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Jialiang Wen,* Lizhi Lin,* Bangyi Lin, Erjie Xia, Jinmiao Qu, Ouchen Wang Department of Thyroid and Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ouchen WangDepartment of Thyroid and Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang, People’s Republic of ChinaTel +86 13957706099Email woc@wmu.edu.cnIntroduction: Breast cancer (BC) is one of the most prevalent malignancies in women and its incidence has increased steadily over recent years (0.3% per year). However, the mechanism of BC tumorigenesis remains elaborate elucidation. With the aid of RNA sequencing technology, we discovered that immortalization-upregulated protein (IMUP) is overexpressed in BC tissues compared to normal breast tissues. Our study is to understand the role of IMUP in BC.Methods: We validated the upregulation of IMUP from multiple public databases. By using quantitative real-time polymerase chain reaction (qRT-PCR), we proved that IMUP is overexpressed in BC tissues and cell lines. We performed proliferation, migration, invasion and apoptosis assays to explore the function of IMUP in BC cell lines (MCF-7 and MDA-MB-231). Besides, we investigated the effect of IMUP silencing on epithelial–mesenchymal transition using Western blotting and qRT-PCR.Results and Discussion: We validated that IMUP expression in BC tissues and cell lines is higher than that in the normal control group. The clinical analysis showed that IMUP is associated with lymph node metastasis and the outcome of neoadjuvant taxol-based therapy. The loss of function assay demonstrated that, with silencing IMUP, the capacities of proliferation, migration, and invasion of BC cell lines were impaired, while the apoptosis rate of cells increased. Meanwhile, the downregulation of IMUP could hinder the procession of epithelial–mesenchymal transition.Conclusion: Our study proved that IMUP plays a vital role in BC and acts as a potential target and marker in future therapy.Keywords: immortalization-upregulated protein, IMUP, breast cancer, BC, progression, epithelial–mesenchymal transition, EMT

Yuefeng Li,1,2,* Xiaofen Zhou,2,* Qiongying Zhang,3 Endong Chen,2 Yihan Sun,2 Danrong Ye,2 Ouchen Wang,2 Xiaohua Zhang,2 Jianxin Lyu1 1Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China; 2Department of Thyroid and Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China; 3Pathology Department, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China *These authors contributed equally to this work Background: Papillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma, which is associated with a high incidence of lymph-node metastasis. Multiple biomarkers have been identified for the precise diagnosis of PTC at an early stage. However, their role in PTC remains poorly elucidated. Previously, we reported that lipase H (LIPH), a membrane-bound protein, was highly expressed in PTC. This study aimed to fully elucidate the causal role of LIPH in the development of PTC and investigated its relationship with lymph-node metastasis in PTC. Materials and methods: Quantitative reverse transcription PCR and immunohistochemistry were used to measure the mRNA and protein expression levels of LIPH in 45 and 6 pairs of PTC tissues and adjacent normal tissues, respectively. Clinical tissue data of 504 PTC tissues and 60 normal thyroid tissues from The Cancer Genome Atlas database were used to analyze the correlation between LIPH expression level and clinical features in PTC. siRNAs were used to knock down genes, while plasmids were used to overexpress genes. Two PTC cell lines (KTC-1 and BCPAP) were used in subsequent cytological function studies. In addition, a hypoxia stress model was constructed using cobaltous chloride hexahydrate reagent, and the protein expression level of the corresponding biomarkers was measured by Western blotting. Results: This study revealed that high expression of LIPH in PTC was closely associated with lymph-node metastasis. Our cellular function experiments indicated that LIPH positively correlated with the malignant behavior of PTC cell lines. We further confirmed the role of LIPH in hypoxia and its relationship with the epithelial–mesenchymal transition pathway in PTC. Conclusion: LIPH plays an important role in PTC oncogenesis and development, especially in lymph-node metastasis. It can be regarded as a biomarker for the diagnosis and treatment of PTC in the near future. Keywords: papillary thyroid carcinoma, hypoxia, metastasis, epithelial–mesenchymal transition

