Storkhead Box Research Articles

STOX1 promotor region -922 T > C polymorphism is associated with Early-Onset preeclampsia

Preeclampsia (PE), affecting 5–8% of pregnancies, is a common pregnancy disease that has harmful effects on mother and foetus. It has been found that the STOX1 (Storkhead Box 1), which is a transcription factor, carries variants associated with PE. Previous studies showed that, there was a strong relationship between PE and STOX1 variants. Therefore, we hypothesised that variants in the promoter region of the gene may be related to the onset of PE. The aim of this study is to investigate the contribution of STOX1 gene promoter region variants to PE. The blood samples taken from 118 PE patients and 96 healthy pregnant women were analysed by Sanger sequencing method. Sequence analysis results showed that, there is a-922 T > C polymorphism (rs884181) in the promoter region of the STOX1 gene. This polymorphism was found to be statistically significant in individuals with early onset PE (p = 0.02) and in PE (p = 0.014) compared to the control group. IMPACT STATEMENT What is already known on this subject? As a result of whole-exon studies on the STOX1 gene, polymorphisms were found to disrupt the structure/expression/function of the gene and strengthen its relationship with PE and HELLP syndrome. A previous study by our team found an association between Y153H, the most common polymorphism of STOX1, and early onset PE. What do the results of this study add? In our study, it was aimed to investigate the effect of genetic modifications in STOX1 gene promoter region on PE through the maternal genotype. Because any change in the promoter region affects the expression level of the gene. Also, for the first time, sequence analysis of the promoter region of STOX1 is investigated in PE. The variations in STOX1 appear to be important in PE especially in Early Onset PE. What are the implications of these findings for clinical practice and/or further research? Although PE is a disease that occurs with pregnancy and shows its effects most during this period, women and children with a history of PE are more prone to various disorders, especially cardiovascular diseases in the following years. Therefore, understanding the pathogenesis of the disease is important for both prevention and treatment process. Variations on STOX1 appear to be important in terms of disease risk.

MiR-30a targets STOX2 to increase cell proliferation and metastasis in hydatidiform moles via ERK, AKT, and P38 signaling pathways

BackgroundA hydatidiform mole is a condition caused by abnormal proliferation of trophoblastic cells. MicroRNA miR-30a acts as a tumor suppressor gene in most tumors and participates in the development of various cancers. However, its role in hydatidiform moles is not clear.MethodsQuantitative real-time reverse transcription PCR was used to verify the expression level of miR-30a and STOX2 (encoding storkhead box 2). Flow cytometry assays were performed to detect the cell cycle in cell with different expression levels of miR-30a and STOX2. Cell Cycle Kit-8, 5-ethynyl-2′-deoxyuridine, and colony formation assays were used to detect cell proliferation and viability. Transwell assays was used to test cell invasion and migration. Dual-luciferase reporter assays and western blotting were used to investigate the potential mechanisms involved.ResultLow miR-30a expression promoted the proliferation, migration, and invasion of trophoblastic cells (JAR and HTR-8). Dual luciferase assays confirmed that STOX2 is a target of miR-30a and resisted the effect of upregulated miR-30a in trophoblastic cells. In addition, downregulation of STOX2 by miR-30a could activate ERK, AKT, and P38 signaling pathways. These results revealed a new mechanism by which ERK, AKT, and P38 activation by miR-30a/STOX2 results in excessive proliferation of trophoblast cells in the hydatidiform mole.ConclusionsIn this study, we found that miR-30a plays an important role in the development of the hydatidiform mole. Our findings indicate that miR-30a might promote the malignant transformation of human trophoblastic cells by regulating STOX2, which strengthens our understanding of the role of miR-30a in regulating trophoblastic cell transformation.

Investigation of the STOX1 polymorphism on lumbar disc herniation.

BackgroundLumbar disc herniation (LDH) is a common musculoskeletal disorder affliction and associated with several genes polymorphism. Storkhead box 1 (STOX1) gene is a transcriptional factor related with several signaling pathways including inflammatory pathway. However, little is known about single‐nucleotide polymorphisms (SNPs) of STOX1 associated with LDH risk.MethodsWe conducted a case–control study among 508 LDH cases and well‐matched 508 controls, and six candidate SNPs in STOX1 were genotyped by Agena MassARRAY. Chi‐squared test, genetic model, and haploview analysis were used to evaluate associations. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by unconditional logistic regression.ResultsIn the allelic model analysis, we found the minor allele “T” of rs7903209 and “A” of rs4472827 were associated with an increased risk of LDH (p = .029, p = .016). Furthermore, in the genotype model analysis, rs7903209 polymorphism was associated with the increased susceptibility of LDH based on dominant (p = .033) and additive model (p = .024); and rs4472827 variant was found to play a harmful role in the LDH risk based on genotype (p = .014), dominant (p = .012), and additive model (p = .015). In the haplotype analysis, the haplotype “GT” in block (rs10998461 and rs10998468) decreased LDH risk (OR = 0.7, 95% CI = 0.52–0.93, p = .016). Functional assessment indicated that rs7903209 and rs4472827 polymorphisms may influence the expression of STOX1.ConclusionOur results provide evidence for polymorphisms of rs7903209 and rs4472827 in STOX1 associated with LDH risk in Chinese Han population.

