FOXO3a Expression Research Articles

Abstract P5-12-06: Nuclear p-ser394-FOXO3a/FOXO3a ratio is associated with an improved survival and response to therapy in breast cancer

Abstract Background:Forkhead box O (FOXO) transcription factors, one of large forkhead family members, control a wide spectrum of carcinogenesis, therapy response and progression in human cancer. Phosphorylation of FOXO3a by ERK1/2 at ser294, ser344, and ser425 induces its nuclear exclusion and sequestration in the cytosol, therefore avoiding FOXO3a transcriptional activity and consequently promoting cell proliferation and tumorigenesis. We aimed to explore the prognostic significance of subcellular p-ser294-FOXO3a distribution patterns in breast cancer. Methods: Immunohistochemistry was used to explore expression patterns of ERK1/2, FOXO3a and p-Ser294-FOXO3a in 216 breast cancer lesions. The obtained information was statistically correlated with clinicopathological parameters including tumor size, lymph node metastasis, tumor stage, histological grade, estrogen receptor α (ER), progesterone receptor (PR), HER2 and patients' follow-up data. The median follow-up period was 118 months. Kaplan-Meier survival curves were analyzed using with log-rank test and Cox-regression model was used to determine their independent prognosis values. Results: Our results showed that nuclear FOXO3a and p-ser394-FOXO3a expression were progressively increased from normal, early to advanced breast tumor. Nuclear p-ser394-FOXO3a and FOXO3a expression was positively correlated (p=0.021). Nerveless, cytoplasmic ERK expression was inversely correlated with nuclear FOXO3a expression (p=0.037), but not its phosphorylation at the ser394 residue. To determine the real impact of p-ser394-FOXO3a, we redefined the ratio of p-ser394-FOXO3a over FOXO3a as p-FOXO3a index. Using χ2 analysis, we found nuclear p-FOXO3a index in tumor tissue was positively associated with lymph node metastasis (p=0.035) and inversely correlated with recurrence rate (p=0.035) in breast cancer patients. The Kaplen-Meier survival curve showed that nuclear p-FOXO3a index, but not cytoplasmic p-FOXO3a index, improved the overall survival rate (p=0.037). In addition, those patients who received hormone therapy would have a better survival rate when they had high nuclear p-FOXO3a index (p=0.005), suggesting that nuclear p-ser394-FOXO3a expression might be required for the response of hormone therapy. Intriguingly, low nuclear p-FOXO3a index significantly associated with the worse overall survival rate of patients who didn't receive chemotherapy or radiotherapy (p=0.049 and p=0.049, respectively), implicating that nuclear p-ser394-FOXO3a expression might also modulate the response of chemotherapy and radiotherapy. Multivariate Cox regression analysis demonstrated that the nuclear p-FOXO3a index (HR=0.46, 95% CI=0.22-0.97, p=0.040), stage (HR=2.48, 95% CI=1.04-5.96, p=0.041), and recurrence (HR=5.69, 95% CI=2.69-12.04, p<0.001) were independent prognostic factors for the overall survival rate of breast cancer patients. Conclusions: In conclusion, this study highlights that the phosphorylation of FOXO3a at the ser394 residue may play a crucial role in treatment response of breast cancer. Increased p-FOXO3a index may serve as a prognostic factor for an improved survival rate of breast cancer patients. Citation Format: Hou MF, Dai HY, Yan LY, Yang SF, Chen FM, Yeh YT. Nuclear p-ser394-FOXO3a/FOXO3a ratio is associated with an improved survival and response to therapy in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-06.

Evaluation of the antioxidant effects of different histone deacetylase inhibitors (HDACis) on human lens epithelial cells (HLECs) after UVB exposure

