Development Of Papillary Thyroid Cancer Research Articles

Hsa_circ_0101050 regulated by ZC3H13 enhances tumorigenesis in papillary thyroid cancer via m6A modification

While the regulatory roles of circular RNAs (circRNAs) and zinc finger CCCH-type containing 13 (ZC3H13) were previously reported in various human cancers, the mechanisms underlying their interaction in papillary thyroid cancer (PTC) remain unclear. We aimed to determine the role of hsa_circ_0101050 and its regulatory relationship with ZC3H13 in PTC. The expression levels of hsa_circ_0101050 and ZC3H13 were determined in tumor samples and adjacent normal tissues from 46 patients with PTC and in two PTC cell lines (IHH-4 and PTC-1) using quantitative reverse transcription–polymerase chain reaction. The roles of hsa_circ_0101050 and ZC3H13 in cell viability, wound healing, and migration were determined using knockdown and overexpression approaches in PTC cell lines, and a xenograft model in nude mice was used to determine their role in vivo. Methylated RNA immunoprecipitation assay was used to analyze N6-methyladenosine (m6A) modification of hsa_circ_0101050 by ZC3H13. We found hsa_circ_0101050 overexpression and ZC3H13 downregulation in PTC samples and PTC cell lines. In PTC cell lines, silencing hsa_circ_0101050 reduced cell viability and migration whereas its overexpression promoted an aggressive PTC phenotype. ZC3H13 increased the m6A modification of hsa_circ_0101050 and repressed its expression. ZC3H13 overexpression inhibited PTC cell viability, migration, and invasion, which were reversed in cells overexpressing hsa_circ_0101050. Taken together, these results suggested that the downregulation of hsa_circ_0101050 mediated by ZC3H13 through m6A modification contributed to its oncogenic effect in PTC development, revealing the ZC3H13–m6A–hsa_circ_0101050 as a potential therapeutic target in PTC.

A Specificity Protein 1 assists the progression of the papillary thyroid cell line by initiating NECTIN4.

Papillary thyroid cancer (PTC) is one of the subtypes of thyroid cancer with increasing incidence worldwide, but the molecular mechanism is still unclear. Papillary thyroid cancer (PTC) is one of the subtypes of thyroid cancer with increasing incidence worldwide, but the molecular mechanism is still unclear. Studies have indicated that nectin cell adhesion molecule 4 (NECTIN4) was an oncogene and played an important role in the development and progression of PTC. Meanwhile, specificity protein 1 (SP1) expresses many important oncogenes and tumor suppressor genes. However, the relationship between NECTIN4 and SP1 in regulating PTC growth is unclear. In the present study, reverse transcription PCR was utilized to detect the mRNA expression of NECTIN4 and SP1 in thyroid cancer cell lines and normal thyroid cell lines. Chromatin immunoprecipitation assays and luciferase reporter assays were used to study whether SP1 could bind to the promoter region of NECTIN4 and activate its transcription. The biological functions of SP1 correlated with NECTIN4 were also performed in TPC-1 and KTC1 cell lines. The study revealed that the mRNA expression level of SP1 and NECTIN-4 showed a positive correlation and were upregulated in PTC cell lines. Moreover, the results of ChIP and luciferase reporter assays showed that SP1 could bind to the NECTIN4 promoter regions and activate the transcriptional level of NECTIN4. The experiments in vitro showed that SP1 could promote cell proliferation, colony formation, migration, and invasion by regulating NECTIN4 in PTC cells. In conclusion, our study, for the first time, demonstrated that SP1 could control the transcriptional regulation of NECTIN4 and accelerate the growth of PTC, which may provide a new potential therapeutic target for PTC patients.

Network Pharmacological Analysis and Experimental Validation of the Effects of Silybin on Proliferation, Migration, and Immunotherapy of Papillary Thyroid Cancer.

