LncRNAs in the Dlk1-Dio3 Domain Are Essential for Mid-Embryonic Heart Development.

The Dlk1-Dio3 domain is important for normal embryonic growth and development. The heart is the earliest developing and functioning organ of the embryo. In this study, we constructed a transcriptional termination model by inserting termination sequences and clarified that the lack of long non-coding RNA (lncRNA) expression in the Dlk1-Dio3 domain caused the death of maternal insertion mutant (MKI) and homozygous mutant (HOMO) mice starting from E13.5. Parental insertion mutants (PKI) can be born and grow normally. Macroscopically, dying MKI and HOMO embryos showed phenomena such as embryonic edema and reduced heart rate. Hematoxylin and eosin (H.E.) staining showed thinning of the myocardium in MKI and HOMO embryos. In situ hybridization (IHC) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) showed downregulation of lncGtl2, Rian, and Mirg expression in MKI and HOMO hearts. The results of single-cell RNA sequencing (scRNA-Seq) analysis indicated that the lack of lncRNA expression in the Dlk1-Dio3 domain led to reduced proliferation of epicardial cells and may be an important cause of cardiac dysplasia. In conclusion, this study demonstrates that Dlk1-Dio3 domain lncRNAs play an integral role in ventricular development.

Short photoperiod inhibited gonadal growth and elevated hypothalamic Dio3 expression unrelated to promoter DNA methylation in young Brandt's voles.

Photoperiod, the length of daylight, has a significant impact on the physiological characteristics of seasonal breeding animals, including their somatic and gonadal development. In rodents, expression of deiodinase type II (Dio2) and III (Dio3) in the hypothalamus is crucial for responding to photoperiodic signals. However, research on the photoperiodism of hypothalamic gene expression and the corresponding regulatory mechanism in Brandt's voles living in the Mongolian steppes is limited. In this study, we gradually changed day length patterns to simulate spring (increasing long photoperiod, ILP) and autumn (decreasing short photoperiod, DSP). We compared the somatic and gonadal development of voles born under ILP and DSP and the expression patterns of five reproduction-related genes in the hypothalamus of young voles. The results showed that DSP significantly inhibited somatic and gonadal development in both female and male offspring. Compared with ILP, Dio3 expression was significantly upregulated in the hypothalamus under DSP conditions and remained elevated until postnatal week 8 in both males and females. However, there was no significant difference in the methylation levels of the proximal promoter region of Dio3 between ILP and DSP, suggesting that methylation in the proximal promoter region may not be involved in regulating the expression of Dio3. These findings suggest that hypothalamic expression of Dio3 plays a key role in the photoperiodic regulation of gonadal activity in Brandt's voles. However, it appears that CpGs methylation in the promoter region is not the main mechanism regulating Dio3 expression.

Different Effects of Obesity and Fasting on the Expression of Type 3 Deiodinase and Thyroid Hormone Receptors in the Liver and Visceral Adipose Tissue of C57BL/6 Male Mice.

Introduction: Energy status can alter thyroid hormone signalling in different tissues. Little is known about the effect of fasting on the local thyroid hormone metabolism under high-fat diet (HFD)-induced obesity. We aimed to investigate the fasting effect on deiodinase type 3 (DIO3) and thyroid hormone receptors (TRs) expression in liver and visceral adipose tissue (VAT) of HFD-induced obese mice. Methods: The 30 male C57BL/6 mice were divided into three groups (n = 10/group): control (CON) group, obese (OB) group, and fasted obese (OBF) group. Materials: In a 14-week study, the expression levels of DIO3 and TRs in the liver and VAT of mice were measured by real-time polymerase chain reaction. Gene expression results were shown as fold changes defined by 2-ΔΔct. Comparison between groups was performed by using one-way-ANOVA or Kruskal-Wallis ANOVA test. Results: In the liver, there was a significantly lower expression of DIO3 and higher expression of TRs in obese fasted mice compared to obese mice. Compared to the lean mice, OBF mice had significantly lower expression of DIO3 and higher expression of TRß. In the VAT, mRNA expression of DIO3 was significantly increased in OBF and OB groups compared to the CON group. There were no significant differences in the mRNA expression of TRs between groups. Conclusion: Our findings suggest that fasting may be more effective in improving thyroid hormone metabolism in the liver rather than the VAT of obese mice.

