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Daniel Patiño-García, Leonor Cruz-Fernandes, Julio Buñay, Renán Orellana, and Ricardo D. Moreno

Reproductive hormone imbalance in infertile women is correlated to high levels of phthalates and alkylphenols, which are among endocrine-disrupting chemicals (EDCs). Previous studies have shown that they interfere with gene expression by deregulating levels of microRNAs (miRs), small non-coding RNAs targeting mRNAs encoding enzymes in the hormone biosynthesis pathway. However, this effect depends on the target organ, dose and whether or not they are alone or in mixtures. Our goal was to study whether the biosynthesis, and a specific group of miRs targeting mRNAs encoding enzymes in steroid hormone biosynthesis, are deregulated in the ovaries of female mice chronically exposed to a mixture of three phthalates (DEHP+DBP+BBP) and two alkylphenols (NP+OP) at a human environmentally relevant dose. We performed qPCR and western blot assays along with a bioinformatics approach and found that this mixture modified the biogenesis machinery of miRs, inducing an increase in the mRNA levels of Drosha and Dicer1 and DROSHA protein levels. In addition, we found changes in the precursor and mature forms of miR-96-5p, miR-200b-3p, miR-365-3p, miR-378a-3p and miR-503-5p which target steroidogenic pathway enzymes. Finally, using primary granulosa cell culture, we confirmed that miR-200b-3p targets Cyp19a1, transcript encoding CYP19A1, the enzyme that produces estradiol (E2). These results indicate that chronic exposure to phthalates and alkylphenols mixture alters the biogenesis of ovary miRs and increases the expression of miRs implicated in the control of steroidal hormone synthesis in female mice, thus contributing to reproductive pathologies.

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Shuai Shao, Hui Wang, Wei Shao, and Na Liu

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and one of the most common causes of infertility in women. PCOS patients have been found with dysregulated microRNAs (miRNAs or miRs), which is indicative of their roles as noninvasive biomarkers and novel therapeutic targets in PCOS. Herein, this study sets out to explore the mechanism of action of miR-199a-5p in PCOS in relation to the janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway via Wilms' tumor 1 (WT1) regulation in a rat model of PCOS. The expression of miR-199a-5p was highly expressed in ovarian cortical tissues and serum of PCOS patients as examined by RT-qPCR. Ovarian granulosa cells (GCs) were harvested from PCOS rat model, followed by subsequent purification. Gain-and loss-of-function experiments of miR-199a-5p were performed to determine its functions in PCOS. Cell viability, cell apoptosis and serum hormone levels were assessed, the results of which showed that down-regulation of miR-199a-5p contributed to the promotion of GC viability and inhibition of apoptosis, while simultaneously inducing the elevation of serum E2 level and reduction of serum AMH, PG, LH and FSH levels in the PCOS rat model. WT1 was identified as a target gene of miR-199a-5p by dual-luciferase reporter gene assay, and inhibition of miR-199a-5p resulted in the activation of WT1-mediated JAK/STAT3 pathway. The activated JAK/STAT3 pathway suppressed the development of PCOS by miR-199a-5p, indicating a mechanism by which miR-199a-5p could potentially prevent PCOS through the WT1-mediated JAK/STAT3 pathway.

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Ruiqi Ma, Lu Gan, Hui Ren, Andrew Harrison, and Jiang Qian

The study aimed to investigate the role of pyruvate dehydrogenase kinase (PDK) in regulating glycolysis and proliferation of perimysial orbital fibroblasts (pOFs) during the pathogenesis of thyroid-associated ophthalmopathy (TAO). EdU and BrdU incorporation assays were performed to examine cell proliferation. Lactate production and oxygen consumption assays were conducted to evaluate glycolysis. Real-time PCR was adapted to quantify PDK mRNA levels. Capillary western immunoassay was adapted to quantify PDK2, Akt, pAkt308 and GAPDH in protein samples. The TAO pOFs exhibited stronger proliferation activity, higher intracellular lactate concentration, and lower oxygen consumption rate than the control pOFs. The PDK inhibitor dichloroacetic acid (DCA) dose-dependently suppressed proliferation of both TAO and control pOFs. DCA reduced lactate production and promoted oxygen consumption in the TAO pOFs but showed no significant effects on glycolysis in the control pOFs. Among four PDK isotypes, PDK2 was overexpressed in the TAO pOFs. The potential PDK signaling mediator, cytoplasmic Akt, was more abundant in TAO pOFs than control pOFs. Knockdown of PDK2 resulted in lower lactate production, stronger oxygen consumption, weaker proliferation activity, and less cytoplasmic Akt in the TAO pOFs but showed no significant effects in the control pOFs. The Akt inhibitor MK2206 suppressed proliferation in both TAO and control pOFs, and lactate production was inhibited by MK2206 in the TAO OFs but not the control pOFs. To conclude, PDK2 overexpression enhances glycolysis and promotes proliferation via Akt signaling in the TAO pOFs. These findings yield insights that PDK2 is a potential therapeutic target for TAO treatment.