BackgroundTo compare the protective effects of the histone deacetylase inhibitors (HDACis) β-hydroxybutyrate (βOHB), trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) on human lens epithelial cells(HLECs) following ultraviolet-B (UVB) exposure.MethodsHLECs were divided into subgroups: four HDACi groups, a control group, a UVB-treated group and a DMSO group (cells treated with DMSO and UVB irradiation). In the HDACi groups, HLECs were cultured with different concentrations of HDACis 12 h prior to UVB irradiation. The protective effects of the HDACis were evaluated by assessing apoptosis rates, cell activity and expression levels of genes associated with apotosis (caspase-3, Bcl-2, BAX, SOD1, FOXO3A and MT2). The levels of superoxide dismutase (SOD), reactive oxygen species (ROS), malondialdehyde (MDA) and total antioxidant capacity (T-AOC) were detected in order to evaluate oxidative stress.ResultsThe results showed that SAHA (1 μmol/L, 2 μmol/L) and TSA (0.2 μmol/L) had mild protective effects on cell viability. βOHB (4 mmol/L) and TSA (0.2 mol/L) demonstrated protective effects on BCL-2 expression. TSA (0.2 mol/L) showed protective effects on SOD1 expression. TSA (0.2 mol/L) and SAHA (1 μmol/L) suppressed BAX and caspase-3 expression. TSA (0.2 mol/L, 0.8 mol/L) and SAHA (1 μmol/L, 2 μmol/L) suppressed the expression of FOXO3A and MT2. SOD levels were increased after treatment with βOHB (4 mmol/L), SAHA (8 μmol/L) and TSA (0.1 mol/L, 0.2 mol/L). T-AOC levels were increased in UVB-treated HLECs after treatment with SAHA (2 μmol/L). MDA levels decreased in UVB-treated HLECs following treatment with TSA (0.2 mol/L, 0.8 mol/L). ROS levels decreased in UVB-treated HLECs following treatment with βOHB (4 mmol/L), SAHA (1 μmol/L, 2 μmol/L) and TSA (0.2 mol/L). Western blotting results demonstrated that SOD1 levels significantly increased in the βOHB (4 mmol/L), SAHA (1 μmol/L, 2 μmol/L), TSA (0.1 mol/L, 0.2 mol/L) and VPA (5 mmol/L) groups. Only SAHA (1 μmol/L) had an anti-apoptotic effect on UVB-treated HLECs.ConclusionsOur findings indicate that low concentrations of HDACis (1 μmol/L of SAHA) mildly inhibit oxidative stress, thus protecting HLECs from oxidation. These results may suggest that there is a possibility to explore the clinical applications of HDACis for treatment and prevention of cataracts.

ING4 suppresses hepatocellular carcinoma via a NF-κB/miR-155/FOXO3a signaling axis.

The tumor suppressor ING4 has been shown to be reduced in human HCC. The alteration of ING4 contributes to HCC progression. However, its effect in HCC and the potential mechanism is largely unclear. Herein, we found that downregulation of ING4 in HCC tumor tissues was closely associated with cancer staging, tumor size and vascular invasion. Lentivirus-mediated ING4 overexpression significantly inhibited proliferation, migration and invasion, and induced cell cycle G1 phase arrest and apoptosis in MHCC97H human HCC cells. Moreover, overexpression of ING4 dramatically suppressed MHCC97H tumor cell growth and metastasis to lung in vivo in athymic BALB/c nude mice. Mechanistic studies revealed that overexpression of ING4 markedly increased expression of FOXO3a both at the mRNA and protein level as well as enhanced nuclear level and transcriptional activity of FOXO3a in MHCC97H tumor cells. In addition, ING4 repressed transcriptional activity of NF-κB and expression of miR-155 targeting FOXO3a. Knockdown of ING4 exhibited opposing effects in MHCC97L human HCC cells. Interestingly, knockdown of FOXO3a attenuated not only ING4-elicited tumor suppression but also ING4-mediated regulatory effect on FOXO3a downstream targets, confirming that FOXO3a is involved in ING4-directed tumor-inhibitory effect in HCC. Overexpression of miR-155 attenuated ING4-induced upregulation of FOXO3a, whereas inhibition of miR-155 blunted ING4 knockdown-induced reduction of FOXO3a. Furthermore, inhibition of NF-κB markedly impaired ING4 knockdown-induced upregulation of miR-155 and downregulation of FOXO3a. Taken together, our study provided the first compelling evidence that ING4 can suppress human HCC growth and metastasis to a great extent via a NF-κB/miR-155/FOXO3a pathway.

Perfluorooctane sulfonate induces apoptosis via activation of FoxO3a and upregulation of proapoptotic Bcl-2 proteins in PC12 cells.

