【作者】 张晓军；

【导师】 葛红；

【作者基本信息】 郑州大学 ， 肿瘤学（专业学位）， 2022， 博士

【摘要】 背景:甲状腺乳头状癌（papillary thyroid carcinoma,PTC）近年来发病率持续增高,在近30年来增加了3倍,引起世界的广泛关注。尽管PTC整体预后较好,10年生存率超过90%至95%,但是,约3%-10%的PTC可呈侵袭性生长或出现广泛远处转移,该类患者通过手术+碘¹³¹治疗无法治愈,而且部分肿瘤在其发展的不同阶段出现碘治疗抵抗,预后极差（10年生存率不足10%,中位生存期不足5年）。目前,PTC的基因组图谱已完成绘制,系统展示了PTC的基因表达形态,确定的几个最常见的驱动子的突变:主要富集在MAPK信号通路上的BRAF（>60%）和RAS（13%）。另外,最新被证实与PTC侵袭转移相关的突变基因有PPM1D、CHEK2、mi R-21、TERT以及DDR通路等。上述研究对认识PTC的发生发展有很大益处,但以上研究均是基于PTC的整体水平,而整块组织测序（bulky sequencing）的基因表达提供的信息是有限的,主要是由于其可能掩盖一些肿瘤内关键细胞之间的基因表达差异,即肿瘤内异质性,而肿瘤的异质性是PTC十分显著的特质之一。肿瘤的进化和异质性的存在导致临床病理同为PTC的患者,出现的临床病理特征和治疗效果不一。因此,在细胞水平建立PTC基因图谱对于理解PTC的异质性、进化特点及侵袭转移特征极为重要,新出现的单细胞转录组测序技术在一定程度上可以解决这些问题。单细胞转录组测序是在单个细胞水平上构建每个细胞的表达谱,揭示单个或某一亚群细胞的基因表达信息。近年来,单细胞转录组测序在癌症研究领域揭示转录组异质性、肿瘤侵袭与转移、肿瘤微环境、干细胞特性、耐药性、免疫分型和免疫逃逸等方面取得了关键性的突破。在乳腺癌、肝癌和恶性黑色素瘤等多种恶性肿瘤方面进行了深入的探索和发现。但在PTC上尚未进行深入研究。本研究拟先搜集临床典型的侵袭性PTC组织样本进行单细胞转录组测序,获得PTC单细胞水平的转录组图谱,寻找与侵袭转移相关的关键细胞亚群和基因;然后针对候选基因之一（CHI3L1）进行临床样本的mRNA和蛋白水平验证;最后,体外构建过表达与敲低CHI3L1表达的质粒载体,转染至PTC细胞株（TPC-1）,通过观察细胞在增殖、迁移与侵袭等方面的变化,探讨CHI3L1对PTC细胞生物学功能的影响。目的:1.通过PTC单细胞转录组表达图谱分析说明PTC的肿瘤异质性,同时寻找恶性程度高,有侵袭转移特征的关键细胞亚群及关键的差异表达基因。2.通过检测PTC临床组织标本中关键基因CHI3L1在mRNA和蛋白水平的表达,分析其与PTC临床病理特征的关系,验证其与PTC侵袭转移的相关性。3.通过体外构建过表达与敲低CHI3L1表达的质粒载体,分别转染至TPC-1细胞株,通过观察PTC细胞在增殖、迁移及侵袭等方面的变化,探讨CHI3L1对PTC细胞生物学特性的影响。方法:1.选取临床表现上两种典型的PTC患者:惰性发展型（PTC1）和侵袭进展型（PTC2）,采用组织特异性酶裂解法对PTC新鲜组织标本进行组织消化,制成单细胞悬液,采用10×Genomics平台进行单细胞转录组测序,通过生物信息学分析绘制PTC单细胞水平的转录组表达图谱,进行细胞亚群分类,拟时序分析肿瘤进化的细胞发展轨迹,寻找肿瘤侵袭转移的关键细胞亚群和基因。2.采用实时荧光定量反转录PCR（Reverse transcription Quantitative real-time PCR,q RT-PCR）检测正常甲状腺2例、PTC无转移组2例、颈部淋巴结转移组5例和远处转移组5例共14例PTC组织标本中CHI3L1 mRNA的表达水平,分析CHI3L1 mRNA的表达与PTC转移的相关性。3.采用免疫组织化学染色（Immunohistochemistry,IHC）的方法检测110例不同TNM分期的PTC石蜡包埋的组织切片中CHI3L1蛋白的表达水平,分析CHI3L1蛋白的表达与PTC临床病理特征的关系,探讨CHI3L1蛋白的表达与PTC侵袭转移的相关性。4.分别构建过表达与敲低CHI3L1表达的质粒载体,通过瞬转的方法转染TPC-1细胞株,与转染空白质粒对照相比,通过CCK-8、Ed U检测、平板克隆、Transwell等实验观察TPC-1细胞在增殖、迁移与侵袭等方面的变化,分析CHI3L1在PTC细胞中的生物学功能。结果:1.在建立PTC单细胞转录组图谱基础上,发现Epi02为PTC中恶性程度最高的细胞亚群,CHI3L1为该亚群中表达差异最大的基因。（1）通过对临床PTC样本进行单细胞转录组测序并建立PTC单细胞水平转录组图谱,定义了包括上皮细胞、巨噬细胞和肿瘤相关成纤维细胞等7个细胞亚群,其中上皮细胞亚群占75%,各细胞亚群功能不同,且彼此之间存在广泛的细胞间通讯。进一步对上皮细胞亚群细分后获得5个新的亚群（Epi01-05）,其CNV（copy number variation）水平、基因表达和功能的不同提示PTC内存在显著的肿瘤异质性。（2）Epi02亚群主要富集CNVs相对较高的水平,提示其恶性程度较高,而GSEA（Gene Set Enrichment Analysis）富集分析进一步发现,Epi02富集的多数信号通路均是与肿瘤侵袭转移和分化相关的,包括细胞粘附、ECM-受体相关作用、m TORC1、p53、NF-KB、Wnt和KRAS等。同时,拟时序分析也发现,Epi02存在于细胞发展轨迹的末端,以上分析均表明Epi02是PTC内分化最差,恶性程度最高的细胞亚群。（3）分析Epi02细胞亚群的差异基因发现,其高表达的基因CHI3L1、CXCL14等与多种肿瘤的侵袭侵袭有关,COL1A1与上皮间质转化相关。同时,CHI3L1在PTC2（侵袭进展型）样本中的表达明显高于PTC1（惰性发展型）,在Epi02亚群中差异是最大的。（4）通过GSCA（Gene Set Cancer Analysis）生信分析发现,CHI3L1 mRNA在在多种恶性肿瘤中高表达,且参与多种恶性肿瘤侵袭转移的相关信号通路,如EMT、PI3K/AKT、RAS/MAPK和TSC-m TOR等。2.临床数据显示CHI3L1的高表达与PTC的侵袭转移密切相关（1）通过14例临床PTC组织样本的q RT-PCR发现,在mRNA水平,与正常甲状腺组织相比,PTC组织中CHI3L1的表达均有不同程度的升高,而且远处转移组较无转移组和颈部转移组有着更为显著的升高（P=0.015）。（2）通过110例临床PTC组织样本的免疫组化发现,在蛋白水平,CHI3L1在9例正常或良性甲状腺组织及8.2%（9/110）的PTC组织中呈阴性表达,在55.5%（61/110）的PTC组织中呈低表达,在36.3%（40/110）的PTC组织中呈高表达。而进一步对CHI3L1蛋白的表达与PTC临床病理特征的相关性进行单因素分析发现,CHI3L1蛋白的高表达与年龄（<55 vs≥55,P=0.013）、T分期（P<0.001）、N分期（P<0.001）、M分期（P=0.007）及显著腺体外侵犯（P<0.001）相关。3.功能研究显示CHI3L1的高表达增强PTC的增殖、迁移和侵袭能力通过体外细胞功能实验,分别构建了CHI3L1基因过表达（TPC-1-CHI3L1）和敲低表达的TPC-1细胞（TPC-1-sh CHI3L1）。（1）通过Western Blot检测证实,过表达组TPC-1-CHI3L1的CHI3L1蛋白表达量高于对照组TPC-1-Control（P=0.002）;而敲低组PTC-1-sh CHI3L1的CHI3L1蛋白表达量低于对照组TPC-1-sh Control（P=0.006）。（2）细胞增殖实验（CCK-8检测）显示,与对照组相比,TPC-1-CHI3L1细胞生长出现明显加速（P<0.001）;相反的,TPC-1-sh CHI3L1细胞较对照组细胞（转染空载体）生长出现明显抑制（P<0.001）,并且随时间的延长而日益显著。