The study aimed to use network pharmacology research and in vitro experiments to investigate the material basis and molecular mechanisms of silybin in the treatment of papillary thyroid carcinoma. Papillary thyroid cancer (PTC) has a decent prognosis; however, recurrence and metastasis are the leading causes of death in patients with PTC. A key research focus in thyroid cancer treatment is the inhibition of PTC proliferation, invasion, and migration. Silybin, the major active element in the traditional Chinese herb silymarin, has been used to treat a range of diseases, including cancer, but no study has been undertaken to determine whether it can help prevent PTC. In this study, we attempted to determine through network pharmacology and in vitro experiments if silybin inhibits the development of papillary thyroid cancer by inhibiting cell cycle and invasive migration. To predict the probable targets and underlying mechanisms of silybin against PTC, a network pharmacology research was performed. In vitro experiments were conducted to further evaluate silybin's anti-cancer properties and priority targets against PTC. The datasets revealed a total of 489 silybin targets acting on PTC, and functional enrichment analysis suggested that the target genes were enriched in functions and pathways related to PTC development, invasion, migration, and immunotherapy. By constructing these target PPI networks, the seven hub genes, fibronectin 1 (FN1), tissue inhibitor of metalloproteinases 1 (TIMP1), N-cadherin (CDH2), collagen type III alpha 1 chain (COL3A1), cyclin D1 (CCND1), AP-1 transcription factor subunit (JUN), and hepatocyte growth factor receptor (MET) were found. These hub genes were determined to be highly linked to a worse clinicopathological form, a higher risk of metastatic recurrence, and a worse prognosis of PTC. The common immunological checkpoint gene expression levels were positively correlated with the expression levels of the hub genes. Silybin decreased the proliferative and metastatic capacity of PTC cells, according to in vitro investigations. When PTC was treated with silybin, the FN1/AKT signaling pathway was blocked, CCND1 expression was reduced, and CDH2, Vimentin, Snail, Slug and PD-L1 expressions were dramatically reduced, while E-cadherin expression was significantly elevated. These findings provide preliminary evidence that silybin inhibits PTC cell proliferation, metastasis, and invasion by altering the FN1/AKT signaling pathway and inhibiting the EMT process. Silybin can reverse immunosuppression in papillary thyroid cancer by affecting immunological checkpoint gene expression levels. These studies provide a theoretical and experimental scientific basis for the potential anticancer effects of silybin on PTC.

The Underestimated Impact of Hashimoto Thyroiditis on Thyroid Papillary Carcinoma

It is stated that Hashimoto’s Thyroiditis (HT) is a risk factor for the development of Papillary Thyroid Cancer (PTC). However, the effect of HT on the coexistence of HT and PTC is still controversial. In this study, our aim is to investigate the effect of the presence of HT on clinicopathological data in patients with PTC. All 356 patients whose pathology was reported as PTC who were operated between 2015 and 2023 were included in the study. PTC patients were divided into 2 groups as those with and without HT. The effect of HT association on clinicopathological features was investigated. In 356 PTC patients, the rate of HT was 31.2%. PTC patients with HT had less multifocality (p < 0.05), more lymph node metastases (LNM) (p < 0.01) compared to PTC patients without HT. The presence of HT did not affect the bilaterality of the tumor, tumor diameter, lymphovascular invasion, or capsule invasion. While multifocality was observed less frequently in PTC patients with HT, lymph node metastasis rates were higher.

Abstract 6859: A novel immune checkpoint CD97/CD55 in the initiation and progression of papillary thyroid cancer by scRNA-seq

