Highly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancer.

BACKGROUND: Thyroid cancer is the most frequent malignancy of the endocrine system, of which papillary thyroid cancer (PTC) is the predominant form with a rapid increasing incidence worldwide. Rearranged during transfection (RET) fusions are common genetic drivers of PTC and the potent RET inhibitor selpercatinib has been recently approved for treating advanced or metastatic RET fusion-positive thyroid cancer. In this study we aimed to develop a droplet digital PCR (ddPCR) system to accurately detect RET fusion in PTC samples. METHODS: The frequency and distribution of RET fusions in PTC were analyzed using genomic data of 402 PTC patients in The Cancer Genome Atlas (TCGA) database. To establish the ddPCR system for detecting CCDC6::RET fusion, a plasmid containing CCDC6::RET infusion fragment was constructed as standard template, the annealing temperature and concentrations of primers and probe were optimized. The analytical performance of ddPCR and quantitative reverse transcription PCR (qRT-PCR) were assessed in standard templates and tissue samples from 112 PTC patients. Sanger sequencing was performed in all the RET fusion-positive samples identified by ddPCR. RESULTS: RET fusions were observed in 25 (6.2%) of the 402 TCGA samples, and 15 (60%) of the RET fusion-positive patients had the CCDC6::RET fusion. Compared with qRT-PCR, the ddPCR method showed a lower limit of detection (128.0 and 430.7 copies/reaction for ddPCR and qRT-PCR, respectively). When applying the two methods to 112 tissue samples of PTC, eleven (9.8%) CCDC6::RET fusion-positive samples were detected by qRT-PCR, while ddPCR identified 4 additional positive samples (15/112, 13.4%). All the CCDC6::RET fusion-positive cases identified by ddPCR were confirmed by Sanger sequencing except for one case with 0.14 copies/uL of the fusion. CONCLUSION: The accurate and sensitive ddPCR method reported here is powerful to detection CCDC6::RET fusion in PTC samples, application of this method would benefit more RET fusion-positive patients in the clinic.

Metabolome-Wide Mendelian Randomization Assessing the Causal Relationship Between Blood Metabolites and Bone Mineral Density.

Mounting evidence has supported osteoporosis (OP) as a metabolic disorder. Recent metabolomics studies have discovered numerous metabolites related to bone mineral density (BMD). However, the causal effects of metabolites on BMD at distinct sites remained underexplored. Leveraging genome-wide association datasets, we conducted two-sample Mendelian randomization (MR) analyses to investigate the causal relationship between 486 blood metabolites and bone mineral density at five skeletal sites including heel (H), total body (TB), lumbar spine (LS), femoral neck (FN), and ultra-distal forearm (FA). Sensitivity analyses were performed to test the presence of the heterogeneity and the pleiotropy. To exclude the influences of reverse causation, genetic correlation, and linkage disequilibrium (LD), we further performed reverse MR, linkage disequilibrium regression score (LDSC), and colocalization analyses. In the primary MR analyses, 22, 10, 3, 7, and 2 metabolite associations were established respectively for H-BMD, TB-BMD, LS-BMD, FN-BMD, and FA-BMD at the nominal significance level (IVW, P < 0.05) and passing sensitivity analyses. Among these, one metabolite, androsterone sulfate showed a strong effect on four out of five BMD phenotypes (Odds ratio [OR] for H-BMD = 1.045 [1.020, 1.071]; Odds ratio [OR] for TB-BMD = 1.061 [1.017, 1.107]; Odds ratio [OR] for LS-BMD = 1.088 [1.023, 1.159]; Odds ratio [OR] for FN-BMD = 1.114 [1.054, 1.177]). Reverse MR analysis provided no evidence for the causal effects of BMD measurements on these metabolites. Colocalization analysis have found that several metabolite associations might be driven by shared genetic variants such as mannose for TB-BMD. This study identified some metabolites causally related to BMD at distinct sites and several key metabolic pathways, which shed light on predictive biomarkers and drug targets for OP.

METTL3-m6A-Rubicon axis inhibits autophagy in nonalcoholic fatty liver disease.