（3）Transwell迁移实验显示:与对照细胞相比,TPC-1-CHI3L1跨膜细胞数（248.65±4.56）显著升高（P<0.001）,TPC-1-sh CHI3L1跨膜细胞数（69.56±6.43）显著下降（P<0.001）。（4）Transwell侵袭实验同样显示:与对照细胞相比,TPC-1-CHI3L1侵袭细胞数（247.32±5.59）显著升高（P<0.001）,TPC-1-sh CHI3L1侵袭细胞数（56.77±4.08）显著下降（P<0.001）。结论:1.本研究通过单细胞测序建立的PTC单细胞转录组图谱特征充分展现了PTC内显著的肿瘤异质性,同时发现Epi02是PTC内恶性程度最高的细胞亚群,而在该亚群内,基因CHI3L1是PTC1和PTC2间表达差异最高的基因之一,且与肿瘤侵袭转移相关。2.CHI3L1在mRNA和蛋白水平的高表达均与临床PTC的侵袭转移特征密切相关。3.在甲状腺乳头状癌细胞株中过表达CHI3L1增强了细胞增殖、迁移和侵袭能力,对肿瘤的侵袭和转移起到促进作用;反之,敲低CHI3L1的表达则降低了细胞的增殖、迁移和侵袭能力,对肿瘤的侵袭和转移起到抑制作用。更多还原

【Abstract】 Background:The incidence of papillary thyroid carcinoma(PTC)has continued to increase in recent decades,arousing worldwide concern.PTCs usually have an excellent prognosis,with 10-year survival rates exceeding 90% to 95%.Most patients with PTC are cured by surgery with or without radioactive iodine.Nevertheless,there is a significant morbidity and mortality associated with extensive extrathyroidal extension and distant metastases,which is of major clinical concern because its 10-year survival rate is less than 10%.The molecular and genomic mechanism of PTC aggressiveness should be responsible for this effect.In recent years,The Cancer Genome Atlas(TCGA)has charted the genetic landscape of PTC,identifying the most common driver mutations,BRAF(>60%)and RAS(13%),which converge on the mitogen-activated protein kinase(MAPK)signaling pathway.Beyond the driver mutations,newly identified mutations in PPM1 D,CHEK2,mi R-21,TERT,as well as genes that code for components of the DNA damage response(DDR)pathway,are associated with an aggressive form of PTC.However,most current transcriptomic studies are performed on a bulk level and typically investigate the average of variable transcriptomes from millions of cells,thereby masking critical differences and providing limited insight into cancer cell programs,intratumoral heterogeneity and tumor microenvironment(TME)influences.The evolution and heterogeneity of tumors result in various clinicopathological features and therapeutic effects among PTC patients.Fortunately,these challenges have gradually been overcome by a new technology: single-cell RNA sequencing(sc RNA-seq).ScRNA-seq is a technology that analyzes transcriptomes of complex tissues at the single-cell level.Through the combination of high-throughput sequencing and bioinformatic tools,sc RNA-seq can detect more than 10,000 transcripts in one cell to distinguish cell subsets and dynamic cellular changes.Recent advances in sc RNA-seq have demonstrated a powerful capacity to reveal transcriptomic heterogeneity,metastasis,TME,stemness,signaling pathways related to drug resistance,immunocyte typing and immune escape.Sc RNA-seq has been conducted in many cancers,including breast cancer,lung cancer,liver cancer,melanoma and oligodendrogliomas.However,there have been no in-depth sc RNA-seq studies on PTC until now.Objectives:1.To acquire a transcriptomic atlas of advanced PTC patients at the single-cell level by employing sc RNA-seq in two fresh human PTC tissues,to delineate PTC intratumoral heterogeneity by identifying diverse cell types and analyzing their individual functions and to explore the highest malignant subcluster of epithelial cells and genes possessing metastasis characteristics through pseudotime analysis.2.To