Abstract Thyroid cancer (TC), as the most common endocrine malignancy, has exhibited a significant increasing trend in incidence in China and globally, with papillary thyroid cancer (PTC) being the most common pathological type. However, the molecular mechanisms driving the initiation and progression of TC remains scarce, posing a bottleneck problem for the precise diagnosis and treatment. Tumors, as a complex heterogeneous composite consisting of tumor cells, immune cells, and stromal cells, have long been considered to play a key role in driving tumor occurrence and malignant progression through the proportion, spatial distribution, and interaction of tumor cells and immune cells in the tumor immune microenvironment(TIME).We performed single-cell RNA sequencing (scRNA-seq) on 16 clinical samples, including normal thyroid tissue, benign nodules, PTC, and PTC with cervical lymph node metastasis(LNM), delineating the cell atlas of the tumor microenvironment (TME) at various stages from normal thyroid to PTC development. Among these, we discovered a previously undefined TNFSF9highCD8+ T cell subset with a distinct transcriptional profile from classical exhausted T cells, which was highly enriched in PTC tumor tissues. Further cell interaction analysis revealed that the highly expressed CD97 receptor on these CD8+ T cells binds to the highly expressed CD55 molecule on PTC tumor cells, promoting the exhaustion of CD8+ T cells, suggesting that CD97/CD55 may be a key new immune checkpoint (IC) inducing CD8+ T cell exhaustion and promoting immune escape of PTC tumor cells. We validated the co-localization of CD97/CD55-mediated CD8+ T cell and tumor cell interactions in PTC patient specimens and spontaneous tumor samples from a mouse PTC model using multiplex immunofluorescence (mIHC). Flow cytometry analysis of the proportion of CD97+ CD8+ T cells in patient and mouse specimens revealed a significant increase in the proportion of CD97+ CD8+ T cells in PTC tumor tissues. Co-culture experiments of activated and expanded CD8+ T cells from peripheral blood mononuclear cells(PBMC) of PTC patients with PTC tumor cells showed that CD55 overexpressing tumor cells inhibited the secretion of cytotoxic cytokines IFN-γ and IL-2 by activated CD8+ T cells through the CD97/CD55 IC signal, thereby weakening the cytotoxity on PTC tumor cells. In summary, the IC regulatory signal formed by the highly expressed CD55 molecule on the surface of PTC tumors and the CD97 receptor on the surface of PTC-specific inhibitory TNFSF9highCD8+ T cells can reshape the TIME, promoting the formation of an immunosuppressive microenvironment, which may represent a novel molecular mechanism promoting immune escape of tumor cells during PTC initiation and progression. Citation Format: Xuan Qin, Jia Li, Dapeng Li, Xing Wan, Xianhui Ruan, Yimeng Liu, Weijing Hao, Kejia Xu, Yi Shi, Yongjun Piao, Xiangqian Zheng. A novel immune checkpoint CD97/CD55 in the initiation and progression of papillary thyroid cancer by scRNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6859.

Adiponectin Inhibits the Progression of Obesity-Associated Papillary Thyroid Carcinoma Through Autophagy.

Obesity is a risk factor for the development of papillary thyroid cancer (PTC). However, the molecular mechanisms by which obesity promotes PTC are unclear. This study aims to identify adipokines that are linked to PTC progression. An adipokine antibody array was used to determine the serum levels of 40 adipokines in normal-weight and obese PTC patients. Enzyme-linked immunosorbent assay was used to determine the serum levels of adiponectin. Recombinant human adiponectin was produced by human adipose-derived stem cells and used to treat PTC cells. Cell proliferation and migration were evaluated using the CCK8 and Transwell assays. Bioinformatics analysis was used to predict mechanisms by which adiponectin affects PTC. Adipokines differentially expressed between normal-weight and obese patients showed a gender-dependent pattern. Obese PTC patients had a significantly lower serum adiponectin level than normal-weight patients, especially in female individuals. Adiponectin levels were negatively correlated with aggressive features of PTC, including tumor diameter > 1 cm, extrathyroidal extension, and lymph node metastasis. Recombinant human adiponectin inhibited the proliferation and migration of human PTC cells in vitro. Bioinformatics analysis identified adiponectin receptor 2 (ADIPOR2) and the autophagy pathway as possible mediators of adiponectin function in TC. In vitro experiments confirmed that adiponectin activated autophagy in PTC cells. These findings shed new lights into the role and mechanisms of adiponectin in TC pathogenesis. Adiponectin is involved in development of obesity-related PTC. Adiponectin can directly inhibit thyroid cancer growth and metastasis through the autophagy pathway.

2225P FOX1 (rs965513), NKX2-1 (rs944289) and BRAF (V600E) gene mutations and their role in development of papillary thyroid cancer

2225P FOX1 (rs965513), NKX2-1 (rs944289) and BRAF (V600E) gene mutations and their role in development of papillary thyroid cancer

Activation of mitogen-activated protein kinase signaling and development of papillary thyroid carcinoma in thyroid-stimulating hormone receptor D633H knockin mice.