N6-methyladenosine (m6A) mRNA modification plays critical roles in various biological events and is involved in multiple complex diseases. However, the role of m6A modification in autophagy in nonalcoholic fatty liver disease (NAFLD) remains largely unknown. Here, we report that m6A modification was increased in livers of NAFLD mouse models and in free fatty acid (FFA)-treated hepatocytes, and the abnormal m6A modification was attributed to the upregulation of methyltransferase like 3 (METTL3) induced by lipotoxicity. Knockdown of METTL3 promoted hepatic autophagic flux and clearance of lipid droplets (LDs), while overexpression of METTL3 inhibited these processes. Mechanistically, METTL3 directly bound to Rubicon mRNA and mediated the m6A modification, while YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), as a partner of METTL3, interacted with the m6A-marked Rubicon mRNA and promoted its stability. Subsequently, RUBICON inhibited autophagosome-lysosome fusion and further blocked clearance of LDs. Taken together, our results showed a critical role of METTL3 and YTHDF1 in regulating lipid metabolism via the autophagy pathway and provided a novel insight into m6A mRNA methylation in NAFLD.

Lipotoxicity-induced circGlis3 impairs beta cell function and is transmitted by exosomes to promote islet endothelial cell dysfunction.

AIMS/HYPOTHESIS: Lipotoxicity constitutes the major driving force for type 2 diabetes. Circular RNAs (circRNAs) play important roles in regulating beta cell function and exosomes are essential mediators of intercellular communication. The role of exosomal circRNAs in type 2 diabetes remains largely unknown. We aimed to examine whether lipotoxicity induces dysregulation of circRNAs in beta cell-derived exosomes and to determine the contribution of exosomal circRNAs to the development of type 2 diabetes. METHODS: Exosomes were extracted from MIN6 cells treated with palmitate or BSA, and RNA sequencing was performed. CircGlis3 (Gli-similar 3) expression level was validated by qPCR. The impact of circGlis3 on beta cell function and the deleterious effects of exosomal circGlis3 on islet endothelial cells (islet ECs) were investigated in vitro and in vivo in human and mouse models by gain or loss of function assays. The molecular mechanism of circGlis3 was explored by RNA pull-down and immunoprecipitation assays. RESULTS: Beta cell-derived exosomal circGlis3 was significantly upregulated under lipotoxic conditions, and exosomal circGlis3 levels were also elevated in the serum of mouse models of diabetes and participants with type 2 diabetes. CircGlis3 participated in lipotoxicity-induced beta cell dysfunction in vitro and in vivo. Moreover, beta cell-derived exosomal circGlis3 could be transferred to islet ECs and reduce the cell viability, cell migration and angiogenesis of islet ECs. Mechanistically, circGlis3 promoted the degradation of glucocorticoid modulatory element-binding protein 1 (GMEB1) by facilitating the interaction between GMEB1 and mindbomb E3 ubiquitin protein ligase 2 (MIB2), thus suppressing the phosphorylation of heat shock protein 27 (HSP27). CONCLUSIONS/INTERPRETATION: Our study points to the involvement of circGlis3 in diabetes development, and exosomal circGlis3 transfer as a communication mode between beta cells and islet ECs, suggesting that circGlis3 might be a potential biomarker and therapeutic target for type 2 diabetes. DATA AVAILABILITY: The RNA-sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) database, with accession number PRJNA689673. Mass spectrometry data are available via ProteomeXchange with identifier PXD024693.

STRA6 regulates tumor immune microenvironment and is a prognostic marker in BRAF-mutant papillary thyroid carcinoma.

Background: BRAF mutation is one of the most common genetic alterations contributing to the initiation and progression of papillary thyroid carcinoma (PTC). However, the prognostic value of BRAF mutation for PTC is limited. Novel markers are needed to identify BRAF-mutant patients with poor prognosis. Methods: Transcriptional expression data were downloaded from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Pathway enrichment was performed by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set enrichment analysis (GSEA). Protein-protein interaction networks were predicted by the GeneMANIA. The correlation between STRA6 expression and immune infiltration was analyzed by tumor immune estimation resource (TIMER) and tumor-immune system interaction database (TISIDB). Immunohistochemistry was used to detect the STRA6 protein expression level of PTC. Infiltration of regulatory T cells (Tregs) and CD8+ T cells in tumor samples were analyzed by fluorescent multiplex immunohistochemistry. Results: In BRAF-mutant PTC, STRA6 was extremely upregulated and predicted unfavorable survival, which was an independent risk factor for increased mortality risk. Bioinformatic analyses indicated that STRA6 might activate the MAPK pathway synergistically with BRAFV600E. The expression of STRA6 was associated with immune infiltrates and T cell exhaustion. Fluorescent multiplex immunohistochemistry showed that STRA6 increased Tregs abundance and decreased CD8+ T cells infiltration in PTC. Moreover, STRA6 promoted epithelial-mesenchymal transition via increased cancer-associated fibroblasts infiltration. Conclusions: Our study demonstrates STRA6 may serve as a prognostic marker for BRAF-mutated PTC, which may drive thyroid carcinogenesis via activation of oncogenic pathway and regulation of tumor immunosuppressive microenvironment.

