Expression Of Foxa2 Research Articles

Unveiling novel double-negative prostate cancer subtypes through single-cell RNA sequencing analysis

Recent advancements in single-cell RNA sequencing (scRNAseq) have facilitated the discovery of previously unrecognized subtypes within prostate cancer (PCa), offering new insights into cancer heterogeneity and progression. In this study, we integrated scRNAseq data from multiple studies, comprising publicly available cohorts and data generated by our research team, and established the Human Prostate Single cell Atlas (HuPSA) and Mouse Prostate Single cell Atlas (MoPSA) datasets. Through comprehensive analysis, we identified two novel double-negative PCa populations: KRT7 cells characterized by elevated KRT7 expression and progenitor-like cells marked by SOX2 and FOXA2 expression, distinct from NEPCa, and displaying stem/progenitor features. Furthermore, HuPSA-based deconvolution re-classified human PCa specimens, validating the presence of these novel subtypes. We then developed a user-friendly web application, “HuPSA–MoPSA” (https://pcatools.shinyapps.io/HuPSA-MoPSA/), for visualizing gene expression across all newly established datasets. Our study provides comprehensive tools for PCa research and uncovers novel cancer subtypes that can inform clinical diagnosis and treatment strategies.

Mediation of FOXA2/IL-6/IL-6R/STAT3 signaling pathway mediates benzo[a]pyrene-induced airway epithelial mesenchymal transformation in asthma

Benzo [a]pyrene (BaP), a toxic pollutant, increases the incidence and severity of asthma. However, the molecular mechanisms underlying the effects of BaP in asthma remain unclear. In terms of research methods, we used BaP to intervene in the animal model of asthma and the human bronchial epithelial (16HBE) cells, and the involved mechanisms were found from the injury, inflammation, and airway epithelial to mesenchymal transition (EMT) in asthma. We also constructed small interfering RNAs and overexpression plasmids to knockdown/overexpress IL-6R and FOXA2 in 16HBE cells and a serotype 9 adeno-associated viral vector for lung tissue overexpression of FOXA2 in mice to determine the mechanism of action of BaP-exacerbated asthma airway EMT. We observed that BaP aggravated inflammatory cell infiltration into the lungs, reduced the Penh value, increased collagen fibres in the lung tissue, and increased serum IgE levels in asthmatic mice. After BaP intervention, the expression of FOXA2 in the lung tissue of asthmatic mice decreased, the production and secretion of IL-6 were stimulated, and STAT3 phosphorylation and nuclear translocation increased, leading to changes in EMT markers. However, EMT decreased after increasing FOXA2 expression and decreasing that of IL-6R and was further enhanced after low FOXA2 expression. Our results revealed that BaP exacerbated airway epithelial cell injury and interfered with FOXA2, activating the IL-6/IL-6R/STAT3 signaling pathway to promote airway EMT in asthma. These findings provide toxicological evidence for the mechanism underlying the contribution of BaP to the increased incidence of asthma and its exacerbations.

Mouse neural tube organoids self-organize floorplate through BMP-mediated cluster competition

During neural tube (NT) development, the notochord induces an organizer, the floorplate, which secretes Sonic Hedgehog (SHH) to pattern neural progenitors. Conversely, NT organoids (NTOs) from embryonic stem cells (ESCs) spontaneously form floorplates without the notochord, demonstrating that stem cells can self-organize without embryonic inducers. Here, we investigated floorplate self-organization in clonal mouse NTOs. Expression of the floorplate marker FOXA2 was initially spatially scattered before resolving into multiple clusters, which underwent competition and sorting, resulting in a stable "winning" floorplate. We identified that BMP signaling governed long-range cluster competition. FOXA2+ clusters expressed BMP4, suppressing FOXA2 in receiving cells while simultaneously expressing the BMP-inhibitor NOGGIN, promoting cluster persistence. Noggin mutation perturbed floorplate formation in NTOs and in the NT invivo at mid/hindbrain regions, demonstrating how the floorplate can form autonomously without the notochord. Identifying the pathways governing organizer self-organization is critical for harnessing the developmental plasticity of stem cells in tissue engineering.