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Maya Elena Lee, Aisha Aderayo Tepede, Adel Mandl, Lee Scott Weinstein, Jaydira del Rivero, Sunita K Agarwal, and Jenny E Blau

Gastroenteropancreatic neuroendocrine tumors (GEP NETs) comprise a heterogenous and diverse group of neoplasms arising from a common neuroendocrine cell origin. The majority of these tumors occur sporadically while ~20% manifest within the context of hereditary syndromes. Germline MEN1 mutations cause a syndrome with an increased susceptibility to multifocal primary GEP NETs. In addition, somatic MEN1 mutations also occur in these sporadic lesions. MEN1 alterations are the most frequent somatic mutation found in pancreatic neuroendocrine tumors. In this review, we explore the implication of the loss of the MEN1-encoded protein menin as a key pathogenic driver in subsets of GEP NETs with downstream consequences including upregulation of the oncogenic receptor c-MET (hepatocyte growth factor receptor). Furthermore, the review will summarize the data related to the clinical presentation, therapeutic standards, and outcomes of these tumors in both sporadic and germline MEN1 mutation-associated contexts. Finally, we present the data on c-MET expression in GEP NETs, clinical trials using c-MET inhibitors and provide an overview of the molecular mechanisms by which c-MET inhibition in these lesions represents a potential precision-medicine targeted approach.

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Leonard Y M Cheung and Karine Rizzoti

In the last 15 years, single-cell technologies have become robust and indispensable tools to investigate cell heterogeneity. Beyond transcriptomic, genomic and epigenome analyses, technologies are constantly evolving, in particular toward multi-omics, where analyses of different source materials from a single cell are combined, and spatial transcriptomics, where resolution of cellular heterogeneity can be detected in situ. While some of these techniques are still being optimized, single-cell RNAseq has commonly been used because the examination of transcriptomes allows characterization of cell identity and, therefore, unravel previously uncharacterized diversity within cell populations. Most endocrine organs have now been investigated using this technique, and this has given new insights into organ embryonic development, characterization of rare cell types, and disease mechanisms. Here, we highlight recent studies, particularly on the hypothalamus and pituitary, and examine recent findings on the pancreas and reproductive organs where many single-cell experiments have been performed.

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Yujia Pan, Weikang Yun, Bingshuai Shi, Rongjun Cui, Chi Liu, Zhong Ding, Jialin Fan, Wenqian Jiang, Jiebing Tang, Tianhu Zheng, Xiaoguang Yu, and Ying Liu

miR-146b-5p is overexpressed in papillary thyroid carcinoma (PTC) and is thought to be a related diagnostic marker. Previous studies have indicated the effects of iodine on oncogenic activation. However, the effect of iodine on the proliferation of PTC cells and the associated underlying mechanisms remain unclear. We found that miR-146b-5p was downregulated and smad4 was upregulated in patients exposed to high iodine concentration by in situ hybridisation (ISH) and immunohistochemical (IHC). NaI (10−3 M) treatment downregulated miR-146b-5p and upregulated Smad4 in PTC cell lines. Luciferase assay was used to confirm that Smad4 is a target of miR-146b-5p. Furthermore, MTT assay and cell cycle analysis indicated that 10−3 M NaI suppressed cell proliferation and caused G0/G1 phase arrest. Real-time PCR and Western blotting demonstrated that 10−3 M NaI increased p21, p27, and p57 levels and reduced cyclin D1 levels in PTC cells. Our findings suggest that 10−3 M NaI increases Smad4 levels through repression of miR-146b-5p expression, curbing the proliferation in PTC.

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Kai Huang, Gezi Chen, Wenqian Fan, and Linli Hu

A receptive endometrium is required in a successful embryo implantation. The ubiquitination-induced β-catenin degradation is related to the implantation failure.This study aimed to elucidate whether miR-23a-3p regulates endometrial receptivity via the modulation of β-catenin ubiquitination.The expressions of miR-23a-3p and CUL3 were detected in endometrial epithelial cells (EECs) isolated from pregnant mice and in hormone-induced EEC-like Ishikawa cells. The ubiquitination experiment was performed to explore the effect of CUL3 and miR-23a-3p on β-catenin ubiquitination level. The trophoblast attachment was detected by co-culturing JAR (choriocarcinoma cell line) spheroids with Ishikawa cell monolayers. miR-23a-3p was upregulated while CUL3 was downregulated in EECs at day 4 after pregnancy compared with day 1, as well as in hormone-induced Ishikawa cells. miR-23a-3p positively regulated the protein level of β-catenin without affecting the mRNA level. The ubiquitination and degradation of β-catenin was suppressed by miR-23a-3p, while it was promoted by CUL3. Immunoprecipitation confirmed the binding between CUL3 and β-catenin. Luciferase reporter assay confirmed the target relationship between miR-23a-3p and CUL3. The ubiquitination of β-catenin was modulated by the miR-23a-3p/CUL3 pathway. The overexpression of miR-23a-3p promoted JAR spheroid attachments in Ishikawa cells. miR-23a-3p is beneficial for the endometrial receptivity and embryo implantation, whose mechanism is partly through the modulation of CUL3/β-catenin.