Perfluorooctane sulfonate (PFOS), a kind of organic pollutant widely found in the environment and biota, could alter normal brain development and produce cognitive dysfunction. For the past years, the neurotoxic effects of PFOS have been shown. Recent studies have proven that PFOS can induce neuronal apoptosis and cause neurotoxicity, but the regulatory proteins referred to the process have not been clarified. In this study, PC12 cells were used to investigate the changes of the expression of apoptosis-related proteins, forkhead box O3 (FoxO3a) and pro-apoptotic Bcl-2 proteins. We detected that the levels of cleaved caspase-3 and cleaved PARP were up-regulated obviously in PFOS-treated PC12 cells by using Western blotting, and that the apoptotic rate of PC12 cells was increased significantly by using flow cytometry, verifying that PFOS could induce neuronal apoptosis. Western blot analysis and immunofluorescence revealed obvious up-regulation of the expression of FoxO3a and proapoptotic Bcl-2 proteins. In addition, knockdown of FoxO3a gene inhibited Bim expression and apoptosis. According to the data, we believe that FoxO3a may play a crucial role in PFOS-induced neurotoxicity.

Activation of the AKT/FOXO3a Signaling Pathway during Breast Cancer Inhibition In Vivo and In Vitro by Amauroderma rude (Agaricomycetes).

We previously reported that Amauroderma rude polysaccharides (AR) displayed strong immunomodulatory tumor-suppressive effects in mice. The current study was designed to explore the potential mechanism by which AR polysaccharides inhibit tumor growth. We found that AR could effectively induce cell death in 4T1 and MDA-MB-231 breast cancer cells. AR could also inhibit tumor cell migration and invasion, significantly promote apoptosis in 4T1 cells, and significantly increase CDK4, CDK6, cylinD1, and P27 mRNA expression in mice. Additionally, AR was able to significantly increase FOXO3a expression and decrease p-AKT expression both in vitro and in vivo, indicating that the AKT/FOXO3a signaling pathway had been activated during the inhibitory process.

HCRP-1 regulates EGFR\u2013AKT\u2013BIM-mediated anoikis resistance and serves as a prognostic marker in human colon cancer

Hepatocellular carcinoma-related protein-1 (HCRP-1), a subunit of mammalian endosomal sorting complex required for transport-I (ESCRT-I), is frequently downregulated in various kinds of malignant tumors. The role of HCRP-1 in colorectal cancer (CRC) remains unknown. We investigate the clinical value of HCRP-1 and its impact on anoikis in CRC. The negative expression of HCRP-1 was significantly correlated with tumor size (P = 0.033), PT status (P = 0.001), TNM stage (P = 0.039), and histological grade (P = 0.01). Univariate and multivariate analyses revealed that HCRP-1 was an independent prognostic factor for CRC (hazard ratio (HR) = 0.237, P < 0.001 for 5-year overall survival). In the in vitro assay, we found that HCRP-1 depletion resulted in cell anoikis resistance. Knockdown of HCRP-1 suppressed Bcl-2 interacting mediator of cell death (BIM) expression, with phosphorylation of AKT and p-FoxO3a, which was reversed by AKT siRNA or AKT inhibitor. Further analysis showed that loss of HCRP-1 obviously increased the activation of EGFR. Inhibition of EGFR blocked si-HCRP1-mediated phosphorylation of EGFR, AKT, FoxO3a, and BIM expression. Moreover, the in vivo results revealed that loss of HCRP-1 promoted cancer metastasis. Our findings implied that reduced HCRP-1 expression in CRC resulted in anoikis resistance and contributed to CRC metastasis and poor prognosis. These data may help design effective therapy targeting HCRP-1 pathway to control colon cancer growth and metastasis.

Metformintreatment Overcomes ATRA-Resistance in Acute Promyelocytic Leukemia and Increases FOXO3A Expression