Characteristics of expression of proteomic markers in women with “thin” endometrium and their importance for the therapeutic effects of the use of exogenous estradiol: A prospective cohort comparative study

Aim. To analyze the relationship between the expression of proteomic markers [estrogen (ER) and progesterone (PR) receptors, LIF, FOXA1, FOXA2] of endometrial receptivity and indicators of the levels of sex steroids [estradiol (E2) and progesterone (P)] in the peripheral blood during the “implantation window” in women with a history of reproductive dysfunction with “thin” endometrium (TE) and evaluate their significance for the therapeutic effect of using estradiol drugs. Materials and methods. The prospective cohort comparative study was conducted. The main group included 52 patients with TE (7 mm according to ultrasound on preovulatory days), the comparison group consisted of 62 women with normal endometrial thickness (≥7 mm according to ultrasound), women of both groups with reproductive dysfunctions of unknown reason; the control group included 16 healthy women. Aspiration biopsy of the endometrium was performed on the 6–8 days after ovulation (LH +6–8), as well as venipuncture to obtain a sample of peripheral blood to determine the E2, P levels. A combined histological and immunohistochemical study of endometrial samples was performed (ER, PR, LIF, FOXA1, FOXA2). The therapeutic effects of estradiol drugs were assessed in a cohort of women from the main group with TE (n=16 out of 26, those who have not changed their reproductive plans). Results. All women had ovulatory values of progesterone [P≥16.1 nmol/l (LH +6–8)] and normoestrogenemia in the blood (p0.05). According to the results of the correlation analysis, there were no associations between the E2, P levels and the value of M-echo, and no relationships were found between the E2, P levels and the expression of ER, PR, LIF, FOX-proteins. It was revealed that there are significant positive relationships between M-echo and FOXA2 expression in the endometrium (r=0.422; p0.001). Conditional threshold values for the ER, PR expression (LH +6–8) were determined, the excess of which is associated with abnormal expression of LIF, FOX-proteins: for PR in the glands – 105 and 285, for ER in glands – 145, for ER in the stroma – 155. It was found that usual doses of estradiol drugs (1–1.5 mg/day) are capable of increasing endometrial proliferation in women with TE, provided that the expression of ER, PR, LIF, FOX-proteins in their endometrium is similar to healthy women. Increased doses of estradiol drugs (4 mg/day) in women with TE had a significant effect in the form of an increase M-echo value and/or pregnancy in cases where the endometrial expression of LIF, FOX-proteins was comparable with healthy women, even in the presence of hormone-receptor interactions in the endometrium that are different from healthy women. Spontaneous pregnancy in participants of the main group with hypoplastic endometrium in our study occurred in 5 women in whom the expression characteristics of FOXA1 and/or FOXA2 in the endometrium were comparable to healthy women. Conclusion. TE is not the only marker of an imbalance of proteomic molecules, but it determines a greater likelihood of impaired expression of ER, PR, LIF, FOX-proteins in the endometrium. In women with an unclear reason of fertility disorders, it is necessary to conduct an in-depth examination of endometrial samples. Characteristics of endometrial expression ER, PR, LIF, FOX-proteins and the usefulness of hormone-receptor interactions in the TE in women with a history of reproductive disorders can be markers for predicting the effectiveness of the use of exogenous estradiol drugs in women in order to enhance endometrial proliferation with an initially low M-echo.

Direct reprogramming of fibroblasts into functional hepatocytes via CRISPRa activation of endogenous Gata4 and Foxa3.

The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments. Clustered regularly interspaced short palindromic repeats (CRISPR) activator (CRISPRa) is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously, providing a new strategy for cell fate determination. The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis. The differentiation of mouse embryonic fibroblasts (MEFs) into functional induced hepatocyte-like cells (iHeps) was achieved by utilizing the CRISPRa synergistic activation mediator (SAM) system, which drove the combined expression of three endogenous transcription factors- Gata4, Foxa3 , and Hnf1a -or alternatively, the expression of two transcription factors, Gata4 and Foxa3 . In vivo , we injected adeno-associated virus serotype 6 (AAV6) carrying the CRISPRa SAM system into liver fibrotic Col1a1-CreER ; Cas9fl/fl mice, effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts. The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction (RT-qPCR) and RNA-seq, and the morphology and characteristics of the induced hepatocytes were observed through microscopy. The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining, while the improvement of liver fibrosis is evaluated through Sirius red staining, alpha-smooth muscle actin (α-SMA) immunofluorescence staining, and blood alanine aminotransferase (ALT) examination. Activation of only two factors, Gata4 and Foxa3 , via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps. These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes, such as drug metabolism and glycogen storage. Additionally, AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis. After 8 weeks of induction, the reprogrammed hepatocytes comprised 0.87% of the total hepatocyte population in the mice, significantly reducing liver fibrosis. CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.