CircGLIS3 inhibits thyroid cancer invasion and metastasis through miR-146b-3p/AIF1L axis.

PURPOSE: Studies have shown that circRNA is involved in the occurrence and development of human cancers. However, it remains unclear that the contribution of circRNA in thyroid carcinoma and its role in the process of tumorigenesis. METHODS: The expression profile of circRNA-miRNA-mRNA in thyroid carcinoma was detected by RNA sequencing and verified by qRT-PCR. The characteristics of circGLIS3 were verified by RNase R and actinomycin assays, subcellular fractionation, and fluorescence in situ hybridization. The functions of circGLIS3 and AIF1L were detected by wound healing, transwell, 3D culture and Western blot. RNA Immunoprecipitation (RIP), RNA pulldown and dual-luciferase reporter assays were used to verify the target genes of circGLIS3 and downstream miRNAs. Functional rescue experiments were performed by transfecting miRNA mimics or siRNA of target genes. Finally, metastatic mouse models were used to investigate circGLIS3 function in vivo. RESULTS: In this study, we discovered a novel circRNA (has_circ_0007368, named as circGLIS3) by RNA sequencing. CircGLIS3 was down-regulated in thyroid carcinoma tissues and cells line, and was negatively associated with malignant clinical features of thyroid carcinoma. Functional studies found that circGLIS3 could inhibit the migration and invasion of thyroid carcinoma cells, and was related to the EMT process. Mechanistically, circGLIS3 can upregulate the expression of the AIF1L gene by acting as a miR-146b-3p sponge to inhibit the progression of thyroid carcinoma. CONCLUSION: Our study identified circGLIS3 as a novel tumor suppressor in thyroid cancer, indicating the potential of circGLIS3 as a promising diagnostic and prognostic marker for thyroid cancer.

STRA6 Promotes Thyroid Carcinoma Progression via Activation of the ILK/AKT/mTOR Axis in Cells and Female Nude Mice.

BACKGROUND: Metastasis has emerged to be an important cause for poor prognosis of thyroid carcinoma (TC) and its molecular mechanisms are not fully understood. STRA6 is a multifunctional membrane protein widely expressed in embryonic and adult tissues. The function and mechanism of STRA6 in TC remain elusive. OBJECTIVE: We aimed to explore the role of STRA6 in TC progression and provide a therapeutic target for TC. METHODS: The expression and clinicopathological relevance of STRA6 were explored in TC. Stable STRA6-knockdown TC cells were established and used to determine the biological function of STRA6 in vitro and in vivo. RNA sequencing and co-immunoprecipitation were performed to unveil the molecular mechanism of STRA6 in TC progression. The potential of STRA6 as a therapeutic target was evaluated by lipid nanoparticles (LNPs) containing siRNA. RESULTS: STRA6 was upregulated in TC and correlated with aggressive clinicopathological features, including extrathyroidal extension and lymph node metastasis, which contributed to the poor prognosis of TC. STRA6 facilitated TC progression by enhancing proliferation and metastasis in vitro and in vivo. Mechanistically, STRA6 could interact with integrin-linked kinase (ILK) and subsequently activate the protein kinase B/mechanistic target of rapamycin (AKT/mTOR) signaling pathway. We further unveiled that STRA6 reprogrammed lipid metabolism through SREBP1, which was crucial for the metastasis of TC. Moreover, STRA6 siRNA delivered by LNPs significantly inhibited cell growth in xenograft tumor models. CONCLUSIONS: Our study demonstrates the critical roles of STRA6 contributing to TC progression via the ILK/AKT/mTOR axis, which may provide a novel prognostic marker as well as a promising therapeutic target for aggressive TC.

Distinct molecular subtypes of papillary thyroid carcinoma and gene signature with diagnostic capability.