Transgenerational epigenetic self-memory of Dio3 dosage is associated with Meg3 methylation and altered growth trajectories and neonatal hormones.

Intergenerational and transgenerational epigenetic effects resulting from conditions in previous generations can contribute to environmental adaptation as well as disease susceptibility. Previous studies in rodent and human models have shown that abnormal developmental exposure to thyroid hormone affects endocrine function and thyroid hormone sensitivity in later generations. Since the imprinted type 3 deiodinase gene (Dio3) regulates sensitivity to thyroid hormones, we hypothesize its epigenetic regulation is altered in descendants of thyroid hormone overexposed individuals. Using DIO3-deficient mice as a model of developmental thyrotoxicosis, we investigated Dio3 total and allelic expression and growth and endocrine phenotypes in descendants. We observed that male and female developmental overexposure to thyroid hormone altered total and allelic Dio3 expression in genetically intact descendants in a tissue-specific manner. This was associated with abnormal growth and neonatal levels of thyroid hormone and leptin. Descendant mice also exhibited molecular abnormalities in the Dlk1-Dio3 imprinted domain, including increased methylation in Meg3 and altered foetal brain expression of other genes of the Dlk1-Dio3 imprinted domain. These molecular abnormalities were also observed in the tissues and germ line of DIO3-deficient ancestors originally overexposed to thyroid hormone in utero. Our results provide a novel paradigm of epigenetic self-memory by which Dio3 gene dosage in a given individual, and its dependent developmental exposure to thyroid hormone, influences its own expression in future generations. This mechanism of epigenetic self-correction of Dio3 expression in each generation may be instrumental in descendants for their adaptive programming of developmental growth and adult endocrine function.

DLK1-DIO3 region as a source of tumor suppressor miRNAs in papillary thyroid carcinoma.

BACKGROUND: In previous studies, we demonstrated the downregulation of several miRNAs from the DLK1-DIO3 genomic region in papillary thyroid carcinoma (PTC). Due to the large number of miRNAs within this region, the individual contribution of these molecules to PTC development and progression remains unclear. OBJECTIVE: In this study, we aimed to clarify the contribution of DLK1-DIO3-derived miRNAs to PTC. METHODS: We used different computational approaches and in vitro resources to assess the biological processes and signaling pathways potentially modulated by these miRNAs. RESULTS: Our analysis suggests that, out of more than 100 mature miRNAs originated from the DLK1-DIO3 region, a set of 12 miRNAs accounts for most of the impact on PTC development and progression, cooperating to modulate distinct cancer-relevant biological processes, such as cell migration, extracellular matrix remodeling, and signal transduction. The restoration of the expression of one of these miRNAs (miR-485-5p) in a BRAFT199A-positive PTC cell line impaired proliferation and migration, suppressing the expression of GAB2 and RAC1, validated miR-485-5p targets. CONCLUSIONS: Overall, our results shed light on the role of the DLK1-DIO3 region, which harbors promising tumor suppressor miRNAs in thyroid cancer, and open prospects for the functional exploration of these miRNAs as therapeutic targets for PTC.

microRNA and the Post-Transcriptional Response to Oxidative Stress during Neuronal Differentiation: Implications for Neurodevelopmental and Psychiatric Disorders.