Chromosome 20p11.2 deletions cause congenital hyperinsulinism via the loss of FOXA2 or its regulatory elements.

Persistent congenital hyperinsulinism (HI) is a rare genetically heterogeneous condition characterised by dysregulated insulin secretion leading to life-threatening hypoglycaemia. For up to 50% of affected individuals screening of the known HI genes does not identify a disease-causing variant. Large deletions have previously been used to identify novel regulatory regions causing HI. Here, we used genome sequencing to search for novel large (>1 Mb) deletions in 180 probands with HI of unknown cause and replicated our findings in a large cohort of 883 genetically unsolved individuals with HI using off-target copy number variant calling from targeted gene panels. We identified overlapping heterozygous deletions in five individuals (range 3-8 Mb) spanning chromosome 20p11.2. The pancreatic beta-cell transcription factor gene, FOXA2, a known cause of HI was deleted in two of the five individuals. In the remaining three, we found a minimal deleted region of 2.4 Mb adjacent to FOXA2 that encompasses multiple non-coding regulatory elements that are in conformational contact with FOXA2. Our data suggests that the deletions in these three children may cause disease through the dysregulation of FOXA2 expression. These findings provide new insights into the regulation of FOXA2 in the beta-cell and confirm an aetiological role for chromosome 20p11.2 deletions in syndromic HI.

9-HODE and 9-HOTrE alter mitochondrial metabolism, increase triglycerides, and perturb fatty acid uptake and synthesis associated gene expression in HepG2 cells

Non-Alcoholic Fatty Liver Disease (NAFLD) prevalence is rising and can lead to detrimental health outcomes such as Non-Alcoholic Steatohepatitis (NASH), cirrhosis, and cancer. Recent studies have indicated that Cytochrome P450 2B6 (CYP2B6) is an anti-obesity CYP in humans and mice. Cyp2b-null mice are diet-induced obese, and human CYP2B6-transgenic (hCYP2B6-Tg) mice reverse the obesity or diabetes progression, but with increased liver triglyceride accumulation in association with an increase of several oxylipins. Notably, 9-hydroxyoctadecadienoic acid (9-HODE) produced from linoleic acid (LA, 18:2, ω-6) is the most prominent of these and 9-hydroxyoctadecatrienoic acid (9-HOTrE) from alpha-linolenic acid (ALA, 18:3, ω-3) is the most preferentially produced when controlling for substrate concentrations in vitro. Transactivation assays indicate that 9-HODE and 9-HOTrE activate PPARα and PPARγ. In Seahorse assays performed in HepG2 cells, 9-HOTrE increased spare respiratory capacity, slightly decreased palmitate metabolism, and increased non-glycolytic acidification in a manner consistent with slightly increased glutamine utilization; however, 9-HODE exhibited no effect on metabolism. Both compounds increased triglyceride and pyruvate concentrations, most strongly by 9-HOTrE, consistent with increased spare respiratory capacity. qPCR analysis revealed several perturbations in fatty acid uptake and metabolism gene expression. 9-HODE increased expression of CD36, FASN, PPARγ, and FoxA2 that are involved in lipid uptake and production. 9-HOTrE decreased ANGPTL4 expression and increased FASN expression consistent with increased fatty acid uptake, fatty acid production, and AMPK activation. Our findings support the hypothesis that 9-HODE and 9-HOTrE promote steatosis, but through different mechanisms as 9-HODE is directly involved in fatty acid uptake and synthesis; 9-HOTrE weakly inhibits mitochondrial fatty acid metabolism while increasing glutamine use.