Nonautoimmune hyperthyroidism (NAH) is rare and occurs due to a constitutively activating thyroid stimulating hormone receptor (TSHR) mutation. In contrast to other thyroid nodules, no further evaluation for malignancy is recommended for hot thyroid nodules. In the first model for NAH in mice nearly all homozygous mice had developed papillary thyroid cancer by 12 months of age. To further evaluate these mice, whole exome sequencing and phosphoproteome analysis were employed in a further generation of mice to identify any other mutations potentially responsible and to identify the pathways involved in thyroid carcinoma development. Only three genes (Nrg1, Rrs1, Rasal2) were mutated in all mice examined, none of which were known primary drivers of papillary thyroid cancer development. Wild-type and homozygous TSHR D633H knockin mice showed distinct phosphoproteome profiles with an enrichment of altered phosphosites found in ERK/mitogen-activated protein kinase (MAPK) signaling. Most importantly, phosphosites with known downstream effects included BRAF p.S766, which forms an inhibitory site: a decrease of phosphorylation at this site suggests an increase in MEK/ERK pathway activation. The decreased phosphorylation at BRAF p.S766 would suggest decreased AMP-activated protein kinase (AMPK) signaling, which is supported by the decreased phosphorylation of STIM1 p.S257, a downstream AMPK target. The modified phosphoproteome profile of the homozygous mice in combination with human literature suggests a potential signaling pathway from constitutive TSHR signaling and cAMP activation to the activation of ERK/MAPK signaling. This is the first time that a specific mechanism has been identified for a possible involvement of TSH signaling in thyroid carcinoma development.

PIWI interacting RNA-13643 contributes to papillary thyroid cancer development through acting as a novel oncogene by facilitating PRMT1 mediated GLI1 methylation

Recently, aberrant expression of PIWI-interacting RNAs (piRNAs) has been discovered in a variety of cancer cells. However, the roles of PIWI proteins and piRNAs in papillary thyroid carcinoma (PTC) are still elusive. RT-qPCR and Northern blotting were used to evaluate piR-13643 levels in PTC and para-carcinoma tissues, as well as in PTC cell lines. piR-13643 mimic and piR-13643 inhibitor were transfected into K-1 and B-CPAP cells. CCK-8, Transwell, annexin V-FITC/PI, flow cytometry and Western blot assays were performed to measure cell proliferation, invasion, apoptosis, cell cycle and E-cadherin and Vimentin proteins, respectively. Total RNA from B-CPAP cells was pulled down with PIWIL1, PIWIL2, or PIWIL3 specific antibodies or IgG as a control, respectively, followed by detection of piR-13643 expression with RT-qPCR. Immunoblotting of PRMT1 was detected in piR-13643 / PIWIL1 complex immune-precipitates by Co-IP assay. Subsequently, PRMT1 protein expression was detected by stably transfection of Flag tagged GLI1 (Flag-GLI1) into B-CPAP cells. Methylation assay with PRMT1 and wild-type or R597 lysine (R597K)-mutant GLI1. Then rescue experiments were applied to explore effects of piR-13643 and GLI1 on the malignant behavior of PTC cells. B-CPAP cells transfected with piR-13643 inhibitor were subcutaneously injected into nude mice to evaluate the effect of piR-13643 knockdown on the xenograft tumor growth of PTC. piR-13643 was elevated in PTC patient specimens and cell lines. piR-13643 overexpression facilitated cell proliferation, invasion and Vimentin level, and restrained apoptosis and E-cadherin expression, whereas piR-13643 knockdown showed the opposite results. Mechanically, piR-13643 could bind to PIWIL1 to form the PIWIL1/piR-13643 complex, and PRMT1 enhanced GLI1 transcription by methylating GLI1 at R597. Further, PIWIL1/piR-13643 promoted PRMT1-mediated GLI1 methylation. GLI1 knockdown countered the effects of piR-13643 mimic on cell malignant behaviors. piR-13643 knockdown preeminently prevented the xenograft tumor growth of PTC in vivo. This study confirmed that piR-13643 facilitates PTC malignant behaviors in vitro and in vivo by promoting PRMT1-mediated GLI1 methylation via forming a complex with PIWIL1, which may provide a novel insight for PTC treatment.