Oxidative stress is one of the most important environmental exposures associated with psychiatric disorders, but the underlying molecular mechanisms remain to be elucidated. In a previous study, we observed a substantial alteration of the gene expression landscape in neuron-like cells that were differentiated from SH-SY5Y cells after or during exposure to oxidative stress, with a subset of dysregulated genes being enriched for neurodevelopmental processes. To further explore the regulatory mechanisms that might account for such profound perturbations, we have now applied small RNA-sequencing to investigate changes in the expression of miRNAs. These molecules are known to play crucial roles in brain development and response to stress through their capacity to suppress gene expression and influence complex biological networks. Through these analyses, we observed more than a hundred differentially expressed miRNAs, including 80 previously reported to be dysregulated in psychiatric disorders. The seven most influential miRNAs associated with pre-treatment exposure, including miR-138-5p, miR-96-5p, miR-34c-5p, miR-1287-5p, miR-497-5p, miR-195-5p, and miR-16-5p, supported by at least 10 negatively correlated mRNA connections, formed hubs in the interaction network with 134 genes enriched with neurobiological function, whereas in the co-treatment condition, miRNA-mRNA interaction pairs were enriched in cardiovascular and immunity-related disease ontologies. Interestingly, 12 differentially expressed miRNAs originated from the DLK1-DIO3 location, which encodes a schizophrenia-associated miRNA signature. Collectively, our findings suggest that early exposure to oxidative stress, before and during prenatal neuronal differentiation, might increase the risk of mental illnesses in adulthood by disturbing the expression of miRNAs that regulate neurodevelopmentally significant genes and networks.

Effect of immunization against OPN5 on the reproductive performance in Shan Partridge ducks under different photoperiods.

Photoperiod is an important environmental factor that influences seasonal reproduction behavior in birds. Birds translate photoperiodic information into neuroendocrine signals through deep brain photoreceptors (DBPs). OPN5 has been considered candidate DBPs involved in regulating seasonal reproduction in birds. We found that OPN5 could mediate light to regulate the follicle development in ducks. In this study, we further verified the effect of OPN5 on follicular development in Shan Partridge ducks by immunizing against the extracellular domain (ECD) of OPN5. We investigated the specific regulatory mechanism of photoperiod mediated by OPN5 on the reproductive activity of ducks. The trial randomly divided 120 Shan Partridge ducks into 3 groups with different treatments: the immunization of OPN5 group was done at d0, d15, d30, and d40 with 1 mL of vaccine containing OPN5 protein (thus containing 1, 1, 0.5, and 0.5 mg of OPN5-KLH protein), and the control group (CS and CL groups) was injected at the same time with the same dose of OPN5-uncontained blank vaccine. The group of CS (900 lux), OPN5 (600 lux), and CL (600 lux) lasted for 40 d in 12 L:12 D photoperiods, respectively. Then, the groups of CS, OPN5, and CL subsequently received 12 L:12 D, 12 L:12 D, and 17 L:7 D light treatments for 33 d, respectively. The ducks were caged in 3 constant rooms with the same feeding conditions for each group, free water, and limited feeding (150 g per duck each day). Duck serum and tissue samples were collected at d 40, d 62, and d 73 (n = 12). It was found that before prolonged light, the group of immunization (group OPN5) and the group of strong light intensity (group CS) were higher than the group of CL in egg production. Subsequent to prolonged light, the group CL in egg production rose about the same as the group immunization, while the strong light group (group CS) was lower. Group OPN5 increased the ovarian index of ducks, and both the immunization of group OPN5 and group CL (extended light) increased the thickness of the granular layer and promoted the secretion of E2, P4, LH, and PRL hormones. Compared with group CS, group CL and OPN5 increased the mRNA level and protein expression of OPN5 in the hypothalamus on d 62 and d 73 (P < 0.05). The gene or protein expression patterns of GnRH, TRH, TSHß, DIO2, THRß, VIP, and PRL were positively correlated with OPN5, whereas the gene expression patterns of GnIH and DIO3 were negatively correlated with OPN5. The results showed that immunization against OPN5 could activate the corresponding transmembrane receptors to promote the expression of OPN5, up-regulate the expression of TSHß and DIO2, and then regulate the HPG axis-related genes to facilitate the follicular development of Shan Partridge ducks. In addition, in this experiment, prolonging the photoperiod or enhancing the light intensity could also enhance follicle development, but the effect was not as significant as immunizing against OPN5. Our results will offer beneficial data and more supportive shreds of evidence in favor of elucidating the role of OPN5 in relation to photoperiods and reproduction.

Epigenetic control and genomic imprinting dynamics of the Dlk1-Dio3 domain.