P157 Differentiation of Tonsil-derived mesenchymal stem cells to intestinal stem cells-like cells for cell therapy of inflammatory bowel disease

Abstract Background Tonsil-derived mesenchymal stem cells (TMSCs) were found to exhibit a faster growth rate compared to mesenchymal stem cells derived from other tissues. This is attributed to the relatively younger age of the tonsil tissue donors. Moreover, it has been verified that TMSCs possess the capability to undergo differentiation into endoderm lineages, giving rise to cell types like parathyroid glands and insulin-producing cells. In this study, we explored the possibility of differentiation of TMSCs into intestinal stem cells-like cells (ISC-like cells), a cell type of endoderm lineages for utilizing these cells for therapeutic applications in inflammatory bowel disease (IBD). Methods TMSCs were cultivated in DMEM (10% FBS) and used in the experiment when 90-100% confluency was observed. Adipose-derived mesenchymal stem cells (AMSCs) were used as the control for assessing the differentiation efficacy of TMSCs. The morphology of cells was verified at each differentiation stage, and the expression of endoderm and intestinal stem cell markers was confirmed using real-time PCR (qPCR), western blotting, and immunofluorescence (IF) staining. Results Subjected to 100ng/ml of Activin A for endoderm differentiation and cultured for 2 to 5 days, TMSCs exhibited an upregulation in endoderm markers (SOX17, FOXA2), as confirmed by real-time PCR. Specifically, under the influence of Activin A at 100ng/ml and cultured for 5 days, there was a significant increase in the expression of SOX17 (pre-differentiation vs. endoderm-differentiation, 0.999 vs. 3.610, P=0.0394) and FOXA2 (1.047 vs. 3.912, P=0.0297). Following the endoderm differentiation phase, the medium was changed to include 500ng/ml of FGF4 and 500ng/ml of Wnt3a for a 7-day period. Post-culture, the expression level of Lgr5, an intestinal stem cell (ISC) marker, was significantly increased in TMSCs (1.061 vs. 134.0, P<0.001). Notably, AMSCs displayed a lower expression rate compared to TMSCs (TMSCs vs. AMSCs, 134.0 vs. 9.819, P<0.001). Moreover, the spheroids formation was observed 7 days after the initiation of FGF4 and Wnt3a treatment, a phenomenon absent in AMSCs differentiation. Immunofluorescence staining for differentiation markers further validated these findings: the expression of SOX17, an endoderm marker, and the fluorescence density of LGR5, an ISC marker, were notably higher in TMSCs compared to AMSCs. Protein expression levels also confirmed the upregulation of each marker in TMSCs. Conclusion This study validated the potential of TMSCs to differentiate into ISC-like cells, with a higher efficiency observed compared to AMSCs. The prospect of differentiating into ISC positions TMSCs as promising candidates for use as a tool in IBD.

Retracted: Long Noncoding RNA SAMMSON Promotes Melanoma Progression by Inhibiting FOXA2 Expression.

[This retracts the article DOI: 10.1155/2023/8934210.].

NatD epigenetically activates FOXA2 expression to promote breast cancer progression by facilitating MMP14 expression

N-α-acetyltransferase D (NatD) mediates N-α-terminal acetylation of histone H4 (Nt-Ac-H4), but its role in breast cancer metastasis remains unknown. Here, we show that depletion of NatD directly represses the expression of FOXA2, and is accompanied by a significant reduction in Nt-Ac-H4 enrichment at the FOXA2 promoter. We show that NatD is commonly upregulated in primary breast cancer tissues, where its expression level correlates with FOXA2 expression, enhanced invasiveness, and poor clinical outcomes. Furthermore, we show that FOXA2 promotes the migration and invasion of breast cancer cells by activating MMP14 expression. MMP14 is also upregulated in breast cancer tissues, where its expression level correlates with FOXA2 expression and poor clinical prognosis. Our study shows that the NatD-FOXA2-MMP14 axis functions as a key signaling pathway to promote the migratory and invasive capabilities of breast cancer cells, suggesting that NatD is a critical epigenetic modulator of cell invasion during breast cancer progression.

Analysis of GATA3 and FOXA2 expression suggests that downregulation of genes involved in the maintenance of a mature yolk sac tumor phenotype may underlie sarcomatoid transformation.