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Dimitrios Doultsinos and Ian Mills

Prostate cancer is a high-incidence male cancer, which is dependent on the activity of a nuclear hormone receptor, the androgen receptor (AR). Since the AR is required for both normal prostate gland development and for prostate cancer progression, it is possible that prostate cancer evolves from perturbations in AR-dependent biological processes that sustain specialist glandular functions. The archetypal example of course is the use of prostate specific antigen (PSA), an organ-type specific component of the normal prostate secretome, as a biomarker of prostate cancer. Furthermore, localised prostate cancer is characterised by a low proliferative index and a heterogenous array of somatic mutations aligned to a multifocal disease pattern. We and others have identified a number of biological processes that are AR dependent and represent aberrations in significant glandular processes. Glands are characterised by high rates of metabolic activity including protein synthesis supported by co-dependent processes such as glycosylation, organelle biogenesis and vesicle trafficking. Impairments in anabolic metabolism and in protein folding/processing will inevitably impose proteotoxic and oxidative stress on glandular cells and, in particular, luminal epithelial cells for which secretion is their primary function. As cancer develops there is also significant metabolic dysregulation including impaired negative feedback effects on glycolytic and anabolic activity under conditions of hypoxia and heightened protein synthesis due to dysregulated PI 3-kinase/mTOR activity. In this review we will focus on the components of the AR regulome that support cancer development as well as glandular functions focussing on the unfolded protein response and on regulators of mTOR activity.

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Rebecca Roy, Caitlyn Nguyen-Ngo, and Martha Lappas

Gestational diabetes mellitus (GDM) affects up to 16% of pregnant women and is associated with significant long-term health detriments for the mother and her offspring. Two central features of GDM are low-grade inflammation and maternal peripheral insulin resistance, therefore therapeutics which target these may be most effective at preventing the development of GDM. Short-chain fatty acids (SCFAs), such as butyrate and propionate, are metabolites produced from the fermentation of dietary fibre by intestinal microbiota. SCFAs possess anti-inflammatory, anti-obesity and anti-diabetic properties. Therefore, this study aimed to investigate the effect of SCFAs on inflammation and insulin signalling defects in an in vitro model of GDM. Human placenta, visceral adipose tissue (VAT) and s.c. adipose tissue (SAT) were stimulated with either the pro-inflammatory cytokine TNF or bacterial product lipopolysaccharide (LPS). The SCFAs butyrate and propionate blocked TNF- and LPS-induced mRNA expression and secretion of pro-inflammatory cytokines and chemokines in placenta, VAT and SAT. Primary human cells isolated from skeletal muscle were stimulated with TNF to assess the effect of SCFAs on inflammation-induced defects in the insulin signalling pathway. Butyrate and propionate were found to reverse TNF-induced increases in IRS-1 serine phosphorylation and decreases in glucose uptake. Butyrate and propionate exerted these effects by preventing ERK activation. Taken together, these results suggest that the SCFAs may be able to improve insulin sensitivity and prevent inflammation induced by sterile or bacterial inflammation. Future in vivo studies are warranted to investigate the efficacy and safety of SCFAs in preventing insulin resistance and inflammation associated with GDM.

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Hong Zhu, Wei Cao, Peng Zhao, Jieyu Wang, Yuying Qian, and Yun Li

The excessive activation of renin-angiotensin system (RAS) is one of key pathophysiological characteristics in the development of cardiac remodelling. Angiotensin (Ang) II, as a main active peptide in RAS, induces cardiac structural disorders and dysfunction. However, the molecular mechanisms are still not fully disclosed. Present study aimed to determine the role and potential mechanisms of cardiac TIR-domain-containing adapter-inducing interferon-β (TRIF) in Ang-II-mediated cardiac remodelling in mice. In vitro and in vivo studies showed Ang II and downstream aldosterone obviously increased the expression of TRIF, accompanied with cardiac structural abnormalities and functional injuries. Specific blockage of cardiac TRIF effectively decreased Ang-II/aldosterone-induced cardiac inflammation, fibrosis, hypertrophy and dysfunction in mice. Mechanistically, the TRIF triggered the activation of EGF receptor (EGFR) signalling by nuclear factor (NF)-κB transcriptional regulation and downstream EGFR ligands. Taken together, present study supported that cardiac TRIF was a potential therapeutic target for attenuating cardiac pathophysiological remodelling. The TRIF/EGFR axis partially explained the molecular mechanism of Ang-II/aldosterone-induced cardiac inflammation, fibrosis, hypertrophy and dysfunction in mice.