investigate the mRNA and protein expression of CHI3L1 in clinical PTC tissues and to analyze the relationship between the expression of CHI3L1 and clinicopathologic characteristics,including metastasis.3.To investigate the biological function of CHI3L1 in TPC-cell(a PTC cell line)treated with CHI3L1 overexpression or silencing by testing cell proliferation,invasion and metastasis.Methods:1.After fresh PTC tissue dissociation,cell purification and single-cell transcriptomic sequencing conducted at 10 × Genomics,we acquired a single-cell transcriptomic atlas of PTC by bioinformatics analysis.Here,T-distributed stochastic neighbor embedding(t-SNE)revealed main cluster classification based on the expression of marker genes;the function of these cell clusters in PTC was investigated by enrichment analysis;and pseudotime analysis was performed to determine the development of epithelial cells in PTC and investigate metastatic subclusters and genes.2.Reverse transcription Quantitative real-time PCR(q RT-PCR)was carried out in5 PTC tissues with cervical lymph node metastases,5 PTC tissues with distant metastases,2 PTC tissues without metastasis and 2 normal thyroid tissues to analyze the relationship between the mRNA expression of CHI3L1 and metastases.3.Immunohistochemistry(IHC)was carried out in 110 paraffin-embedded PTC tissues to analyze the relationship between the protein expression of CHI3L1 and clinicopathologic characteristics,including metastases.4.We constructed plasmid vectors that interfere with and overexpress CHI3L1 respectively,and then transfected the TPC-1 cell line by transient method.Then,we examined the impact of interference and overexpression of CHI3L1 on the proliferation,migration and invasion ability of TPC-1 cells through CCK-8 kit,Ed U,plate colony formation and Transwell chamber assays.Results:1.Single-cell transcriptomic sequencing of two samples from different patients was completed based on 10×Genomics.After quality control,we obtained a total of3,497 single-cell transcriptomes(1,705 samples,PTC1;1792 samples,PTC2).T-SNE revealed seven main clusters based on the expression of marker genes,and the seven clusters were epithelial,macrophage,dendritic cell 2(DC2),T-cell,inflammatory cancer-associated fibroblasts(i CAFs),myo-cancer-associated fibroblasts(m CAFs)and endothelial cells.Among these clusters,the epithelial cluster was the dominant cluster,and approximately 75% of the total number of cells was classified as this cluster.The inferred copy number variation(CNV)analysis revealed a higher burden of CNVs(aneuploid)in the epithelial cluster,which identified the malignancy.The epithelial cluster was then divided into five subclusters(from Epi01 to 05)by t-SNE.The differences in gene expression and function revealed the intra-tumoral heterogeneity in PTC.Inferred CNV analysis and gene set enrichment analysis(GSEA)were carried out and revealed that Epi02 was the most malignant subcluster,and most of its enriched pathways were related to tumor invasion,metastasis and differentiation.Pseudotime analysis showed that Epi01 and Epi03,Epi04,Epi05 and Epi02 existed in the front,middle,and end of the cell development trajectory of the tree structure,respectively,indicating that Epi02 had low differentiation and a high degree of malignancy.Collectively,the above results