In the post-chemotherapy setting, germ cell tumors of the testis (GCTT) that resemble non-specific sarcomas and co-express cytokeratins and glypican-3 (GPC3) are diagnosed as "sarcomatoid yolk sac tumor postpubertal-type (YSTpt)". The diagnosis of sarcomatoid YSTpt is clinically relevant but challenging due to its rarity, non-specific histology, and negative α-fetoprotein (AFP) staining. Recently, FOXA2 has emerged as a key-gene in the reprogramming of GCTT (activating the transcription of several genes, among which GATA3), and immunohistochemical studies showed that GATA3 and FOXA2 have a higher sensitivity for non-sarcomatoid YSTpt than GPC3 and AFP. We found that sarcomatoid YSTpt did not express FOXA2 [0: 14/14 (100%)] and showed focal expression of GATA3 [0: 12/14 (85.7%), 1 + : 2/14 (14.3%)], thus suggesting that these markers are not useful in diagnosing this tumor. Furthermore, we proposed a potential mechanism of sarcomatoid transformation in the post-chemotherapy setting of GCTT, mediated by the downregulation of FOXA2 and GATA3.

FXR controls insulin content by regulating Foxa2-mediated insulin transcription

Farnesoid X receptor (FXR) is a nuclear ligand-activated receptor of bile acids that plays a role in the modulation of insulin content. However, the underlying molecular mechanisms remain unclear. Forkhead box a2 (Foxa2) is an important nuclear transcription factor in pancreatic β-cells and is involved in β-cell function. We aimed to explore the signaling mechanism downstream of FXR to regulate insulin content and underscore its association with Foxa2 and insulin gene (Ins) transcription. All experiments were conducted on FXR transgenic mice, INS-1 823/13 cells, and diabetic Goto-Kakizaki (GK) rats undergoing sham or Roux-en-Y gastric bypass (RYGB) surgery. Islets from FXR knockout mice and INS-1823/13 cells with FXR knockdown exhibited substantially lower insulin levels than that of controls. This was accompanied by decreased Foxa2 expression and Ins transcription. Conversely, FXR overexpression increased insulin content, concomitant with enhanced Foxa2 expression and Ins transcription in INS-1 823/13 cells. Moreover, FXR knockdown reduced FXR recruitment and H3K27 trimethylation in the Foxa2 promoter. Importantly, Foxa2 overexpression abrogated the adverse effects of FXR knockdown on Ins transcription and insulin content in INS-1 823/13 cells. Notably, RYGB surgery led to improved insulin content in diabetic GK rats, which was accompanied by upregulated FXR and Foxa2 expression and Ins transcription. Collectively, these data suggest that Foxa2 serves as the target gene of FXR in β-cells and mediates FXR-enhanced Ins transcription. Additionally, the upregulated FXR/Foxa2 signaling cascade could contribute to the enhanced insulin content in diabetic GK rats after RYGB.

FOXA2 activates HIF2α expression to promote tumor progression and is regulated by the E3 ubiquitin ligase VHL in renal cell carcinoma

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system with high mortality and morbidity. However, the molecular mechanisms underlying RCC progression are still largely unknown. In this study, we identified FOXA2, a pioneer transcription factor, as a driver oncogene for RCC. We show that FOXA2 was commonly upregulated in human RCC samples and promoted renal cancer cell proliferation, as evidenced by assays of cell viability, colony formation, migratory and invasive capabilities, and stemness properties. Mechanistically, we found that FOXA2 promoted RCC cell proliferation by transcriptionally activating HIF2α expression in vitro and in vivo. Furthermore, we found that FOXA2 could interact with VHL (von Hippel‒Lindau), which ubiquitinated FOXA2 and controlled its protein stability in RCC cells. We showed that mutation of lysine at position 264 to arginine in FOXA2 could mostly abrogate its ubiquitination, augment its activation effect on HIF2α expression, and promote renal cancer cell proliferation in vitro and RCC progression in vivo. Importantly, elevated expression of FOXA2 in RCC patients positively correlated with the expression of HIF2α and was associated with shorter overall and disease-free survival. Together, these findings reveal a novel role of FOXA2 in RCC development and provide insights into the underlying molecular mechanisms of FOXA2-driven pathological processes in RCC.