Introduction:FOXO3A is a transcription factor shown to be involved in all-trans retinoic acid (ATRA)-induced granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL). Its biological activity may be significantly enhanced upon metformin, raising the possibility that ATRA and Metformin may act synergistically; which could be useful to overcome ATRA resistance. Despite progress in APL treatment, approximately 10-15% of patients will relapse after treatment with ATRA and anthracyclines and frequently present with ATRA resistance. Relapsed patients respond well to arsenic trioxide (ATO) treatment, but the cost of ATO is a significant barrier in many countries. Aims: Here, we investigated the effects of in vitro treatment with ATRA plus metformin in APL cell lines and primary blasts, and quantified FOXO3A expression and correlated its levels with treatment outcome in a cohort of patients enrolled in the International Consortium on Acute Leukemia (ICAPL2006) study. Finally, we evaluated the effect of induced overexpression of FOXO3A gene in regards to cell viability and proliferation. Methods: Primary leukemic blasts from hCG-PMLRARα transgenic mice (TM; n=4) and APL patients (age, 25-47y; n=4) were treated with metformin alone (5mM/ 10mM) or in combination with ATRA (1µM) and evaluated for cell viability. In addition, 106 patients (age, 18-82y) with newly diagnosed APL enrolled in the ICAPL2006 study were included. As controls, eight samples of bone marrow mononuclear cells (BMMC) from healthy volunteers (age, 18-60y) were analyzed. To validate our data, FOXO3A transcript levels from an independent cohort was used (31 patients from Amazonia!, Probe n. 204131_s_at, and five normal BMMC samples included in the same databank). NB4/NB4R2 (ATRA-resistant) cell lines were transduced with FOXO3A or empty vector (control). After synchronization using double thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For dose-response curves, cells were treated with graded concentrations of ATRA, ATO and metformin. For the apoptosis analysis, cells were treated with ATRA (1µM), metformin (5mM) and combination for 24, 48 and 72 hours. The granulocytic differentiation in response to ATRA treatment was evaluated based on the CD11b surface levels. Results: In primary cells (from TM/APL patients) a 2-fold reduction in viable cells was observed after metformin and metformin plus ATRA treatment (P Disclosures Tallman:BioSight: Other: Advisory board; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding; AROG: Research Funding; AbbVie: Research Funding; Cellerant: Research Funding; Orsenix: Other: Advisory board. Ganser:Novartis: Membership on an entity9s Board of Directors or advisory committees. Lowenberg:Editorial Board "European Oncology & Haematology": Membership on an entity9s Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity9s Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity9s Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity9s Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity9s Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity9s Board of Directors or advisory committees; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity9s Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity9s Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity9s Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity9s Board of Directors or advisory committees; Astex: Consultancy; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity9s Board of Directors or advisory committees; Celgene: Membership on an entity9s Board of Directors or advisory committees.

Arsenic Trioxide Suppressed Migration and Angiogenesis by Targeting FOXO3a in Gastric Cancer Cells.

Arsenic trioxide (As2O3), a traditional remedy in Chinese medicine, has been used in acute promyelocytic leukemia (APL) research and clinical treatment. Previous studies have shown that As2O3 exerts its potent antitumor effects in solid tumors by regulating cell proliferation and survival. The aim of this study was to investigate whether As2O3 inhibited gastric cancer cell migration and angiogenesis by regulating FOXO3a expression. We found that As2O3 reduced gastric cancer cell viability in a dose-dependent manner and also inhibited cell migration and angiogenesis in vitro. Western blotting and immunofluorescence showed that As2O3 downregulated the levels of p-AKT, upregulated FOXO3a expression in the nucleus, and attenuated downstream Vascular endothelial growth factor (VEGF) and Matrix metallopeptidase 9 (MMP9) expression. Moreover, we demonstrated that knockdown of FOXO3a significantly reversed the inhibition of As2O3 and promoted cell migration and angiogenesis in vitro. Further, As2O3 significantly inhibited xenograft tumor growth and angiogenesis by upregulating FOXO3a expression in vivo. However, knockdown of FOXO3a attenuated the inhibitory effect of As2O3 in xenograft tumors, and increased microvessel density (MVD) and VEGF expression. Our results demonstrated that As2O3 inhibited migration and angiogenesis of gastric cancer cells by enhancing FOXO3a expression.

