The Role Of CHI3L1 In Invasion And Metastasis In Papillary Thyroid Carcinoma

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.