Foxa2 attenuates steatosis and inhibits the NF-κB/IKK signaling pathway in nonalcoholic fatty liver disease

Objective Forkhead box a2 (Foxa2) is proven to be an insulin-sensitive transcriptional regulator and affects hepatic steatosis. This study aims to investigate the mechanism by which Foxa2 affects nonalcoholic fatty liver disease (NAFLD). Methods Animal and cellular models of NAFLD were constructed using high-fat diet (HFD) feeding and oleic acid (OA) stimulation, respectively. NAFLD mice received tail vein injections of either an overexpressing negative control (oe-NC) or Foxa2 (oe-Foxa2) for four weeks. HepG2 cells were transfected with oe-NC and oe-Foxa2 for 48 h before OA stimulation. Histological changes and lipid accumulation were assessed using hematoxylin-eosin staining and oil red O staining, respectively. Expression of Foxa2, NF-κB/IKK pathway proteins, lipid synthesis proteins, and fatty acid β-oxidation protein in HFD mice and OA-induced HepG2 cells was detected using western blot. Results Foxa2 expression was downregulated in HFD mice and OA-induced HepG2 cells. Foxa2 overexpression attenuated lipid accumulation and liver injury, and reduced the levels of aspartate aminotransferase, alanine aminotransferase, total cholesterol, or triglyceride in HFD mice and OA-induced HepG2 cells. Moreover, Foxa2 overexpression decreased the expression of lipid synthesis proteins and increased fatty acid β-oxidation protein expression in the liver tissues. Furthermore, overexpression of Foxa2 downregulated the expression of p-NF-κB/NF-κB and p-IKK/IKK in OA-induced HepG2 cells. Additionally, lipopolysaccharide (NF-κB/IKK pathway activator) administration reversed the downregulation of lipid synthesis proteins and the upregulation of fatty acid β-oxidation protein. Conclusion Foxa2 expression is downregulated in NAFLD. Foxa2 ameliorated hepatic steatosis and inhibited the activation of the NF-κB/IKK signaling pathway.

Bone marrow stem cell-derived β-cells: New issue for diabetes cell therapy

This investigation aimed to establish the promising role of insulin-producing cells (IPCs) growing from bone marrow-mesenchymal stem cells (BM-MSCs) in relieving hyperglycemia induced in rats. BM-MSCs were differentiated into IPCs using three different protocols. The efficiency of BM-MSCs differentiation into IPCs in vitro was confirmed by detecting IPCs specific gene expression (Foxa-2, PDX-1 and Ngn-3) and insulin release assay. The in vivo study design included 3 groups of male Wistar rats; negative control group, diabetic group and IPCs-transfused group (5 ×106 cells of the most functional IPCs/rat). One month after IPCs infusion, serum glucose, insulin, c-peptide and visfatin levels as well as pancreatic glucagon level were quantified. Gene expression analysis of pancreatic Foxa-2 and Sox-17, IGF-1 and FGF-10 was done. Additionally, histological investigation of pancreatic tissue sections was performed. Our data clarified that, the most functional IPCs are those generated from BM-MSCs using differentiation protocol 3 as indicated by the significant up-regulation of Foxa-2, PDX-1 and Ngn-3 gene expression levels. These findings were further emphasized by releasing of a significant amount of insulin in response to glucose load. The transplantation of the IPCs in diabetic rats elicited significant decline in serum glucose, visfatin and pancreatic glucagon levels along with significant rise in serum insulin and c-peptide levels. Moreover, it triggered significant up-regulation in the expression levels of pancreatic Foxa-2, Sox-17, IGF-1 and FGF-10 genes versus the untreated diabetic counterpart. The histopathological examination of pancreatic tissue almost assisted the biochemical and molecular genetic analyses. These results disclose that the cell therapy holds potential to develop a new cure for DM based on the capability of BM-MSCs to generate β-cell phenotype using specific protocol.

CK1α deficiency impairs mouse uterine adenogenesis by inducing epithelial cell apoptosis through GSK3β pathway and inhibiting Foxa2 expression through p53 pathway†.