Coiled-coil domain-containing 80 accelerates atherosclerosis development through decreasing lipoprotein lipase expression via ERK1/2 phosphorylation and TET2 expression

Recent studies showed that coiled-coil domain-containing 80 (CCDC80) has a positive link with atherosclerosis and that plasma CCDC80 levels are positively correlated with the levels of fasting plasma triglycerides (TG) in obese individuals. The underlying mechanisms, however, are unclear. Using Hematoxylin-eosin (H&E) and Oil Red O staining, we found that CCDC80 overexpression in vivo significantly increased plasma lipid contents, decreased the expression and activity of lipoprotein lipase (LPL), and accelerated the development of atherosclerosis. Conversely, knockdown of CCDC80 decreased plaque lesions area. In vitro, qRT-PCR and western blot results showed that CCDC80 overexpression significantly decreased, while CCDC80 knockdown increased, LPL expression in cultured vascular smooth muscle cells (VSMCs). Further, we found that CCDC80 reduced LPL expression via inhibiting the phosphorylation of extracellular regulated protein kinase 1/2 (ERK1/2) and also increased the methylation of LPL promoter via down-regulating Tet methylcytosine dioxygenase 2 (TET2). Our results also revealed that CCDC80 significantly down-regulated TET2 expression through decreasing the phosphorylation of ERK1/2. In addition, we found that CCDC80 decreased binding of TET2 to forkhead box O3 (FOXO3a) but had no effect on FOXO3a expression. On the other hand, and that FOXO3a was partially involved in TET2-regulated LPL expression. CCDC80 down-regulated ERK1/2 phosphorylation and decreased expression of TET2 and its interaction with FOXO3a, leading to a reduction of LPL expression and acceleration of atherosclerosis.

SIRT1 knock-in mice preserve ovarian reserve resembling caloric restriction

Previous studies have proposed that caloric restriction (CR) regulates many cell functions and prolongs the lifespan of an organism. Our previous studies proposed that CR also prevents follicular activation and preserves the ovarian reserve in mice by activating SIRT1. To test if SIRT1 preserves the ovarian reserve and prolongs the ovarian longevity, we generated SIRT1 knock-in mice that can overexpress SIRT1 in oocytes of the mouse. Ovaries of the mice at ages 35 days and 15 months were collected, and the follicular development and follicular reserve were examined. The vaginal opening and onset of estrus of transgenic female mice (both the homozygous and heterozygous for SIRT1 overexpression) were later than that of wild-type mice. Both the homozygous and heterozygous SIRT1-overexpressing mice had a larger and stronger reproductive capacity than wild-type mice. Moreover, 35-day-old and 15-month-old homozygous and heterozygous SIRT1-overexpressing mice also had a higher mean number and percentage of healthy follicles, fewer atretic follicles than wild-type mice, and the mean number and percentage of primordial follicles in both the homozygous and heterozygous SIRT1-overexpressing mice were higher than wild-type mice at the same age. However, the phenotypes of heterozygous and homozygous transgenic mice came no difference. Immunohistochemistry showed increased expression of SIRT1 and FOXO3a, and decreased expression of mTOR in both the homozygous and heterozygous SIRT1-overexpressing mice compared with wild-type mice. Thus, oocyte-specific SIRT1-overexpressing mice continuously activate FOXO3a and suppress mTOR and have a larger reproductive capacity, larger follicle reserve and longer ovarian lifespan.

MiR-34a regulates the glucose metabolism of Blunt snout bream (Megalobrama amblycephala) fed high-carbohydrate diets through the mediation of the Sirt1/FoxO1 axis

To investigate the regulatory effect of miR-34a on the sirtuin 1(sirt1) / forkhead box O 1 (foxo1) axis in Megalobrama amblycephala under high carbohydrate metabolism, M. amblycephala fish were fed either a control or a high glucose diet for eight weeks. Metabolic models of the high glucose group, normal glucose group, miR-34a inhibition group, and miR-34a overexpression group were constructed using miR-34a inhibitor and miR-34a mimic injection (where appropriate) in M. amblycephala. The results showed that the hepatic expression of SIRT1 was decreased by the high glucose diet after eight weeks, which promoted the expression of foxo1a. Additionally, hepatic pepck and g6pase expression were upregulated, thereby promoting gluconeogenesis. The upregulation of pparα increased the expression of lipid metabolic enzymes such as fas, lps, and acc; therefore, excessive glycogen was deposited as lipid in the adipose tissue. Furthermore, we found that SIRT1 expression increased following miR-34a inhibitor injection, activating foxo1a expression levels, reducing hepatic gluconeogenesis, decreasing hepatic glycogen accumulation, and accelerating hepatic fat decomposition and fatty acid synthesis. In a nutrient metabolic homeostatic state, injection of miR-34a mimics promoted the expression of foxo1a and pepeck, enhanced fat metabolism and fatty acid synthesis, and inhibited SIRT1 protein expression. These results revealed that miR-34a mediates regulation of metabolic enzymes related to hepatic glucose and lipid metabolism by targeting SIRT1/FoxO1 in M. amblycephala under high carbohydrate diet.