all indicated that the Epi02 subcluster may play a key role in PTC metastasis.The highly expressed genes in Epi02 included CHI3L1 and CXCL14,which are related to the metastasis and invasion of a variety of tumors;COL1A1,suggesting the existence of epithelial-mesenchymal transition.These results suggested that CHI3L1,CXCL14 and COL1A1 were potential regulators of PTC metastasis and malignant progression.GSCA(Gene Set Cancer Analysis)analysis showed that CHI3L1 mRNA was highly expressed in a variety of malignant tumors,and was involved in a variety of signal pathways related to tumor invasion and metastasis,such as EMT,PI3 KAKT,RASMAPK,TSCm TOR and so on.2.q RT–PCR results showed that the mRNA expression of CHI3L1 in the malignant PTC groups was significantly higher than that in the benign PTC group.In addition,in the three malignant groups,the expression of CHI3L1 in the distant metastasis group was significantly higher than that in the no metastasis group or cervical lymph node metastasis group.The results suggested that the expression of CHI3L1 in PTC is associated with the metastasis of PTC and that CHI3L1 is an important positive regulator of the metastasis of PTC.Through IHC,univariate analysis showed a statistically significant association between the expression of CHI3L1 protein and patient age(<55 vs.≥55,P=0.013),T stage(P<0.001),N stage(P<0.001),M stage(P=0.007)and gross extrathyroidal extension(P<0.001).This result suggested that the expression of CHI3L1 is associated with the advanced stage,invasion and metastasis of PTC.3.In vitro,CHI3L1 overexpression and interference vector plasmids were constructed separately and transfected into the TPC-1 cell line.Cell proliferation,invasion and metastasis were tested.The results showed that CHI3L1 expression,cell proliferation,cell invasion and cell metastasis were significantly increased in cells in which CHI3L1 was overexpressed(TPC-1-CHI3L1)and significantly decreased in cells treated with CHI3L1 interference(TPC-1-sh CHI3L1).These results suggest that CHI3L1 is an important regulator of the malignant development of PTC cells and promotes proliferation,invasion and metastasis in TPC-1 cells cultured in vitro.This result was consistent with the results of single-cell sequencing and clinical samples shown above.Conclusions1.The single-cell transcriptomic atlas of PTC charted in this study revealed intratumoral heterogeneity in PTC.Epi02 subcluster had the lowest differentiation and highest degree of malignancy.CHI3L1 was the most different gene in expression and associated with metastasis and malignant progression.2.Further study of the metastatic potential of the CHI3L1 gene discovered by sc RNA-seq in clinical PTC specimens revealed that CHI3L1 is closely related to the invasion and metastasis of PTC at both the mRNA and protein levels.This suggested that CHI3L1 may play an important role in the invasion and metastasis of PTC.3.In vitro experiments confirmed that the overexpression of CHI3L1 increased the proliferation,invasion and migration capabilities of PTC cells,and vice versa,interference of CHI3L1 expression reduced cell proliferation,migration and invasion ability.The above studies provide important insight into the molecular metastasis mechanism of PTC and provide more target options for the treatment of advanced PTC.更多还原