Yue Cheng,1 Yiqi Pan,2 Yiyuan Pan,3 Ouchen Wang1 1Department of Thyroid and Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China; 2Wenzhou Medical University, Wenzhou, China; 3Department of Thyroid and Breast Surgery, Wenzhou People’s Hospital, Wenzhou, China Purpose: lncRNAs have recently been identified as key regulators of basic biological processes as well as the pathogenesis of various diseases. Previous studies have shown that lncRNA MNX1-AS1 promotes cell migration and invasion in ovarian cancer; however, its role in regulating breast cancer-associated biological processes remains unclear. Materials and methods: We obtained paired specimens of breast cancer tissues and adjacent normal tissues by modified radical mastectomy from 36 patients, in addition to four breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT-549 and MCF-7). RNA was isolated from these tissues and cell lines and subsequently subjected to quantitative real-time polymerase chain reaction. This was followed by bisulfite deep sequencing. The cells were also transfected with siRNA against MNX1-AS1. The cells were then subject to cell proliferation, Transwell migration and invasion assays. Finally, Western blotting analysis was conducted to determine expression levels of MNX1, 5-cadherin, Snail and Slug. Results: Our results show that MNX1-AS1 expression was significantly higher in breast cancer tissues than adjacent normal tissues. Moreover, knockdown/overexpression of MNX1-AS1 inhibits/promotes proliferation, migration and invasion of breast cancer cells. MNX1-AS1 and its natural sense transcript MNX1 are expressed synergistically in breast tumor tissues. Our results suggest that MNX1-AS1 is a functional oncogene that induces epithelial–mesenchymal transition, in addition to activating AKT/mTOR pathway and its natural sense transcript MNX1 in breast cancer cells. Conclusion: Our data indicate that MNX1-AS1 can serve as a novel therapeutic target in breast cancer. Keywords: MNX1-AS1, breast cancer, oncogene, MNX1, EMT, AKT/mTOR signaling pathway

Endong Chen,1 Qiaolin Wu,2 Yixiang Jin,1 Wenxu Jin,1 Yefeng Cai,1 Qingxuan Wang,1 Xiaohua Zhang,1 Ouchen Wang,1 Quan Li,1 Zhouci Zheng3 1Department of Thyroid and Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 2Department of Anesthesiology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 3Department of Head and Neck Surgery, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China Objectives: Few studies on prognostic indicators for primary thyroid lymphoma (PTL) have been presented due to the uncommon nature of the tumor. This is the first study to explore the independent prognostic factors in the 2 PTL subtypes.Methods: We retrospectively reviewed 1,653 cases of PTL. The cases comprised 28 Chinese patients from a local cohort and 1,625 patients from the Surveillance, Epidemiology, and End Results database from 1973 to 2013. Statistical analysis was performed to determine the demographics and prognostic factors of PTL patients.Results: The disease-specific survival (DSS) and prognostic indicators were significantly different between patients with extranodal marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) and patients with diffuse large B-cell lymphoma (DLBCL). Patients with MALT lymphoma were younger (P=0.011) and had lower clinical stage (P=0.014) compared to patients with DLBCL. Cox regression analysis revealed that age, treatment modalities employed, clinical stage, and number of other types of cancer were independent prognostic factors for DLBCL patients.Conclusion: PTL demonstrates specific clinical features and is associated with a relatively good prognosis. Older age is associated with poor DSS in both MALT patients and DLBCL patients. Additionally, combination of different treatment modalities is associated with improved DSS in DLBCL patients. Keywords: primary thyroid lymphoma, SEER database, disease-specific survival, combined modality treatment, prognosis