Hypoxic papillary thyroid carcinoma cells-secreted exosomes deliver miR-221-3p to normoxic tumor cells to elicit a pro-tumoral effect by regulating the ZFAND5

Papillary thyroid cancer (PTC) has seen a worldwide expansion in incidence in the past three decades. Tumor-derived exosomes have been associated with the metastasis of cancer cells and are present within the local hypoxic tumor microenvironment, where they mediate intercellular communication by transferring molecules including microRNAs (miRNAs) between cells. Although miRNAs have been shown to serve as non-invasive biomarkers for cancer diagnosis, the role of hypoxia-induced tumor-derived exosomes in PTC progression remains unclear. Herein, we investigated the differentially expressed miRNA expression profiles from GEO datasets (GSE191117 and GSE151180) by using the DESeq package in R and identified a novel role for miR-221-3p as an oncogene in PTC development. In vivo and in vitro loss and gain assays were used to clarify the mechanism of hypoxic PTC cells derived exosomal-miR-221-3p in PTC. miR-221-3p was upregulated in human PTC plasma exosomes, tissues and cell lines. We found that hypoxic PTC cells derived exosomal-miR-221-3p promoted normoxic PTC cells proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in vitro, while inhibition of miR-221-3p limited PTC tumor growth in our PTC xenograft model in nude mice. We finally identified ZFAND5, to be a miR-221-3p target. Mechanistically, hypoxic PTC cell lines-derived exosomes carrying miR-221-3p promoted PTC tumorigenesis by regulating ZFAND5. Our findings further the understanding of the underlying mechanisms associated with PTC progression and identify exosomal-miR-221-3p as a potential biomarker for the diagnosis and prognosis of PTC patients. Our study also suggests that miR-221-3p inhibitors could be a potential treatment strategy for PTC.

SRPX2 promotes cancer cell proliferation and migration of papillary thyroid cancer

Thyroid cancer is the endocrine tumor with the highest incidence at present. It originates from the thyroid follicular epithelium or follicular paraepithelial cells. There is an increasing incidence of thyroid cancer all over the world. We found that SRPX2 expression level was higher in papillary thyroid tumors than in normal thyroid tissues, and SRPX2 expression was closely related to tumor grade and clinical prognosis. Previous reports showed that SRPX2 could function by activating PI3K/AKT signaling pathway. In addition, in vitro experiments showed that SRPX2 promoted the proliferation and migration of papillary thyroid cancer (PTC). In conclusion, SRPX2 could promote the malignant development of PTC. This may be a potential treatment target for PTC.

LncRNA GLTC targets LDHA for succinylation and enzymatic activity to promote progression and radioiodine resistance in papillary thyroid cancer.

Dysregulation of long noncoding RNAs (lncRNAs) has been associated with the development and progression of many human cancers. Lactate dehydrogenase A (LDHA) enzymatic activity is also crucial for cancer development, including the development of papillary thyroid cancer (PTC). However, whether specific lncRNAs can regulate LDHA activity during cancer progression remains unclear. Through screening, we identified an LDHA-interacting lncRNA, GLTC, which is required for the increased aerobic glycolysis and cell viability in PTC. GLTC was significantly upregulated in PTC tissues compared with nontumour thyroid tissues. High expression of GLTC was correlated with more extensive distant metastasis, a larger tumour size, and poorer prognosis. Mass spectrometry revealed that GLTC, as a binding partner of LDHA, promotes the succinylation of LDHA at lysine 155 (K155) via competitive inhibition of the interaction between SIRT5 and LDHA, thereby promoting LDHA enzymatic activity. Overexpression of the succinylation mimetic LDHAK155E mutant restored glycolytic metabolism and cell viability in cells in which metabolic reprogramming and cell viability were ceased due to GLTC depletion. Interestingly, GLTC inhibition abrogated the effects of K155-succinylated LDHA on radioiodine (RAI) resistance in vitro and in vivo. Taken together, our results indicate that GLTC plays an oncogenic role and is an attractive target for RAI sensitisation in PTC treatment.

Clinical Implications of mTOR Expression in Papillary Thyroid Cancer-A Systematic Review.