Genomic imprinting is an epigenetic process whereby genes are monoallelically expressed in a parent-of-origin-specific manner. Imprinted genes are frequently found clustered in the genome, likely illustrating their need for both shared regulatory control and functional inter-dependence. The Dlk1-Dio3 domain is one of the largest imprinted clusters. Genes in this region are involved in development, behavior, and postnatal metabolism: failure to correctly regulate the domain leads to Kagami-Ogata or Temple syndromes in humans. The region contains many of the hallmarks of other imprinted domains, such as long non-coding RNAs and parental origin-specific CTCF binding. Recent studies have shown that the Dlk1-Dio3 domain is exquisitely regulated via a bipartite imprinting control region (ICR) which functions differently on the two parental chromosomes to establish monoallelic expression. Furthermore, the Dlk1 gene displays a selective absence of imprinting in the neurogenic niche, illustrating the need for precise dosage modulation of this domain in different tissues. Here, we discuss the following: how differential epigenetic marks laid down in the gametes cause a cascade of events that leads to imprinting in the region, how this mechanism is selectively switched off in the neurogenic niche, and why studying this imprinted region has added a layer of sophistication to how we think about the hierarchical epigenetic control of genome function.

miR-134-3p driven by anisomycin impairs ovarian cancer stem cell activity through inhibiting GPR137 expression.

Background: Ovarian cancer recurrence and metastasis are predominantly attributed to ovarian cancer stem cells; however, the mechanism by which anisomycin regulates human ovarian cancer stem cells (HuOCSCs) remains unclear. Methods: cDNA microArray was used to screen microRNAs (miRNAs) targeted by anisomycin, and RT-qPCR validated the miRNA targets. TargetScan database, GO enrichment analysis, and RT-qPCR, accompanied by a fluorescent reporter system, were employed to verify the miRNA target genes. In vitro experimental cell proliferation inhibition assay, flow cytometry, Transwell, angiogenesis assay, and in vivo transplantation tumor assay were implemented to assess the ability of the overexpressed miRNAs to hinder HuOCSC activity. Western blot, RT-qPCR, and immunofluorescence were applied to measure the transcriptional and protein-level expression of the miRNA target genes and their related genes. Bioinformatic analysis predicted and deciphered the role of the miRNA target genes and related genes in the development and prognosis of ovarian cancer. Results: The expression levels of multiple DLK1-DIO3 imprinted microRNA cluster members were altered by anisomycin, among which miR-134-3p expression was most significantly elevated. miR-134-3p overexpression significantly suppressed HuOCSC activity. The screening and validation of target genes uncovered that miR-134-3p was able to markedly suppress GPR137 expression. Additionally, miR-134-3p regulated the cytoskeleton, migration-related protein in the NDEL1/DYNEIN/TUBA1A axis through targeting GPR137. Bioinformatics prediction unveiled a close association of GPR137, NDEL1, DYNC1H1, and TUBA1A with ovarian cancer development and prognosis. Conclusions: The activity of HuOCSCs may be compromised by anisomycin through the regulation of miR-134-3p, which inhibits the GPR137/NDEL1/DYNEIN/TUBA1A axis.

Long intergenic non-coding RNA DIO3OS promotes osteosarcoma metastasis via activation of the TGF-ß signaling pathway: a potential diagnostic and immunotherapeutic target for osteosarcoma.