Development of acquired resistance to lapatinib may sensitise HER2-positive breast cancer cells to apoptosis induction by obatoclax and TRAIL

BackgroundLapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells.MethodsWe examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer.ResultsIn this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells.ConclusionsOur findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.

The involvement of miR-155 in blood pressure regulation in pregnant hypertension rat via targeting FOXO3a.

Pathogenesis factor of pregnant hypertension is still unclear and lacks of effective treatment. MiR-155 is a recently discovered miRNA molecule with differential expression in pregnant hypertension, which participates in the disease regulation. As a downstream target gene of miR-155, FOXO3a is correlated with blood pressure regulation. We investigated the regulatory role and mechanism of miR-155 in pregnant hypertension. We established a pregnant hypertension rat model, on which miR-155 inhibitor or FOXO3a siRNA was applied, followed by HE staining, 24 h urea protein, blood pressure and serum creatine assay to evaluate disease severity. MiR-155 expression was significantly elevated in model rats, accompanied by a reduction of the FOXO3a level. MiR-155 inhibitor suppressed miR-155 expression, increased FOXO3a level and placental tissue morphology by HE staining, and depressed blood pressure as well as serum creatine level. Downregulation of FOXO3a by specific siRNA resulted in opposite effects. These results illustrated the miR-155 mediated FOXO3a expression in pregnant hypertension. The inhibition of miR-155 improves the damage of pregnant hypertension via the upregulation of FOXO3a, which provides academic leads for the future therapy of pregnant hypertension.

Metabolic gene expression and epigenetic effects of the ketone body \u03b2-hydroxybutyrate on H3K9ac in bovine cells, oocytes and embryos

The rapid decline in fertility that has been occurring to high-producing dairy cows in the past 50 years seems to be associated with metabolic disturbances such as ketosis, supporting the need for research to improve our understanding of the relations among the diet, metabolism and embryonic development. Recently, the ketone body β-hydroxybutyrate (BOHB) was demonstrated to be a potent inhibitor of histone deacetylases (HDACs). Herein, we performed a series of experiments aiming to investigate the epigenetic effects of BOHB on histone acetylation in somatic cells, cumulus-oocyte complexes (COCs) and somatic cell nuclear transfer (SCNT) embryos. Treatment with BOHB does not increase histone acetylation in cells but stimulates genes associated with ketolysis and master regulators of metabolism. We further demonstrated that maturing COCs with high levels of BOHB does not affect their maturation rate or histone acetylation but increases the expression of PPARA in cumulus cells. Treatment of somatic cell nuclear transfer zygotes with BOHB causes hyperacetylation, which is maintained until the blastocyst stage, causing enhanced FOXO3A expression and blastocyst production. Our data shed light on the epigenetic mechanisms caused by BOHB in bovine cells and embryos and provide a better understanding of the connection between nutrition and reproduction.

MiR-223 promotes cardiomyocyte apoptosis by inhibiting Foxo3a expression.

MicroRNAs (miRs) are proven to possess diversified functions in the pathogenesis of cardiac diseases. The current study is designed aiming at determining the effect of miR-223 on oxidative stress induced apoptosis in cardiomyocytes. Mouse model of myocardial infarction (MI) was constructed, and endogenous level of miR-223 in the border zone of infarcted heart tissues was determined. Primarily cultured cardiomyocytes were exposed to H2O2 treatment to mimic the oxidative stress stimulation. Multiple approaches including quantitative reverse transcription polymerase chain reaction (qRT-PCR), cell viability assay, luciferase assay, Western blot assay and flow cytometry assay were employed to determine its expression, function and mechanism in apoptosis. MiR-223 expression was significantly upregulated in the border zone of infarcted heart ventricular tissues and in cardiomyocytes treated with H2O2. Overexpression of miR-223 in cardiomyocytes promoted apoptosis, whereas inhibition of endogenous miR-223 protected cardiomyocytes from oxidative stress induced apoptosis. MiR-223 directly targets the 3'untranslated region (UTR) of Foxo3a mRNA. Overexpression of miR-223 inhibited Foxo3a protein expression, however, inhibition of miR-223 suppressed its expression. Silencing Foxo3a using small interfering RNA (siRNA) mimicked the effect of miR-223, indicating its functional significance. MiR-223 is an important regulator of cardiomyocyte apoptosis under oxidative stress. Inhibition of the miR-223/Foxo3a signaling axis may be a potential therapeutic strategy for cardiac injuries.

