poorly understood.
We have previously performed whole-transcriptome sequencing and bioinformatics analysis on 19 paired PTC tissues and adjacent normal tissues 11 and found that EVA1A, a gene encoding a transmembrane protein, is one of the most significantly up-regulated genes among patients with PTC. Through the gene set rich analysis (GSEA), we have found that the expression of EVA1A expression is correlated to metastasis, proliferation and apoptosis. These phenotypes are related to several pathways described below.
The epithelial-mesenchymal transition (EMT) is a biological phenomenon that plays an essential role in cancer invasion and metastasis. 12 The levels of the related proteins N-cadherin and vimentin are up-regulated during EMT. This process leads to the decrease in cell-cell adhesion and an increase in tumour cell metastasis and invasion. 13 The intrinsic apoptosis pathway is regulated by the Bcl-2 family located in the mitochondria. The antiapoptosis protein Bcl-xL and the proapoptosis protein Bax are prominent cancer makers.
Sufficient evidence has shown that the up-regulation of Bcl-xL and the down-regulation of Bax can suppress the apoptosis of the tumour cells. 14 The Hippo signalling pathway plays an essential role in regulating tumour cell proliferation and apoptosis. 15 As a pathway that suppresses tumours, the Hippo signalling pathway inhibits the transcriptional activity of the Yes-associated protein (YAP) and the transcriptional coactivator with PDZ-binding motif (TAZ). YAP and TAZ are downstream effectors of the Hippo pathway and overexpressed in numerous cancers. 16 In recent years, several studies have found that the Hippo signalling pathway can promote tumour invasion and metastasis via EMT. [16][17][18][19] And the molecular mechanism through which the Hippo pathway activates apoptosis is realized via p73. [20][21][22] According to researchers, such as Yoon et al, 23

| Patients and samples
This study included 43 patients with PTC who underwent surgi-

| Cell proliferation assay
Cell proliferation was determined using the Cell Counting Kit-8 (CCK-

| Colony formation assay
The two transfected or control cells (1.5 × 10 3 cells for KTC-1 and TPC-1) were seeded into six-well plates and incubated for 7-14 days.
Each well was fixed with 4% paraformaldehyde (Sigma-Aldrich Co.) for 30 minutes, stained with 0.01% crystal violet and photographed.
All experiments were performed in triplicate.

| Cell migration and invasion assays
The migration assay was performed using the Transwell chambers (Corning Incorporated). For the wound healing assay, 1.5 × 10 5 cells were cultured into a 24-well plate until 90% confluency was reached.
Scratch wounds were created using 0.

| Protein extraction and Western blot analysis
The

| EVA1A expression is overexpressed in PTC
In our previous study, RNA sequencing analysis is performed on the primary PTC and the adjacent normal tissues obtained from 19 patients with PTC. 11 After analysing the data, we have found that EVA1A was significantly up-regulated (Table 1). This result coincided with the data shown in the TCGA cohort ( Figure 1A). Afterwards, we detected the relative expression of EVA1A in 43 patients via qRT-PCR to verify the sequencing results. The expression level of EVA1A in tumour tissues was significantly higher compared with that in adjacent normal tissues ( Figure 1B). The ROC curve analysis was used to evaluated the diagnostic capability of EVA1A. As shown in Figure 1C,D, EVA1A expression had excellent diagnostic value to distinguish non-cancerous tissue from cancer tissue in the TCGA data set (AUC = 81.2%) and our local cohort (AUC = 94%).

| EVA1A is associated with the clinicopathological features of PTC
TCGA data were analysed to determine the relationship between the expression level of EVA1A and the clinicopathological features and further explore the role of EVA1A in PTC. We divided the patients with PTC into two groups, namely high (n = 251) and low (n = 251) EVA1A expression in accordance with the median value of the EVA1A expression level in TCGA cohort. Results showed that a high EVA1A expression was related to histological type (P < .001), age (P = .025), tumour size (P = .06) and lymph node metastasis (LNM; P < .001; Table 2). Nevertheless, the associations of EVA1A expression with gender, distant metastasis and disease stage (AJCC7) were not significant. In our validated cohort, high EVA1A expression was correlated with LNM (P = .009) and disease stage (AJCC7; P = .011; Table 3).
These results supported that EVA1A gene was an oncogene in PTC.