Uterine glands and their secretions are crucial for conceptus survival and implantation in rodents and humans. In mice, the development of uterine gland known as adenogenesis occurs after birth, whereas the adenogenesis in humans initiates from fetal life and completed at puberty. Uterine adenogenesis involves dynamic epithelial cell proliferation, differentiation, and apoptosis. However, it is largely unexplored about the mechanisms governing adenogenesis. CK1α plays important roles in regulating cell division, differentiation, and death, but it is unknown whether CK1α affects adenogenesis. In the current study, uterus-specific CK1α knockout female mice (Csnk1a1d/d) were infertile resulted from lack of uterine glands. Subsequent analysis revealed that CK1α deletion induced massive apoptosis in uterine epithelium by activating GSK3β, which was confirmed by injections of GSK3β inhibitor SB216763 to Csnk1a1d/d females, and the co-treatment of SB216763 and CK1 inhibitor d4476 on cultured epithelial cells. Another important finding was that our results revealed CK1α deficiency activated p53, which then blocked the expression of Foxa2, an important factor for glandular epithelium development and function. This was confirmed by that Foxa2 expression level was elevated in p53 inhibitor pifithrin-α injected Csnk1a1d/d mouse uterus and in vitro dual-luciferase reporter assay between p53 and Foxa2. Collectively, these studies reveal that CK1α is a novel factor regulating uterine adenogenesis by inhibiting epithelial cell apoptosis through GSK3β pathway and regulating Foxa2 expression through p53 pathway. Uncovering the mechanisms of uterine adenogenesis is expected to improve pregnancy success in humans and other mammals.

Endometrial expression of FOX proteins (FOXA1 and FOXA2) in women of reproductive age with different endometrial thickness: prospective cohort comparative study

Aim. To evaluate the expression of FOX proteins (FOXA1 and FOXA2) in the endometrium during the implantation window in women with a history of reproductive dysfunctions with different thickness of the endometrium.
 Materials and methods. The prospective cohort comparative study was conducted. The main group included patients with "thin" endometrium (7 mm according to ultrasound on preovulatory days; n=52), the comparison group consisted of women with normal endometrial thickness (7 mm according to ultrasound; n=62; women of both groups with reproductive dysfunctions of unknown reason), the control group included 16 healthy fertile women. Aspiration biopsy of the endometrium was performed on the 68 days after ovulation, as well as venipuncture to obtain a sample of peripheral blood to determine the levels of sex steroids (estradiol E2 and progesterone P). A combined histological and immunohistochemical study of endometrial biopsies was performed.
 Results. All women had ovulatory values of progesterone P16.1 nmol/l (68 days after ovulation) and normoestrogenemia (E2, pmol/l) in the blood. E2/P was similar in all groups (p0, 05). A pronounced expression of FOXA1 was noted in women with thin endometrium significantly more often (p0.05) with various hormone-receptor characteristics of the endometrium (42% n=22 out of 52) compared with healthy participants (0%; n=0). Reduced FOXA2 expression in the uterine mucosa was significantly more often detected on 68 days after ovulation in women with "thin" endometrium (56% n=29 of 52) than in women with normal endometrial thickness, both in women from the comparison group and in healthy women from the control group (p0.05). In a generalized analysis of the expression of FOX proteins in the endometrium on days 68 after ovulation, it was generally found that every second (50%; n=57 out of 114) women with a history of reproductive disorders (with a reduced and normal M-echo value) expression of proteomic markers differed from healthy women. In the case of "thin" endometrium, more than two thirds of patients (71%; n=37 out of 52) showed differences in endometrial expression of FOX proteins compared with women without a burdened reproductive history.
 Conclusion. In the majority of women (71%) with a thin endometrium and a history of reproductive dysfunctions, the expression of FOX proteins in the endometrium differed from the control group. Overall, endometrial expression of FOX proteins, which is likely to be different from healthy women, in patients with reproductive dysfunctions of unknown origin is a significant predictor of reproductive failure. At the same time, such an isolated indicator as M-echo value 7 mm according to ultrasound data is not an absolute prognostic marker of endometrial receptivity disorders.

OC 45.2 Knockdown of Zebrafish Tmem242 Enhances Production of ROS thatSignals to Increase Foxa2 and Foxa3 Expression that May Increase Clotting Factors Resulting in Thrombosis and Bleeding

OC 45.2 Knockdown of Zebrafish Tmem242 Enhances Production of ROS thatSignals to Increase Foxa2 and Foxa3 Expression that May Increase Clotting Factors Resulting in Thrombosis and Bleeding

Hepatocyte-specific HDAC3 ablation promotes hepatocellular carcinoma in females by suppressing Foxa1/2