BACKGROUND: The aim of this study was to determine the underlying potential mechanisms and function of DIO3OS, a lincRNA in osteosarcoma and clarify that DIO3OS can be used as a potential diagnostic biomarker and immunotherapeutic target. METHODS: The expression matrix data and clinical information were obtained from XENA platform of UCSC and GEO database as the test cohorts. The external validation cohort was collected from our hospital. Bioinformatics analysis was used to annotate the biological function of DIO3OS. Immune infiltration and immune checkpoint analysis were applied to evaluate whether DIO3OS can be used as an immunotherapeutic target. ROC curves and AUC were established to assess the diagnostic value of DIO3OS for differentiating patients from other subtypes sarcoma. The expression analysis was detected by qRT-PCR, western blot, and immunohistochemical. Wound healing assay and Transwell assay were applied to determine the migration and invasion function of DIO3OS in osteosarcoma cell lines. The tail vein injection osteosarcoma cells metastases model was used in this research. RESULTS: High expression of DIO3OS was identified as a risk lincRNA for predicting overall survival of osteosarcoma in test cohort. The outcomes of experiments in vitro and in vivo showed that low expression of DIO3OS limited osteosarcoma tumor metastasis with inhibiting TGF-ß signaling pathway. Immune checkpoint genes (CD200 and TNFRSF25) expressions were inhibited in the low DIO3OS expression group. The DIO3OS expression can be applied to reliably distinguish osteosarcoma from lipomatous neoplasms, myomatous neoplasms, nerve sheath tumors, and synovial-like neoplasms. This result was further validated in the validation cohort. CONCLUSIONS: In conclusion, our outcomes indicated that DIO3OS is a potential diagnostic and prognostic biomarker of osteosarcoma, emphasizing its potential as a target of immunotherapy to improve the treatment of osteosarcoma through TGF-ß signaling pathway. TRIAL REGISTRATION NUMBER: The present retrospectively study was approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University [Review (2020) No. (115)].

The Conserved LncRNA DIO3OS Restricts Hepatocellular Carcinoma Stemness by Interfering with NONO-Mediated Nuclear Export of ZEB1 mRNA.

Hepatocellular carcinoma (HCC) is an aggressive and fatal disease caused by a subset of cancer stem cells (CSCs). It is estimated that there are approximately 100 000 long noncoding RNAs (lncRNAs) in humans. However, the mechanisms by which lncRNAs affect tumor stemness remain poorly understood. In the present study, it is found that DIO3OS is a conserved lncRNA that is generally downregulated in multiple cancers, including HCC, and its low expression correlates with poor clinical outcomes in HCC. In in vitro cancer cell lines and an in vivo spontaneous HCC mouse model, DIO3OS markedly represses tumor development via its suppressive role in CSCs through downregulation of zinc finger E-box binding homeobox 1 (ZEB1). Interestingly, DIO3OS represses ZEB1 post-transcriptionally without affecting its mRNA levels. Subsequent experiments show that DIO3OS interacts with the NONO protein and restricts NONO-mediated nuclear export of ZEB1 mRNA. Overall, these findings demonstrate that the DIO3OS-NONO-ZEB1 axis restricts HCC development and offers a valuable candidate for CSC-targeted therapeutics for HCC.

Meg8-DMR as the Secondary Regulatory Region Regulates the Expression of MicroRNAs While It Does Not Affect Embryonic Development in Mice.

Meg8-DMR is the first maternal methylated DMR to be discovered in the imprinted Dlk1-Dio3 domain. The deletion of Meg8-DMR enhances the migration and invasion of MLTC-1 depending on the CTCF binding sites. However, the biological function of Meg8-DMR during mouse development remains unknown. In this study, a CRISPR/Cas9 system was used to generate 434 bp genomic deletions of Meg8-DMR in mice. High-throughput and bioinformatics profiling revealed that Meg8-DMR is involved in the regulation of microRNA: when the deletion was inherited from the mother (Mat-KO), the expression of microRNA was unchanged. However, when the deletion occurred from the father (Pat-KO) and homozygous (Homo-KO), the expression was upregulated. Then, differentially expressed microRNAs (DEGs) were identified between WT with Pat-KO, Mat-KO, and Homo-KO, respectively. Subsequently, these DEGs were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) term enrichment analysis to explore the functional roles of these genes. In total, 502, 128, and 165 DEGs were determined. GO analysis showed that these DEGs were mainly enriched in axonogenesis in Pat-KO and Home-KO, while forebrain development was enriched in Mat-KO. Finally, the methylation levels of IG-DMR, Gtl2-DMR, and Meg8-DMR, and the imprinting status of Dlk1, Gtl2, and Rian were not affected. These findings suggest that Meg8-DMR, as a secondary regulatory region, could regulate the expression of microRNAs while not affecting the normal embryonic development of mice.

MiR-24-3p/Dio3 axis is essential for BDE47 to induce local thyroid hormone disorder and neurotoxicity.

