Identification of key functional gene signatures indicative of dedifferentiation in Papillary Thyroid cancer

Papillary thyroid cancer (PTC) is the most common type of thyroid cancer, and the majority of PTCs exhibit a relatively good prognosis. However, it has been observed recently that some PTCs may dedifferentiate in some situations. When PTC appears to be dedifferentiated, its prognosis becomes very poor, and conventional surgical treatment cannot achieve good therapeutic effects. Such patients often experience relapse or metastasis in a short period of time. At present, the treatment methods for poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) are limited, and their progression mechanism is still unclear.It has been shown that many ATC and PDTC result from dedifferentiation of DTC. In addition, some genetic abnormalities such as <i>TERT</i> and <i>TP53</i> mutation may play an important role. A considerable number of studies have shown that occurrence and development of PDTC and ATC are closely related to immune microenvironment and epigenetic changes. Our previous study also revealed that some genes may have a significant impact on the initiation and progression of dedifferentiation thyroid cancer (DDTC) through metabolism related pathways. However, considering that dedifferentiation of DTC is accompanied by a great increase in the degree of malignancy, it is likely that dedifferentiation must involve more than one mechanism. It is not clear which malignant phenotypes are affected by genes closely related to DTC dedifferentiation and this needs to be investigated further. This study was oriented toward mining out differentially expressed genes among PDTC, PTC and normal thyroid (NT) at the level of transcriptome, and then classifying them into different groups based on their biological functions, to explore possible dedifferentiation related processes. We expect that our findings could provide a plausible basis for further study of PDTC, thereby helping to predict prognosis and development of PTC, and exploring the possibility of reversing dedifferentiation or re-differentiation.<b>Sample collection</b> Six NT, five PTC and five PDTC specimens were obtained from eight patients who underwent surgical management in FUSCC . The information of the eight patients and 16 samples was described in our previous study. These 16 samples were included in a discovery cohort, and used for high-throughput RNA sequencing (RNA-seq) to identify differentially expressed genes. Written informed consent was obtained from each patient before his/her specimens were used in this study, and the study was approved by the Medical Ethics Committee of the FUSCC. All procedures performed in this study were in accordance with the Declaration of Helsinki. <b>RNA-seq analysis</b> Total RNA was extracted from all samples using TRIzol reagent (Life Technologies, Carlsbad, CA). We used RiboMinus eukaryote kit (Qiagen, Valencia, CA) to remove ribosomal RNA of total RNA (~3 mg) before RNA-seq libraries construction. Strand-specific RNA-Seq libraries were prepared using the Illumina workflow (New England BioLabs, Beverly, MA).Next, the samples were fragmented, reverse-transcribed, and ligated to Illumina adaptors. We purified the ligated cDNA products to remove second-strand cDNA. After 13-15 cycles of amplification, libraries were controlled for quality and quantified using with an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) and sequenced by a HiSeq 2000 sequencing system (Illumina, SanDiego, CA) on a 100-bp paired-end run. Clustal Omega was used for sequence alignment. Human genome version GRCh38.100 were used throughout. Significant differences were determined by Limma package (version 3.11; https://bioconductor.org/packages/limma/).

乳头状甲状腺癌（Papillary thyroid cancer, PTC）是最常见的甲状腺癌类型，多数患者预后相对良好。但近年研究发现，部分PTC在特定情况下可发生去分化。当PTC出现去分化时，患者预后极差，常规手术治疗难以取得理想疗效，此类患者往往短期内即可出现复发或转移。目前，低分化甲状腺癌（poorly differentiated thyroid cancer, PDTC）和未分化甲状腺癌（anaplastic thyroid cancer, ATC）的治疗手段有限，其进展机制尚未阐明。已有研究表明，多数ATC与PDTC源自分化型甲状腺癌（differentiated thyroid cancer, DTC）的去分化过程。此外，<i>TERT</i>与<i>TP53</i>突变等遗传异常可能发挥重要作用。大量研究证实，PDTC与ATC的发生发展与免疫微环境及表观遗传改变密切相关。我们前期研究还发现，部分基因可通过代谢相关通路对去分化型甲状腺癌（dedifferentiation thyroid cancer, DDTC）的起始与进展产生显著影响。然而，鉴于DTC去分化伴随恶性程度大幅提升，其致病机制很可能不止一种。目前尚不清楚与DTC去分化密切相关的基因会影响哪些恶性表型，有待进一步研究。

本研究旨在从转录组层面挖掘PDTC、PTC与正常甲状腺（normal thyroid, NT）组织间的差异表达基因，并基于其生物学功能进行聚类分类，以探索与去分化相关的潜在生物学过程。我们期望本研究结果可为PDTC的后续研究提供可靠依据，进而辅助预测PTC的预后与进展，并探索逆转去分化或实现再分化的可能性。

<b>样本收集</b> 本研究从复旦大学附属肿瘤医院（FUSCC）接受手术治疗的8例患者中获取了6例正常甲状腺组织、5例PTC组织及5例PDTC组织。8例患者的临床信息与16份样本的相关情况已在我们前期研究中进行了描述。该16份样本纳入发现队列，用于高通量RNA测序（RNA-seq）以筛选差异表达基因。所有患者在样本用于本研究前均签署了书面知情同意书，本研究经复旦大学附属肿瘤医院医学伦理委员会批准，所有实验操作均符合《赫尔辛基宣言》的相关规范。

<b>RNA测序分析</b> 采用TRIzol试剂（Life Technologies，美国加利福尼亚州卡尔斯巴德）从所有样本中提取总RNA。在构建RNA测序文库前，使用RiboMinus真核生物试剂盒（Qiagen，美国加利福尼亚州瓦伦西亚）去除总RNA（约3 mg）中的核糖体RNA。采用Illumina建库流程（New England BioLabs，美国马萨诸塞州贝弗利）制备链特异性RNA-seq文库。具体步骤包括样本片段化、反转录及连接Illumina接头。随后纯化连接后的cDNA产物以去除第二链cDNA。经过13~15个循环的扩增后，使用Agilent 2100生物分析仪（Agilent Technologies，美国加利福尼亚州圣克拉拉）对文库进行质量控制与定量，并通过HiSeq 2000测序系统（Illumina，美国加利福尼亚州圣迭戈）以100 bp双端测序模式进行测序。使用Clustal Omega进行序列比对，全程采用人类基因组版本GRCh38.100作为参考序列。差异显著性分析采用Limma软件包（版本3.11；https://bioconductor.org/packages/limma/）完成。