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/).

甲状腺乳头状癌（Papillary thyroid cancer, PTC）是最为常见的甲状腺癌类型，多数患者的预后相对良好。但近年研究发现，部分甲状腺乳头状癌可在特定情境下发生去分化。当甲状腺乳头状癌呈现去分化表型时，患者预后极差，常规外科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）与未分化甲状腺癌（anaplastic thyroid cancer, ATC）的治疗手段较为有限，其进展机制仍未阐明。已有研究证实，多数未分化甲状腺癌与低分化甲状腺癌源自分化型甲状腺癌（differentiated thyroid cancer, DTC）的去分化；此外，<i>TERT</i>、<i>TP53</i>突变等多种遗传异常可能在其中发挥重要作用。大量研究表明，低分化甲状腺癌与未分化甲状腺癌的发生发展与免疫微环境及表观遗传改变密切相关。我们前期的研究亦发现，部分基因可能通过代谢相关通路对去分化甲状腺癌（dedifferentiated thyroid cancer, DDTC）的起始与进展产生显著影响。鉴于分化型甲状腺癌去分化后恶性程度显著升高，其发生过程可能涉及多种机制；目前尚不清楚与分化型甲状腺癌去分化密切相关的基因可影响哪些恶性表型，有待进一步探究。本研究旨在从转录组层面挖掘低分化甲状腺癌（PDTC）、甲状腺乳头状癌（PTC）与正常甲状腺组织（normal thyroid, NT）间的差异表达基因，再根据其生物学功能进行聚类分组，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低分化甲状腺癌的后续研究提供可靠依据，助力甲状腺乳头状癌的预后与病情发展预测，并为探索逆转去分化或实现再分化的可能性提供参考。<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6例正常甲状腺组织（NT）、5例甲状腺乳头状癌（PTC）标本及5例低分化甲状腺癌（PDTC）标本。8例患者与16份样本的相关信息已在我们前期的研究中详述。该16份样本被纳入发现队列，用于高通量RNA测序（RNA-seq）以筛选差异表达基因。所有患者在其标本用于本研究前均签署书面知情同意书，本研究已通过复旦大学附属肿瘤医院医学伦理委员会审批，所有实验操作均符合《赫尔辛基宣言》。<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）对约3 mg总RNA进行核糖体RNA去除。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。随后对样本进行片段化、反转录，并连接Illumina接头。纯化连接后的cDNA产物以移除第二条链cDNA。经13~15轮扩增后，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，随后采用HiSeq 2000测序系统（Illumina，美国加利福尼亚州圣迭戈）以100 bp双端测序模式开展测序。使用Clustal Omega进行序列比对，全程采用人类基因组版本GRCh38.100。采用Limma软件包（版本3.11；https://bioconductor.org/packages/limma/）进行显著性差异分析。