Inhibition of mTORC2 enhances UVB-induced apoptosis in keratinocytes through a mechanism dependent on the FOXO3a transcriptional target NOXA but independent of TRAIL

The primary cause of non-melanoma skin cancer (NMSC) is ultraviolet B (UVB) radiation. We have shown previously that mTORC2 inhibition sensitizes keratinocytes to UVB-induced apoptosis mediated by the transcription factor FOXO3a. FOXO3a is a key regulator of apoptosis and a tumor suppressor in several cancer types. Activation of FOXO3a promotes apoptosis through the coordinated expression of a variety of target genes, including TRAIL and NOXA. We hypothesized that in the setting of mTORC2 inhibition, the UVB-induced expression of these factors would lead to apoptosis in a FOXO3a-dependent manner. Using spontaneously immortalized human keratinocytes (HaCaT cells), we observed that both TRAIL and NOXA expression increased in cells exposed to UVB and the TOR kinase inhibitor Torin 2. Similar to knockdown of FOXO3a, NOXA knockdown reversed the sensitization to UVB-induced apoptosis caused by mTORC2 inhibition. In contrast, loss of TRAIL by either knockdown or knockout actually enhanced expression of nuclear FOXO3a, which maintained apoptosis. These surprising results are not due to faulty death receptor signaling in HaCaT cells, as we found that the cells undergo extrinsic apoptosis in response to treatment with recombinant TRAIL. Even more striking, TRAIL knockout cells were sensitized to recombinant TRAIL-induced apoptosis compared to wild-type HaCaT cells, with the largest increase occurring in the presence of mTORC2 inhibition. Taken together, these studies provide strong evidence that mTORC2 controls UVB-induced apoptosis by regulating NOXA expression downstream of FOXO3a. Moreover, FOXO3a transcriptional activation by mTORC2 inhibitors may be a valuable target for prevention or therapy of NMSC, especially in cases with low endogenous TRAIL.

Breeders Age Affects Reproductive Success in Nothobranchius furzeri.

The present study was conducted to examine, for the first time in Nothobranchius furzeri, the effects of mating in different aged breeders with particular emphasis on reproductive fitness and the effects of parental aging on offspring gametogenesis. In N. furzeri, the increase of maternal and paternal age is often a predictable indicator of the upcoming deterioration on their natural habitat (i.e., ephemeral puddles) during African dry season. We previously revealed that elderly parents respond to their physiological decline state by decelerating offspring's development and growth rate. In the present study, we focused on the effects of different age parents at the onset of female offspring's sexual maturity since interaction between growth and reproduction traits generally occurs in vertebrates and could represent a life strategy. For this purpose, four different age breeder groups were set up. The age-specific breeder differences in reproductive performance were examined by analyzing the reproductive age-related fitness, the gametogenesis process in F1 females by histology, the offspring oocytes quality analyzing the gene expression of age-related molecular markers, like sirt1 and foxo3a, and the biochemical composition of vitellogenic oocytes using a spectroscopic approach. Results suggest that both maternal and paternal age affect reproductive performance and could influence the onset of sexual maturity in female offspring. In conclusion, these findings highlighted the effects of parental aging on life history traits in the short-lived model N. furzeri. Our results suggested that the advanced sexual maturity in offspring from young parents could be related to an adaptive response to the temporary habitat conditions.