| EVA1A overexpression aggravates the risk of LNM in PTC
Further investigation was carried out to find the relationship of EVA1A expression with LNM. We conducted a univariate logistic regression analysis in TCGA data. Results showed that the significant variables for LNM were EVA1A expression (odds

| EVA1A knockdown suppresses cell proliferation and colony formation
We evaluated the expression level of EVA1A in several PTC and normal thyroid cell lines (HTORI-3) in the mRNA and protein levels.

| EVA1A knockdown inhibits migration and invasion
The migration and invasion assays were performed on TPC-1 and KTC-1 to further confirm the function of EVA1A in PTC. As expected, the PTC cells with underexpressed EVA1A had significantly inhibited ability to migrate ( Figure 4A,C) and invade ( Figure 4E) compared with those in the Si-NC control group.

| EVA1A knockdown induces the activation of the intrinsic apoptosis pathway and suppresses EMT progression via the Hippo signalling pathway
Accumulating number of studies have shown that EMT and apoptosis plays a critical role in PTC metastasis and proliferation. 25,26 GSEA indicated that the expression of EVA1A was related to EMT and apoptosis ( Figure 6A). Related proteins were detected to explore the mechanism of the EVA1A on EMT and apoptosis progression. Recent studies have provided enormous evidences suggesting that the Hippo signalling pathway can induce EMT. 17,27,28 Results revealed that compared with the control group, the Si-EVA1A group showed decreased N-cadherin, vimentin and Bcl-xL expression and increased Bax expression ( Figure 6B). Furthermore, the YAP and TAZ levels in the Si-EVA1A group were lower than those in the control group ( Figure 6B). These results suggested that EVA1A may inhibit apoptosis and promote the metastasis and the proliferation of PTC by suppressing the intrinsic apoptosis pathway and activating the EMT via the Hippo signalling pathway.

| D ISCUSS I ON
With the development of molecular biology, our understanding of PTC molecular mechanisms is gradually developing. Next-generation sequencing has paved the way for the discovery that PTC is driven by several characteristic genetic alterations, such as point mutations in proto-oncogenes and chromosomal rearrangements. 29 However, despite the enormous progress in PTC genetic research, the current non-surgical treatment for thyroid cancer is still limited. Therefore, further research on the pathogenetic mechanism of PTC is essential for the diagnosis and therapy of PTC.
According to Cancer Statistics 2019, thyroid cancer incidence is rising faster than other cancers in men and women and accounts for 4% of the total estimated female cancer burden in 2019. 30 In our previous study, whole-genome sequencing has been performed on 19 pairs of PTC tumours and adjacent normal tissues. 11 We have found that the expression level of EVA1A is up-regulated in patients with PTC in Wenzhou. This finding supports the hypothesis that EVA1A may have a role as an oncogene in PTC. The function of EVA1A in apoptosis is complex and suggested as essential in the process.
However, the molecular mechanisms of EVA1A in thyroid carcinoma are still unclear.
In the present study, the statistical results of the clinicopatho- In summary, we first found that EVA1A in PTC was a potent oncogene of enhanced tumour aggressiveness. This evidence suggested that EVA1A may be an effective molecular marker of PTC for diagnosis and therapy.

ACK N OWLED G EM ENTS
The authors would like to thank all the patients who participated in this study.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
The ethical approval for this study was obtained from the Ethics

Committee of the First Affiliated Hospital of Wenzhou Medical
University.

I N FO R M E D CO N S E NT
Written informed consent was obtained from each participant (approval no. 2012-57).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used during this study and additional images are included in this article. Raw data are available upon reasonable request.