Erjie Xia,* Xiaofen Zhou,* Adheesh Bhandari, Xiaohua Zhang, Ouchen Wang Department of Thyroid & Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China *These authors contributed equally to this work Introduction: Synaptopodin 2 (SYNPO2) is a functioning protein. It has been detected in many malignancies. But the relation between SYNPO2 and breast cancer (BC) is unclear. Materials and methods: In this study, we explored the expression and function of SYNPO2 in BC. We found that SYNPO2 gene in BC was downregulated at the transcriptional level in both validated and TCGA cohorts. Results: The results revealed that age, lymph node metastasis, and clinical stage in the validated cohort were related to the expression of SYNPO2 negatively. Kaplan–Meier analysis showed that patients with lower SYNPO2 expression had a worse overall survival. Discussion: We found that migration and invasion were promoted after knocking down SYNPO2 in MCF-7, MDA-MB-231, BT-549, and MDA-MB-468. Meanwhile, knockdown of SYNPO2 could enhance PI3K/AKT/mTOR signaling pathway, which may induce migration and invasion. Our findings reveal that SYNPO2 was associated with BC. Keywords: breast cancer, SYNPO2, metastasis, PI3K/AKT/mTOR signaling pathway

Quan Li,* Wen-Xu Jin,* Yi-Xiang Jin, Zhou-Ci Zheng, Xiao-Fen Zhou, Qing-Xuan Wang, Dan-Rong Ye, Yi-Han Sun, Xiao-Hua Zhang, Ou-Chen Wang, En-Dong Chen, Ye-Feng Cai Department of Breast and Thyroid Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China *These authors contributed equally to this work Aim: To investigate the clinical effects of MUC1 on papillary thyroid cancer (PTC) and explore the relationship between MUC1 expression and BRAF mutation. Methods: The data of 69 patients subjected to fine-needle aspiration biopsy in our hospital and 486 patient data downloaded from The Cancer Genome Atlas (TCGA) database were used. Univariate and multivariate analyses were performed.Results: The results on the 486 patients recorded in the TCGA indicated that high MUC1 expression was independently related to BRAF mutation, lymph node metastasis (LNM), and unifocal type. In the 69 fine-needle aspiration biopsy patients with PTC, high MUC1 expression was significantly related to LNM and extrathyroid extension (ETE). The result of Pearson’s correlation coefficient showed that BRAF mutation and MUC1 expression were moderately correlated. Moreover, in the subgroup with low MUC1 expression, the patients with BRAF mutation had higher ETE frequency and LNM than those without BRAF mutation. In the subgroup with BRAF mutation, patients with high MUC1 expression exhibited higher ETE frequency than those with low MUC1 expression, and high MUC1 expression occurred in older patients. In the subgroup with BRAF wild-type mutation, patients with high MUC1 expression had a higher incidence of ETE and LNM than those with low expression.Conclusion: We demonstrated that the MUC1 is an important oncogene in PTC and may have great significance on therapeutic cancer vaccine development.Keywords: MUC1, BRAF mutation, papillary thyroid cancer, prognosis, lymph node metastasis, extrathyroid extension

Adheesh Bhandari,* Erjie Xia,* Yuying Zhou, Yaoyao Guan, Jingjing Xiang, Lingguo Kong, Yinghao Wang, Fan Yang, Ouchen Wang, Xiaohua Zhang Department of Thyroid and Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China *These authors contributed equally to this work Background: Breast cancer is the most common malignancy in women and the underlying mechanism of breast cancer cell metastasis is still far from uncover. Integrin subunit alpha 7 (ITGA7) is a functioning protein. It has been detected in many malignancies. But the function of ITGA7 in breast cancer is not clear. Our aim is to explore ITGA7 expression and its role in breast cancer. Methods: Real-time PCR was performed to determine ITGA7 expression in BC tissues and normal adjacent tissues. The specific functions of ITGA7 in breast cancer cell lines (MDA-MB-231 and BT-549) transfected with small interfering RNA were determined through migration, invasion assays. Western blot assays were performed to determine the expression of c-met and vimentin. Results: ITGA7 was down-regulated in breast cancer tissues compared to the adjacent normal tissues (T:N =7.68±27.38: 41.01± 31.47, P