Different roles of insulin receptor a and b in maintaining blood glucose homeostasis in zebrafish

An inability of insulin to signal glycolysis and gluconeogenesis would largely result in type 2 diabetes. In this study, the physiological roles of zebrafish insulin receptor a and b in maintaining blood glucose homeostasis were characterized. We observed that, though blood glucose in insra−/− fish and insrb−/− fish were comparable with the control siblings at 0 h postprandium (hpp), the most evident hyperglycemia have been observed in insra−/− fish from 1 hpp to 3 hpp. A mild increase of blood glucose in insrb−/− fish has been seen only at 1.5 hpp. The down-regulated expressions of glycolytic enzymes were observed in insra−/− fish and insrb−/− fish liver and muscle, together with the significantly decreased activities or concentrations of glycolytic enzymes. These results suggest that both Insra and Insrb were critical in glycolysis. Intriguingly, the up-regulated expressions of gluconeogenic enzymes, pck1 and g6pca.1, along with the elevated enzyme activities, were observed in insra−/− fish liver at 1 hpp and 1.5 hpp. Compared with the control fish, the elevated plasma insulin and lowered phosphorylated AKT were observed in insra−/− fish and insrb−/− fish, suggesting that there is an insulin resistance in insra−/− fish and insrb−/− fish. The increased levels of both transcriptions of foxo1a and Foxo1a protein abundance in the insra−/− fish liver have been found. When insra−/− fish treated with the Foxo1 inhibitor, the postprandial blood glucose levels could be normalized, accompanied with the normalized expression levels and enzyme activities of both pck1 and g6pca.1. Therefore, Insra and Insrb demonstrate a similar role in promoting glycolysis, but Insra is involved in inhibiting gluconeogenesis via down-regulating the expression of foxo1a. Our results indicate that Insra and Insrb exhibit diversified functions in maintaining glucose homeostasis in zebrafish.

RSjP40 suppresses hepatic stellate cell activation by promoting microRNA-155 expression and inhibiting STAT5 and FOXO3a expression.

Activation of hepatic stellate cells (HSCs) is the central event of the evolution of hepatic fibrosis. Schistosomiasis is one of the pathogenic factors which could induce hepatic fibrosis. Previous studies have shown that recombinant Schistosoma japonicum egg antigen P40 (rSjP40) can inhibit the activation and proliferation of HSCs. MicroRNA‐155 is one of the multifunctional noncoding RNA, which is involved in a series of important biological processes including cell development, proliferation, differentiation and apoptosis. Here, we try to observe the role of microRNA‐155 in rSjP40‐inhibited HSC activation and explore its potential mechanisms. We found that microRNA‐155 was raised in rSjP40‐treated HSCs, and further studies have shown that rSjP40 enhanced microRNA‐155 expression by inhibiting STAT5 transcription. Up‐regulated microRNA‐155 can down‐regulate the expression of FOXO3a and then participate in rSjP40‐inhibited expression of α‐smooth muscle actin (α‐SMA) and collagen I. Furthermore, we observed microRNA‐155 inhibitor could partially restore the down‐regulation of FOXO3a, α‐SMA and collagen I expression in LX‐2 cells induced by rSjP40. Therefore, our research provides further insight into the mechanism by which rSjP40 could inhibit HSC activation via miR‐155.

Sirtuin3 protects aged human mesenchymal stem cells against oxidative stress and enhances efficacy of cell therapy for ischaemic heart diseases.

Sirtuin3 (SIRT3) is associated with oxidative stress and lifespan. However, the possible mechanisms underlying its influence are unknown. We hypothesized that SIRT3 increases the antioxidant capacity of aged cells and improves the efficacy of human mesenchymal stem cell (hMSC) therapy for ischaemic heart diseases in aged patients. In vitro, the antioxidant capacity of old hMSCs (O‐hMSCs) was increased after SIRT3 overexpression using a gene transfection technique, while the antioxidant capacity of young hMSCs (Y‐hMSCs) was decreased by SIRT3 silencing. The levels of forkhead box O3a (FoxO3a) in the nucleus, and antioxidant enzymes Mn‐superoxide dismutase (MnSOD) and catalase (CAT) increased in SIRT3‐overexpressed O‐hMSCs while they decreased in SIRT3‐silenced Y‐hMSCs after oxidative stress. Following myocardial infarction in adult rats in vivo, infarct size decreased and cardiac function was significantly enhanced after cell transplantation with SIRT3 overexpressed O‐hMSCs. The number of apoptotic cells decreased and the survival rate of transplanted cells increased following SIRT3 overexpression in O‐hMSCs. SIRT3 protects aged hMSCs against oxidative stress by positively regulating antioxidant enzymes (MnSOD and CAT) via increasing the expression of FoxO3a in the nucleus. The efficacy of aged hMSC transplantation therapy for ischaemic heart diseases can be improved by SIRT3 overexpression.