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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

Yihan Sun,* Danrong Ye,* Yuefeng Li, Endong Chen, Rutian Hao, Yefeng Cai, Qingxuan Wang, Ouchen Wang, Xiaohua Zhang Department of Thyroid and Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 32500, China *These authors contributed equally to this work Background: In recent years, the incidence of thyroid cancer (TC), the most common endocrine malignancy, has been increasing. Emerging evidence indicates that the CUT/CUX/CDP family of proteins can play an important role in tumor development and progression by regulating many cancer-related functions. However, the molecular functions of CUX2 in TC remain unknown. Methods: In this study, we used a series of loss-of-function experiments and Western blot analysis to investigate the function of CUX2 in TC and the mechanisms involved. Results: Our data revealed that CUX2 expression levels were upregulated in papillary thyroid cancer (PTC). Functionally, CUX2 silencing significantly inhibited PTC cell line (KTC-1 and BCPAP) proliferation, colony formation, migration, invasion, and apoptosis. Furthermore, CUX2 induced epithelial–mesenchymal transition (EMT) and influenced the phosphorylation of AKT and mTOR in the PI3K–AKT–mTOR pathways.Conclusion: In summary, CUX2 may function as a tumor promoter in TC. Keywords: papillary thyroid carcinoma, CUX2, oncogene

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

Yinghao Wang,* Yixiang Jin,* Adheesh Bhandari, Zhihan Yao, Fan Yang, Yiyuan Pan, Zhouci Zheng, Shixu Lv, Ouchen Wang Department of Surgical Oncology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China *These authors contributed equally to this work Background: Thyroid cancer is the most commonly reported endocrine malignancy, and its increased incidence has been the highest in all human tumors in recent decades. To investigate the mechanism of papillary thyroid cancer (PTC) occurrence and progression, we performed RNA sequencing and found an upregulated gene, LAMB3. However, the biological function of LAMB3 is still not clear. Materials and methods: We analyzed LAMB3 expression using The Cancer Genome Atlas (TCGA) database and hypothesized LAMB3 to be a gene associated with PTC. To test this hypothesis, we collected 89 pairs of thyroid nodules and adjacent normal thyroid tissues (56 pairs of PTCs, 33 pairs of benign thyroid nodules). Afterward, we performed real-time quantitative polymerase chain reaction (RT-qPCR) to investigate LAMB3 expression in thyroid nodule patients, and then analyzed clinicopathologic features. We performed proliferation, colony formation, migration, and invasion assays to determine the function of LAMB3 in PTC. Results: We demonstrated that LAMB3 plays oncogenic roles in PTC. The relative expression of LAMB3 is significantly upregulated in PTC compared with matched thyroid normal tissues in validated cohort and TCGA cohort (P

Adheesh Bhandari,* Ying-hao Wang,* Shi-xu Lv, Er-jie Xia, Ou-Chen Wang Department of Oncology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China *These authors contributed equally to this work Background: Tracheal and bronchial stenosis is a life-threatening condition causing difficulty in breathing and even severe respiratory distress. The silicone tracheobronchial stents were placed using the rigid bronchoscopy into the trachea of severe dyspneic patients and they exhibited symptomatic improvement as well as a rise in the saturation of oxygen. The bronchial stents were applicable to many extensive malignant airway stenosis patients, such as those with esophageal cancer, lung cancer, and laryngeal cancer. But the effectiveness of bronchial stents for thyroid cancer is not certain.Case presentation: Here, we report 3 emergency patients with a thyroid mass referred to our hospital because of grade 4 dyspnea according to the American Thoracic Society shortness of breath guidelines. The main clinical symptoms were severe dyspnea and stridor. The radiographic examination and tomographic examination showed the narrowing and displacement of the trachea. To the best of our knowledge, ideal airway management for the massive thyroid mass was considered to be temporary tracheobronchial stent placement pre-operation.Conclusion: In our study, we applied the tracheobronchial stent to massive thyroid mass patients with dyspnea and aimed to not only improve preoperative airway obstruction but also to protect the potential airway collapse from post-operative tracheomalacia following extubation. We found that application of tracheobronchial stents may provide a new strategy to dyspneic patients with huge thyroid mass. Keywords: tracheobronchial stents, airway management, tracheal stenosis, dyspnea

Fan Yang, Shi-Xu Lv, Lin Lv, Ye-Huan Liu, Si-Yang Dong, Zhi-Han Yao, Xuan-xuan Dai, Xiao-Hua Zhang, Ou-Chen Wang Department of Surgical Oncology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China Background: Luminal subtype breast cancer accounts for a predominant number of breast cancers. Considering the heterogeneity of the disease, it is urgent to develop novel biomarkers to improve risk stratification and optimize therapy choices. Long non-coding RNA (lncRNA) represents an emerging and understudied class of transcripts that play a significant role in cancer biology. Growing knowledge of cancer-associated lncRNAs contributes to the development of molecular markers for prognosis evaluation and gene therapy.Materials and methods: Three pairs of primary luminal subtype breast cancer tissues and adjacent non-cancerous tissues were collected and sequenced. EBseq algorithm was used to identify differentially expressed lncRNAs. RNA sequencing data from The Cancer Genome Atlas (TCGA) database were used to validate the robustness of our RNA-seq results. Kaplan–Meier and Cox regression analyses were utilized to assess the association between the lncRNAs and overall survival of patients in TCGA cohort.Results: A total of 796 lncRNAs were significantly dysregulated in luminal subtype breast cancer, including 436 upregulated and 360 downregulated lncRNAs. Among them, FAM83H antisense RNA 1 (FAM83H-AS1) was the most upregulated lncRNA, whereas GSN antisense RNA 1 (GSN-AS1) was the most downregulated lncRNA. Moreover, we proved that the high expression level of FAM83H-AS1 indicated unfavorable prognosis not only in luminal subtype breast cancer but also in all subtype breast cancers. To the best of our knowledge, this is the first report indicating that FAM83H-AS1 was involved in luminal subtype breast cancer and was an independent prognostic indicator.Conclusion: Our study provides a rich resource to the research community for further identifying lncRNAs with diagnostic and therapeutic potentials and exploring biological function of lncRNAs in luminal subtype breast cancer. Keywords: breast cancer, long non-coding RNA, FAM83H-AS1, prognosis