BDE47 (2,2,4,4-tetrabromodiphenyl ether) is a member of the most important congeners of polybrominated diphenyl ethers (PBDEs) and has been identified as a developmental, reproductive and nervous system toxicant and endocrine system disruptor due to its frequent detection in human tissue and environmental samples. Our preliminary work suggested that high- and low-level of bromodiphenyl ethers have different effects on neuronal cells with differential targets of actions on neural tissues. In this study, we presented the underlying mechanism of BDE47 neurotoxicity from the perspective of thyroid hormone (TH) metabolism using in vitro model of human SK-N-AS neuronal cells. BDE47 could induce local TH metabolism disorder in neuronal cells by inhibiting the expression of the main enzyme, human type III iodothyronine deiodinase (Dio3). Further elucidation revealed that BDE47 effectively up-regulating miR-24-3p, which binds to the 3'-UTR of Dio3 and inhibits its expression. In addition, BDE47 could also inhibit the deiodinase activity of Dio3. Collectively, our study demonstrates the molecular mechanism of BDE47 regulating Dio3-induced TH metabolism disorder through inducing miR-24-3p, providing new clues for the role of miRNAs in neurodevelopmental toxicity mediated by environmental pollutants.

Epigenetic developmental programming and intergenerational effects of thyroid hormones.

Mounting evidence is showing that altered signaling through the nuclear hormone receptor superfamily can cause abnormal, long-term epigenetic changes which translate into pathological modifications and susceptibility to disease. These effects seem to be more prominent if the exposure occurs early in life, when transcriptomic profiles are rapidly changing. At this time, the coordination of the complex coordinated processes of cell proliferation and differentiation that characterize mammalian development. Such exposures may also alter the epigenetic information of the germ line, potentially leading to developmental changes and abnormal outcomes in subsequent generations. Thyroid hormone (TH) signaling is mediated by specific nuclear receptors, which have the ability to markedly change chromatin structure and gene transcription, and can also regulate other determinants of epigenetic marks. TH exhibits pleiotropic effects in mammals, and during development, its action is regulated in a highly dynamic manner to suit the rapidly evolving needs of multiple tissues. Their molecular mechanisms of action, timely developmental regulation and broad biological effects place THs in a central position to play a role in the developmental epigenetic programming of adult pathophysiology and, through effects on the germ line, in inter- and trans-generational epigenetic phenomena. These areas of epigenetic research are in their infancy, and studies regarding THs are limited. In the context of their characteristics as epigenetic modifiers and their finely tuned developmental action, here we review some of the observations underscoring the role that altered TH action may play in the developmental programming of adult traits and in the phenotypes of subsequent generations via germ line transmission of altered epigenetic information. Considering the relatively high prevalence of thyroid disease and the ability of some environmental chemicals to disrupt TH action, the epigenetic effects of abnormal levels of TH action may be important contributors to the non-genetic etiology of human disease.

Effect of epigenetic activating of Dlk1-Dio3 imprinted cluster on miR-370 expression due to folate deficiency during nerve development.

Proper Dlk1-Dio3 imprinting plays a critical role in embryogenesis, and folic acid deficiency may affect the imprinting of this locus through epigenetic regulation. However, whether and how folic acid directly impacts the imprinting status of Dlk1-Dio3 to affect neural development remain unclear. Here, we found decreased IG-DMR (intergenic -differentially methylated regions) methylation in the folate-deficient encephalocele in humans, suggesting that abnormal Dlk1-Dio3 imprinting status is related to neural tube defects (NTDs) caused by folate deficiency. Similar results were obtained with folate-deficient embryonic stem cells. By miRNA chip analysis, folic acid deficiency led to changes in multiple miRNAs, including the upregulation of 15 miRNAs located in the Dlk1-Dio3 locus. Real-time PCR confirmed that seven of these miRNAs were upregulated, especially miR-370. In contrast to normal embryonic development, in which expression of miR-370 is highest at E9.5, the abnormally high and sustained expression of miRNA-370 in folate-deficient E13.5 embryos may contribute to NTDs. In addition, we found that DNMT3A (de novo DNA methyltransferases 3A) is a direct target gene of miR-370 in neural cells, and DNMT3A participates in the role of miR-370 in inhibiting cell migration. Finally, in the folate-deficient mouse model, Dlk1-Dio3 epigenetic activation was found in fetal brain tissue, along with the upregulation of miR-370 and the downregulation of DNMT3A. Collectively, our findings demonstrate a pivotal role of folate in the epigenetic regulation of Dlk1-Dio3 imprinting during neurogenesis, revealing an elegant mechanism for the activation of Dlk1-Dio3 locus miRNAs in folic acid deficiency.