BackgroundHepatocellular carcinoma (HCC), the most common primary liver cancer, prevails mainly in males and has long been attributed to androgens and higher circumstantial levels of interleukin-6 (IL-6) produced by resident hepatic macrophages.MethodsConstitutively hepatocyte-specific histone deacetylase 3 (HDAC3)-deficient (HDAC3LCKO) mice and constitutively hepatocyte-specific HDAC3 knockout and systemic IL-6 simultaneously ablated (HDAC3LCKO& IL-6−/−) mice were used in our study to explore the causes of sex differences in HCC. Additionally, we performed human HCC tissues with an IHC score. Correlation analysis and linear regression plots were constructed to reveal the association between HDAC3 and its candidate genes. To further elucidate that HDAC3 controls the expression of Foxa1/2, we knocked down HDAC3 in HUH7 liver cancer cells.ResultsWe observed a contrary sex disparity, with an earlier onset and higher incidence of HCC in female mice when HDAC3 was selectively ablated in the liver. Loss of HDAC3 led to constant liver injury and the spontaneous development of HCC. Unlike the significant elevation of IL-6 in male mice at a very early age, female mice exhibit stable IL-6 levels, and IL-6 ablation did not eliminate the sex disparity in hepatocarcinogenesis in HDAC3-deficient mice. Oestrogen often protects the liver when combined with oestrogen receptor alpha (ERα); however, ovariectomy in HDAC3-ablated female mice significantly delayed tumourigenesis. The oestrogen-ERα axis can also play a role in tumour promotion in the absence of Foxa1 and Foxa2 in the receptor complex. Loss of HDAC3 profoundly reduced the expression of both Foxa1 and Foxa2 and impaired the binding between Foxa1/2 and ERα. Furthermore, a more frequent HDAC3 decrease accompanied by the simultaneous Foxa1/2 decline was found in female HCC compared to that in male HCC.ConclusionIn summary, we reported that loss of HDAC3 reduces Foxa1/2 and thus promotes HCC development in females in an oestrogen-dependent manner.

Characterisation of forkhead box protein A3 as a key transcription factor for hepatocyte regeneration

Liver regeneration is vital for the recovery of liver function after injury, yet the underlying mechanism remains to be elucidated. Forkhead box protein A3 (FOXA3), a member of the forkhead box family, plays important roles in endoplasmic reticulum stress sensing, and lipid and glucose homoeostasis, yet its functions in liver regeneration are unknown. Here, we explored whether Foxa3 regulates liver regeneration via acute and chronic liver injury mice models. We further characterised the molecular mechanism by chromatin immunoprecipitation sequencing and rescue experiments invivo and invitro. Then, we assessed the impact of Foxa3 pharmacological activation on progression and termination of liver regeneration. Finally, we confirmed the Foxa3-Cebpb axis in human liver samples. Foxa3 is dominantly expressed in hepatocytes and cholangiocytes and is induced upon partial hepatectomy (PH) or carbon tetrachloride (CCl4) administration. Foxa3 deficiency in mice decreased cyclin gene levels and delayed liver regeneration after PH, or acute or chronic i.p. CCl4 injection. Conversely, hepatocyte-specific Foxa3 overexpression accelerated hepatocytes proliferation and attenuated liver damage in an CCl4-induced acute model. Mechanistically, Foxa3 directly regulates Cebpb transcription, which is involved in hepatocyte division and apoptosis both invivo and invitro. Of note, Cebpb overexpression in livers of Foxa3-deficient mice rescued their defects in cell proliferation and regeneration upon CCl4 treatment. In addition, pharmacological induction of Foxa3 via cardamonin speeded up hepatocyte proliferation after PH, without interfering with liver regeneration termination. Finally, Cebpb and Ki67 levels had a positive correlation with Foxa3 expression in human chronic disease livers. These data characterise Foxa3 as a vital regulator of liver regeneration, which may represent an essential factor to maintain liver mass after liver injury by governing Cebpb transcription. Liver regeneration is vital for the recovery of liver function after chemical insults or hepatectomy, yet the underlying mechanism remains to be elucidated. Herein, via invitro and invivo models and analysis, we demonstrated that Forkhead box protein A3 (FOXA3), a Forkhead box family member, maintained normal liver regeneration progression by governing Cebpb transcription and proposed cardamonin as a lead compound to induce Foxa3 and accelerate liver repair, which signified that FOXA3 may be a potential therapeutic target for further preclinical study on treating liver injury.