Papillary thyroid cancer (PTC) comprises approximately 80% of all thyroid malignancies. Although several etiological factors, such as age, gender, and irradiation, are already known to be involved in the development of PTC, the genetics of cancerogenesis remain undetermined. The mTOR pathway regulates several cellular processes that are critical for tumorigenesis. Activated mTOR is involved in the development and progression of PTC. Therefore, we performed a systematic review of papers studying the expression of the mTOR gene and protein and its relationship with PTC risk and clinical outcome. A systematic literature search was performed using PubMed, Embase, and Scopus databases (the search date was 2012-2022). Studies investigating the expression of mTOR in the peripheral blood or tissue of patients with PTC were deemed eligible for inclusion. Seven of the 286 screened studies met the inclusion criteria for mTOR gene expression and four for mTOR protein expression. We also analyzed the data on mTOR protein expression in PTC. We analyzed the association of mTOR expression with papillary thyroid cancer clinicopathological features, such as the TNM stage, BRAF V600E mutation, sex distribution, lymph node and distant metastases, and survival prognosis. Understanding specific factors involved in PTC tumorigenesis provides opportunities for targeted therapies. We also reviewed the possible new targeted therapies and the use of mTOR inhibitors in PTC. This topic requires further research with novel techniques to translate the achieved results to clinical application.

MicroRNA-96-5p is negatively regulating GPC3 in the metastasis of papillary thyroid cancer

Backgrounds: Papillary thyroid cancer is the most common pathological type of thyroid cancer. miR-96-5p, a member of the miR-183 family, constitute a polycistronic miRNA cluster. In breast cancer, miR-96-5p promotes cell invasion, migration, and proliferation in vitro by inhibiting PTPN9. Moreover, miR-96-5p was reported to function as an oncogene in many cancers. However, whether miR-96-5p is involved in the development of papillary thyroid cancers and its potential mechanism is still unknown. The present study aims to explore the relationship between miR-96-5p and GPC3 expression in the development of papillary thyroid cancers. Methods: Transcriptomic sequencing was carried out using six pairs of papillary thyroid cancer and adjacent normal tissues. Quantitative real-time polymerase chain reaction (PCR) experiments were performed to examine the expression of genes. Results: In total, there were 1588 up-regulated and 1803 down-regulated differentially expressed genes between papillary thyroid cancer and normal tissues. Gene ontology and Kyoto encyclopedia of genes and genomes analysis revealed that extracellular matrix structure and proteoglycans were mainly involved in papillary thyroid cancer. Among the cluster of proteoglycans, GPC3 was significantly down-regulated in papillary thyroid cancer and is a target of miR-96. Conclusion: miR-96-5p participates in the development of papillary thyroid cancer by regulating the expression of GPC3. Thus, targeting miR-96-5p may be a potential therapeutic approach for preventing and treating papillary thyroid cancer.

M6A Writer METTL3-Mediated lncRNA LINC01125 Prevents the Malignancy of Papillary Thyroid Cancer.

Long non-coding RNA (lncRNA) LINC01125 is an anti-tumor factor in a variety of tumors, and regulates cancer cell function. However, its function and mechanism of N6-methyladenosine (m6A) modification in papillary thyroid cancer (PTC) tumorigenesis remain unclear. This study aimed to reveal the function and m6A modification of LINC01125 in PTC tumorigenesis. The LINC01125 and methyltransferase-like 3 (METTL3) levels in PTC cells and tissues was assessed by qRT-PCR. The binding relationship among LINC01125 and METTL3 was determined by MeRIP, Pearson, bioinformatics, and RNA stabilization analysis. Transwell assays were performed to confirm the changes of PTC cell migration and invasion. Cell proliferation was revealed by CCK-8 as well as colony formation assays. Low expression of LINC01125 and METTL3 was identified in PTC. LINC01125 was a downstream target of METTL3-mediated m6A modification and was stably upregulated via METTL3. Cell invasion, migration, viability, and colony formation levels were decreased when LINC01125 or METTL3 was upregulated. Inhibition of LINC01125 had the opposite impact, promoting cell proliferation and metastasis, and reversing METTL3 overexpression-resulted cell malignancy suppression. Overall, this study proved that the m6A modification of LINC01125 was mediated by METTL3 and LINC01125 inhibited cell invasion, migration and proliferation, thereby suppressing the development of PTC. This points to the LINC01125-m6A-METTL3 axis as a possible prospective target for future treatment of PTC.