Papillary thyroid carcinoma (PTC) is heterogeneous and its molecular characteristics remain elusive. We integrated transcriptomic sequencing, genomic analysis and clinicopathologic information from 582 tissue samples of 216 PTC and 75 benign thyroid nodule (BTN) patients. We discovered four subtypes of PTC including Immune-enriched Subtype, BRAF-enriched Subtype, Stromal Subtype and CNV-enriched Subtype. Molecular subtypes were validated in an external cohort of 497 PTC cases from the TCGA. Tumors in the Immune-enriched Subtype showed higher immune infiltration and overexpression of immune checkpoints, whilst BRAF-enriched Subtype showed a higher tendency for extrathyroidal extension and more advanced TNM stage. Key oncogenes including LRRK2, SLC34A2, MUC1, FOXQ1 and KRT19 were overexpressed and enriched in oncogenic MAPK and PI3K/AKT signaling pathways in BRAF-enriched subtype. Further analysis of BRAF-enriched Subtype identified three subclasses with different degrees of malignancies. We also uncovered the molecular link of the initiation and progression from BTN to subtypes of PTC using trajectory analysis. Moreover, a 20-gene expression signature was generated for differential diagnosis of PTC from BTN patients. Together, our work identified previously unreported molecular subtypes of PTC, offering opportunities to stratify patients into optimal treatment plans based on molecular subtyping.

Selenite Induces Cell Cycle Arrest and Apoptosis via Reactive Oxygen Species-Dependent Inhibition of the AKT/mTOR Pathway in Thyroid Cancer.

Thyroid cancer is the most common endocrine malignancy, and its incidence has increased in the past decades. Selenium has been shown to have therapeutic effects against several tumors. However, its role in thyroid cancer and its underlying molecular mechanism remains to be explored. In the present study, we demonstrated that sodium selenite significantly decreased cell viability and induced G0/G1 cell cycle arrest and apoptosis in thyroid cancer cells in a dose-dependent manner. Transcriptomics revealed that sodium selenite induced intracellular reactive oxygen species (ROS) by promoting oxidative phosphorylation. Increased intracellular ROS levels inhibited the AKT/mTOR signaling pathway and upregulated EIF4EBP3. Intracellular ROS inhibition by N-acetylcysteine (NAC) ameliorated the cellular effects of sodium selenite. The in vitro findings were reproduced in xenograft thyroid tumor models. Our data demonstrated that sodium selenite exhibits strong anticancer effects against thyroid cancer cells, which involved ROS-mediated inhibition of the AKT/mTOR pathway. This suggests that sodium selenite may serve as a therapeutic option for advanced thyroid cancer.

LncRNA-Malat1 is Involved in Lipotoxicity-Induced ß-cell Dysfunction and the Therapeutic Effect of Exendin-4 via Ptbp1.

Increasing evidence indicates that long noncoding RNAs (lncRNAs) have crucial roles in various biological processes. However, the contribution of lncRNAs to ß-cell dysfunction and their roles in diabetes therapeutics remain poorly understood. The aim of this study was to identify the lncRNAs dysregulated in diabetic islets and to explore the lncRNAs involved in ß-cell function as potential therapeutic targets. By using RNA sequencing and real-time PCR, we identified thousands of lncRNAs in the islets of db/db mice and db/m littermate mice. Among the differentially expressed lncRNAs, lncRNA-Malat1 (metastasis-associated lung adenocarcinoma transcript 1) was reduced in the islets of db/db mice and palmitate-treated MIN6 cells. The results of TUNEL, Western blot and flow cytometric analyses, and GSIS assays revealed that Malat1 knockdown significantly induced ß-cell apoptosis and inhibited insulin secretion. Mechanistically, RNA immunoprecipitation showed that Malat1 enhanced polypyrimidine tract-binding protein 1 (Ptbp1) protein stability by direct interaction, thereby adjusting the ratio of pyruvate kinase muscle (PKM) isoforms 1 and 2 (PKM1/PKM2). Moreover, luciferase assay and chromatin immunoprecipitation indicated that Malat1 was transcriptionally activated by pancreatic and duodenal homeobox 1 (Pdx1), through which exendin-4 alleviated lipotoxicity-induced ß-cell damage. In summary, our findings suggested the involvement of Malat1 in ß-cell dysfunction under diabetic conditions via the Malat1/Ptbp1/PKM2 pathway. In addition, exendin-4 ameliorated ß-cell impairment by Pdx1-mediated Malat1 upregulation. Hence, Malat1 may serve as a therapeutic target for the treatment of type 2 diabetes.