Si Yang Dong,* Hao Chen,* Li Zhi Lin, Lingli Jin, Dan Xiang Chen, Ou Chen Wang, Zhi Qiang Ye Department of Thyroid and Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhi Qiang Ye; Ou Chen WangDepartment of Thyroid and Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000 People’s Republic of ChinaTel/Fax +86 13867705524Email yezhiqiang1982@126.com woc@wmu.edu.cnBackground: Microfibril-associated protein 2 (MFAP2) is a protein coding gene that exerts important phenotypic effects on cell motility, and increasing research has indicated that MFAP2 was correlated with many cancers. However, the functional and potential clinical role of MFAP2 in papillary thyroid cancer (PTC) has not yet been verified.Materials and Methods: We performed whole transcriptome sequencing on 78 paired PTC tissues and corresponding adjacent normal tissues and found that MFAP2 was highly expressed in PTC tissues. Then, we analyzed the expression of MFAP2 and its relation with the clinicopathological features of PTC in The Cancer Genome Atlas (TCGA) PTC genomic dataset. We detected MFAP2 expression in 40 paired PTC tissues and corresponding adjacent normal tissues through RT-qPCR (real time-quantitative polymerase chain reaction) to validate the sequencing data and TCGA cohort. Cell functional assays were performed to elucidate the function of MFAP2 in PTC cells, Western blot assay was performed to explore the correlation between MFAP2 and EMT (epithelial-mesenchymal transition)-related proteins.Results: Statistical analysis showed that MFAP2 was obviously upregulated in PTC tissues compared to matched normal tissues, and the expression levels of MFAP2 in PTC tissues were strongly related with lymph node metastasis (p=0.016). The results of RT-qPCR of our own tissue specimens showed the same conclusions as that in TCGA dataset. The results of functional assays in PTC cell lines showed that MFAP2 could promote proliferation, colony formation, migration and invasion abilities and decrease the apoptotic rate in PTC cells. Western Blot assay showed that MFAP2 could regulate the expression of EMT-related proteins.Conclusion: MFAP2 increases the proliferation, motility and decreases the apoptosis of PTC cells, and might be a potential therapeutic target for papillary thyroid cancer.Keywords: biomarker, proliferation, migration, epithelial-mesenchymal transition, EMT, apoptosis

Wen-Xu Jin,1,2,* Dan-Rong Ye,2,* Yi-Han Sun,2,* Xiao-Fen Zhou,2 Ou-Chen Wang,2 Xiao-Hua Zhang,2 Ye-Feng Cai2 1Department of Vascular Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, 325000, China; 2Department of Breast and Thyroid Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, 325000, China *These authors contributed equally to this work Background: Preoperative diagnosis of central lymph node metastasis (CLNM) poses to be a challenge in clinical node-negative papillary thyroid microcarcinoma (PTMC). This research work aims at investigating the association existing between BRAF mutation, clinicopathological factors, ultrasound characteristics, and CLNM, in addition to establishing a predictive model for CLNM in PTMC.Materials and methods: The study included 673 PTMC patients, already undergone total thyroidectomy or lobectomy with prophylactic central lymph node dissection. The predictor factors were identified through univariate and multivariate analyses. The support vector machine was put to use to develop statistical models, which could predict CLNM on the basis of independent predictors.Results: Tumor size (>5 mm), lower location, no well-defined margin, contact of >25% with the adjacent capsule, display of enlarged lymph nodes, and BRAF mutation were independent predictors of CLNM. Through the use of the predictive model, 79.6% of the patients were classified accurately, the sensitivity and specificity amounted to be 85.1% and 75.8%, respectively, and the positive predictive value and negative predictive value stood at 71.6% and 87.6%, respectively.Conclusions: We established a predictive model in order to predict CLNM preoperatively in PTMC when preoperative diagnosis of CLNM was not clear. Keywords: papillary thyroid microcarcinoma, central lymph node, predictive factor, support vector machine