IncRNA XIST Stimulates Papillary Thyroid Cancer Development through the miR-330-3p/PDE5A Axis

Long non-coding RNAs (lncRNAs) possess both tumor suppressive and oncogenic functions in papillary thyroid cancer (PTC). Among all the thyroid cancers, PTC is the most prevalent form. Herein, we aim to determine the regulatory mechanisms and functions of lncRNA XIST in the multiplication, invasion, and survival of PTC. Quantitative reverse transcription polymerase chain reaction and Western blot experiments were performed to determine the patterns of lncRNA XIST, miR-330-3p, and PDE5A expressions. The subcellular localization of XIST was determined through subcellular fractionation. Bioinformatics analyses were performed to determine miR-330-3p's relationships with XIST and PDE5A, which were further confirmed through luciferase reporter assays. Loss-of-function combined with Transwell, CCK-8, and caspase-3 activity experiments were performed to determine the mechanism of the XIST/miR-330-3p/PDE5A axis in regulating the malignancy of PTC cells. Xenograft tumor experiment was employed to study the influence of XIST on tumor development in vivo. The PTC cell lines and tissues manifested considerably high levels of lncRNA XIST expression. The XIST knockdown inhibited proliferation, blocked migration, and strengthened apoptosis among PTC cells. Moreover, its knockdown suppressed PTC tumor development in vivo. XIST repressed miR-330-3p to stimulate the malignant behaviors of PTC. Through the downregulation of PDE5A, miR-330-3p attenuated the capability of PTC cells to grow, migrate, and survive. lncRNA XIST promotes tumor development in PTC through the regulation of the miR-330-3p/PDE5A axis. The findings from this study provide new insights into the treatment of PTC.

Integrative Analysis of Potential Biomarkers Involved in the Progression of Papillary Thyroid Cancer.

This study aims to explore key prognostic and diagnostic biomarkers involved in the pathogenesis of papillary thyroid cancer (PTC) which is one of the most common endocrine cancers and whose occurrence is rapidly increasing. Papillary thyroid cancer datasets containing normal and tumor samples were collected from Gene Expression Omnibus. Protein-protein interaction (PPI) network for common upregulated differentially expressed genes (DEGs) was constructed, and hub genes were studied. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed to identify the vital biological behaviors and pathways involved in PTC. PPI network analysis demonstrated the interaction between 134 common upregulated DEGs, and top 15 pivotal genes with highest degree of connectivity were retrieved. Three of the hub genes (DPP4, ITGA2, FN1) were linked to the prognosis of PTC patients and considered clinically relevant core genes via survival analysis. We suggest that the identification of key genes associated with PTC development help us in understanding molecular mechanisms related to disease. These genes could also be considered the diagnostic biomarkers or as therapeutic targets in the future treatment for PTC.

OR09-4 Novel Murine Models of BrafV600E driven Papillary Thyroid Cancer

Abstract Thyroid cancer is the most prevalent endocrine malignancy and is estimated to have affected 52,890 individuals in the United States in the year 2020. Papillary thyroid cancer (PTC) accounts for up to 80% of all thyroid cancer diagnoses. Within PTC, BRAFV600E is the most common mutation in adults and the second most common mutation in pediatrics. While there are several validated human thyroid cancer cell lines harboring BRAF mutations, no pediatric derived cell line has been established to date, limiting generalizations in pediatric disease. Patient-derived pre-clinical models are valuable tools but are limited due to their need to be studied in vitro or in an immunocompromised host. However, in vivo mouse models recapitulate the complex interactions between tumor cells, the immune system, and components of the microenvironment, allowing more comprehensive investigation of thyroid oncogenesis and response to therapy. In this study we describe a new subcutaneous mouse model that can be used to understand differences between pediatric onset and adult onset PTC. We have developed multiple independent, congenic murine cell lines from different stages of thyroid cancer progression. Two independent BRAFV600E-driven cell lines harboring the same genetic mutations were utilized in this study, one representing a well-differentiated tumor (WD) and one representing a poorly-differentiated tumor (PD). Despite coming from different stages of disease, both cell lines showed similar expression of the thyroid specific genes: Pax8, Ttf1, Tg, and Slc5a5, but expression of Pax8, Tg, and Slc5a5 was lower compared to WT thyrocytes. Activation of the Pi3Kinase pathway and the Akt Pathway were assessed via western blot analysis. The WD cell line had increased pERK activation compared to the PD cell line, and the PD cell line had increased pAKT compared to WD cell line. Both cell lines were injected subcutaneously into the hind flank of Wt SJV129 mice of pediatric age (4-5 weeks) and adult age (20-22 weeks). These animals were monitored for 10 weeks post injection. While the PD cell line developed tumors at approximately the same rate and penetrance in both age groups, tumors reached end point more rapidly in the adult cohort. Adult hosts injected with the WD cell line developed tumors more rapidly than in pediatric hosts, however, tumors receded in both cohorts. While adult and pediatric thyroid cancer share common driving mutations, there are distinct differences in disease pathogenesis between these patient populations. There are known differences in the immune system, metabolism, and other variables between pediatrics and adults. However, it is unknown what causes the difference in PTC pathogenesis between pediatric versus adult patients. We believe these new models provide a great opportunity to evaluate the role of age in PTC development and progression. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