Decreased DIO3OS Expression Predicts Poor Prognosis in Hepatocellular Carcinoma and is Associated with Immune Infiltration.

Hepatocellular carcinoma has become one of the most shared cancers in the whole world because of its high morbidity, poor survival rate, and low recovery rate. LncRNA DIO3 opposite strand upstream RNA (DIO3OS) has been reported to be obviously important in several human cancers, while its biological function in hepatocellular carcinoma (HCC) remains unclear. Here, DIO3OS gene expression data and clinical information of HCC patients were extracted from the Cancer Genome Atlas (TCGA) database and the university of California Santa Cruz (UCSC) Xena database. In our study, the Wilcoxon rank sum test was used to compare DIO3OS expression between healthy individuals and HCC patients. It was found that patients with HCC had significantly lower DIO3OS expression than healthy individuals. Furthermore, Kaplan-Meier curves and Cox regression analysis showed that high DIO3OS expression tended to predict better prognosis and higher survival rate in HCC patients. In addition, the gene set enrichment analysis (GSEA) assay was used to annotate the biological function of DIO3OS. It was found that DIO3OS was significantly correlated with immune invasion in HCC. This was also aided by the subsequent ESTIMATE assay. Our study provides a novel biomarker and therapeutic strategy for patients with hepatocellular carcinoma.

Developmental thyroid hormone action on pro-opiomelanocortin-expressing cells programs hypothalamic BMPR1A depletion and brown fat activation.

Thyroid hormone excess secondary to global type 3 deiodinase (DIO3) deficiency leads to increased locomotor activity and reduced adiposity, but also to concurrent alterations in parameters of the leptin-melanocortin system that would predict obesity. To distinguish the underlying contributions to the energy balance phenotype of DIO3 deficiency, we generated mice with thyroid hormone excess targeted to pro-opiomelanocortin (POMC)-expressing cells via cell-specific DIO3 inactivation. These mice exhibit a male-specific phenotype of reduced hypothalamic Pomc expression, hyperphagia, and increased activity in brown adipose tissue, with adiposity and serum levels of leptin and thyroid hormones remained normal. These male mice also manifest a marked and widespread hypothalamic reduction in the expression of bone morphogenetic receptor 1a (BMPR1A), which has been shown to cause similar phenotypes when inactivated in POMC-expressing cells. Our results indicate that developmental overexposure to thyroid hormone in POMC-expressing cells programs energy balance mechanisms in a sexually dimorphic manner by suppressing adult hypothalamic BMPR1A expression.

Exosomal microRNAs in the DLK1-DIO3 imprinted region derived from cancer-associated fibroblasts promote progression of hepatocellular carcinoma by targeting hedgehog interacting protein.

BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most commonly diagnosed cancer and third leading cause of cancer-related death worldwide in 2020. Exosomes derived from cancer-associated fibroblasts (CAFs-exo) can promote tumor progression in various human cancers. However, the underlying regulatory mechanism controlling how CAFs-exo can promote HCC progression remains poorly understood. METHODS: CAFs and para-cancer fibroblasts (PAFs) were isolated from HCC tissues and corresponding para-cancer tissues, then were cultured in vitro. CAFs and PAFs were characterized by immunofluorescence and western blot (WB) assays. Exosomes were isolated by ultracentrifugation, and characterized by transmission electron microscopy, nanoflow cytometry, and WB assay. The internalization of exosomes by HCC cells was observed under a fluorescence microscope. Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell proliferation. Wound healing and transwell assays were used for migration and invasion experiments. RT-PCR assay was used to examine differentially expressed microRNAs (miRNAs) in exosomes and HCC cells. The TargetScan database was used to predict miRNA target genes. Hedgehog interacting protein (HHIP) expression analysis, prognostic analysis, and enrichment analysis of HHIP-related co-expressed genes were performed using the TIMER, UALCAN, Kaplan-Meier plotter, and LinkedOmics databases. RESULTS: CAFs-exo were internalized by HCC cells. CAFs-exo contributed to the aggressive phenotype of HCC cells, while inhibiting exosome secretion reversed these effects. Mechanistically, miRNAs in the DLK1-DIO3 imprinted region (miR-329-3p, miR-380-3p, miR-410-5p, miR-431-5p) were increased in HCC cells co-cultured with CAFs-exo compared with PAFs-exo. Expression of HHIP, a possible miR-431-5p target gene, was significantly downregulated in HCC cells. Low HHIP expression level in tumor tissues could predict poor prognosis in HCC patients. HHIP-related co-expressed genes were mainly associated with cell adhesion molecules. CONCLUSIONS: CAFs-exo can promote HCC progression by delivering miRNAs in the DLK1-DIO3 imprinted region to HCC cells, subsequently inhibiting HHIP expression. HHIP is a potential prognostic biomarker in HCC.

Alterations of DNA methylation and expression of genes related to thyroid hormone metabolism in colon epithelium of obese patients.

BACKGROUND: Colorectal cancer is common among obese individuals. The purpose of the current study was to determine changes in DNA methylation status and mRNA expression of thyroid hormone receptor beta (THRB), as a tumor suppressor, and thyroid hormone inactivating enzyme, type 3 deiodinase (DIO3) genes, in human epithelial colon tissues of healthy obese individuals. METHODS: Colon biopsies were analyzed by methylation sensitive-high resolution melting (MS-HRM) to investigate promoter methylation of DIO3 and THRB, and by quantitative real-time polymerase chain reaction to assay expression of DIO3 and THRB mRNA on eighteen obese and twenty-one normal-weight healthy men. RESULTS: There was no significant difference in mean methylation levels at the THRB promoter region between the two groups. Nevertheless, obesity decreased THRB expression levels, significantly (P < 0.05; fold change: 0.19). Furthermore, obesity attenuated DNA methylation (P < 0.001) and enhanced mRNA expression of DIO3 (P < 0.05; fold change: 3). CONCLUSIONS: Our findings suggest that obesity may alter expression of THRB and DIO3 genes through epigenetic mechanism. Alterations of THRB and DIO3 expressions may predispose colon epithelium of obese patients to neoplastic transformation.

C/D box snoRNA SNORD113-6 guides 2'-O-methylation and protects against site-specific fragmentation of tRNALeu(TAA) in vascular remodeling.

C/D box small nucleolar RNAs (snoRNAs) of the DLK1-DIO3 locus are associated with vascular remodeling and cardiovascular disease. None of these snoRNAs has any known targets yet except for one, AF357425/SNORD113-6. We previously showed that this snoRNA targets mRNAs of the integrin signaling pathway and affects arterial fibroblast function. Here, we aimed to identify whether AF357425/SNORD113-6 can also target small RNAs. We overexpressed or inhibited AF357425 in murine fibroblasts and performed small RNA sequencing. Expression of transfer (t)RNA fragments (tRFs) was predominantly regulated. Compared with overexpression, AF357425 knockdown led to an overall decrease in tRFs but with an enrichment in smaller tRFs (<30 nucleotides). We focused on tRNA leucine anti-codon TAA (tRNALeu(TAA)), which has a conserved predicted binding site for AF357425/SNORD113-6. Adjacent to this site, the tRNA is cleaved to form tRFLeu 47-64 in both primary murine and human fibroblasts and in intact human arteries. We show that AF357425/SNORD113-6 methylates tRNALeu(TAA) and thereby prevents the formation of tRFLeu 47-64. Exposing fibroblasts to oxidative or hypoxic stress increased AF357425/SNORD113-6 and tRNALeu(TAA) expression, but AF357425/SNORD113-6 knockdown did not increase tRFLeu 47-64 formation under stress even further. Thus, independent of cellular stress, AF357425/SNORD113-6 protects against site-specific fragmentation of tRNALeu(TAA) via 2'O-ribose-methylation.