Silencing circ_0000644 inhibits papillary thyroid cancer cell malignancy by combining with miR-671-5p to release the inhibition on ANXA2.

Papillary thyroid cancer (PTC) is life-threatening due to its malignant progression. Considerable evidence demonstrates that circular RNA (circRNA) regulates PTC development. This study aims to explore the mechanism of circ_0000644 modulating PTC malignant progression. The RNA levels of circ_0000644, microRNA-671-5p (miR-671-5p) and annexin A2 (ANXA2) were detected by quantitative real-time polymerase chain reaction. Western blot was performed to check protein expression. Cell proliferation and cell apoptosis were investigated by 5-ethynyl-29-deoxyuridine and flow cytometry. Angiogenic capacity, migration and invasion were analyzed by tube formation assay and transwell assay. The interaction between miR-671-5p and circ_0000644 or ANXA2 was identified by dual-luciferase reporter assay. Xenograft mouse model assay was performed to analyze the effect of circ_0000644 on tumor formation in vivo. Circ_0000644 and ANXA2 expression was significantly upregulated, while miR-671-5p was downregulated in PTC tissues and cells when compared with control groups. Circ_0000644 knockdown inhibited PTC cell proliferation, tube formation, migration, and invasion, but induced apoptosis in vitro. Moreover, circ_0000644 knockdown led to delayed tumorigenesis in vivo. In addition, circ_0000644 acted as a miR-671-5p sponge and mediated PTC cell tumor properties through miR-671-5p. ANXA2 was identified as a target gene of miR-671-5p, and its overexpression relieved miR-671-5p-induced effects in PTC cells. Furthermore, circ_0000644 depletion inhibited ANXA2 production by combining with miR-671-5p. Circ_0000644 depletion repressed PTC cell tumor properties through the miR-671-5p/ANXA2 axis.

AXL, along with PROS1, is overexpressed in papillary thyroid carcinoma and regulates its biological behaviour

BackgroundAXL, a TAM tyrosine kinase receptor, plays an essential role in the pathogenesis of various solid tumours. This study explores the role of AXL and its ligand PROS1 in the generation and biological behaviour of papillary thyroid cancer (PTC).MethodsThe expression levels of AXL in PTC cancer tissue were analysed using immunohistochemistry (IHC) staining. The expression levels of AXL in PTC and normal thyroid cell lines were analysed using real-time quantitative polymerase chain reaction (RT-qPCR). CCK-8 was used to assess the proliferation of the PTC cell line with and without the effect of the AXL inhibitor (R428). Scratching assays played a role in evaluating the cell migration rate.ResultsPROS1 and AXL were expressed in TPC-1, B-CPAP, and Nthy-Ori 3–1 cells at different levels. Expression was significantly higher in PTC cell lines (TPC-1 and B-CPAP) than in the normal thyroid cell line (Nthy-Ori 3–1) (p < 0.05). In addition, AXL expression in PTC tissues was significantly higher than in adjacent normal tissues (p < 0.05). CCK-8 experiments confirmed that R428 suppresses the proliferation of PTC cell lines in a dose-dependent manner, with an increase in concentration from 0.5 to 4 μM, decreasing the inhibitory effect (p < 0.01). In addition, R428 inhibited PTC cell line migration to different degrees in a range of concentrations from 0.5 to 2 μM compared to control cells (p < 0.01).ConclusionPROS1 and its downstream receptor AXL expression were significantly higher in PTC than in normal thyroid cells. AXL expression was also higher in human PTC tissues than in normal thyroid tissues. Inhibiting the PROS1-AXL-mediated TAM signaling pathway via the AXL blocker R428 suppressed the proliferation and migration of human PTC cells, highlighting the role of this cascade in human PTC development and progression.