Stox1 induced the proliferation and cell cycle arrest in pulmonary artery smooth muscle cells via AKT signaling pathway

BackgroundPulmonary arterial hypertension (PAH) is a life-threatening disease characterized by the vascular remodeling that also involves proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). Overexpression of Storkhead box (STOX1) regulates genes involved hypoxia, redox balance, nitric oxide, and energy metabolism. In this study, we supposed Stox1 adjusted cells proliferation and migration in PASMCs development and played an important role in the pulmonary arterial vascular remodeling. MethodsHemodynamic assay and Right ventricular morphometric assay were used to check the rat model of PAH. HE staining was used to examine the arterial wall thickness. Masson staining showed that the deposition of collagen was significantly increased in PAH. In addition, Stox1 were assessed by immunofluorescence and immunohistochemistry staining. The effect of Stox1 on PASMCs was assessed by cell counting Kit-8 assay (CCK-8 assay), Scratch-Wound assay, EdU staining assay, Cell cycle analysis and Western blot. ResultsRight ventricular systolic pressure (RVSP) and right ventricular were significantly increased in hypoxia group and monocrotaline group compared to control group. The expression of Stox1 was increased in lung tissues in PAH rats. In vitro, the expression of Stox1 was up-regulated with time-dependent manner in hypoxia condition. Meanwhile, Stxo1 promoted the proliferation and migration in hypoxia-treated PASMCs. Moreover, we found that hypoxia promoted the expression of PCNA, Cyclin E and Cyclin A, increased more cells from G0/G1 phase to S phase and induced the activation of AKT proteins, which was significantly attenuated by inhibition of Stox1 expression in PASMCs. ConclusionThese findings indicated that Stox1 induced proliferation of PASMCs and the effect is, at least in part, mediated through AKT signaling pathway.

Alpha-1 microglobulin as a potential therapeutic candidate for treatment of hypertension and oxidative stress in the STOX1 preeclampsia mouse model

Preeclampsia is a human placental disorder affecting 2–8% of pregnancies worldwide annually, with hypertension and proteinuria appearing after 20 weeks of gestation. The underlying cause is believed to be incomplete trophoblast invasion of the maternal spiral arteries during placentation in the first trimester, resulting in oxidative and nitrative stress as well as maternal inflammation and organ alterations. In the Storkhead box 1 (STOX1) preeclampsia mouse model, pregnant females develop severe and early onset manifestations as seen in human preeclampsia e.g. gestational hypertension, proteinuria, and organ alterations. Here we aimed to evaluate the therapeutic potential of human recombinant alpha-1 microglobulin (rA1M) to alleviate the manifestations observed. Human rA1M significantly reduced the hypertension during gestation and significantly reduced the level of hypoxia and nitrative stress in the placenta. In addition, rA1M treatment reduced cellular damage in both placenta and kidneys, thereby protecting the tissue and improving their function. This study confirms that rA1M has the potential as a therapeutic drug in preeclampsia, and likely also in other pathological conditions associated with oxidative stress, by preserving normal organ function.

Effect of STOX1 on recurrent spontaneous abortion by regulating trophoblast cell proliferation and migration via the PI3K/AKT signaling pathway.

STOX1 is a transcription factor that is implicated in the high prevalence of human gestational diseases. It has been studied in various types of gestational diseases using different molecular and cellular biological technologies. However, the effect and detailed mechanism of storkhead box 1 (STOX1) in recurrent spontaneous abortion (RSA) remain unknown. This study aimed to explore the effect and detailed mechanism of STOX1 in human trophoblast cells. The result showed that downregulation of STOX1 by short hairpin RNA (shRNA) led to a decrease in proliferation and migration in HTR‐8/SVneo cells, while it induced the apoptosis of HTR‐8/SVneo cells. Moreover, the result showed that trophoblast cells expressed lower levels of pAKT and p85 subunits after treatment with STOX1 shRNA when compared with control. However, overexpression of STOX1 obviously increased the pAKT and p85 protein expressions. Transfection of pcDNA‐AKT plasmid increased cell proliferation and migration in HTR‐8/SVneo cells while suppressed the apoptosis of HTR‐8/SVneo cells. Furthermore, inhibition of the PI3K/Akt pathway by a specific inhibitor promoted cell apoptosis and aggravatedly suppressed cell proliferation and migration of HTR‐8/SVneo cells. On the other hand, upregulation of the PI3K/Akt pathway could increase the relative expression level of Bcl‐2 and decrease the relative expression levels of Bax and Bim, while inhibition of the PI3K/Akt pathway led to adverse results. Our results demonstrated that inhibition of STOX1 could suppress trophoblast cell proliferation and migration, while promote apoptosis through inhibiting the PI3K/Akt signaling pathway. These findings might provide a new fundamental mechanism for regulating RSA and could be used to prevent and treat RSA in clinic.

63. Molecular and functional long-term effects of preeclampsia on the cardiovascular system assessed on a mouse model of severe preeclampsia

Preeclampsia (PE) is a characterized by de novo hypertension and proteinuria. Women affected with PE have an increased risk of chronic hypertension and premature cardiovascular disease years later. Mice bearing transgenic fetuses overexpressing the human transcription factor STOX1 (storkhead box1) develop a severe preeclamptic phenotype. The molecular analysis of long term cardiovascular risks induced by preeclampsia will be studied using our mouse model. We started with 10 eight to ten-months-old mice, 5 had control gestations and 5 preeclamptic gestations 6 to 8 months earlier. We analyzed heart functional parameters by ultrasonography, in normal and stressed conditions. Then we carried out a transcriptional profile of endothelial cells and hearts of the mice. Fibrosis was evaluated by a histology analysis of the hearts. Finally the cytokine levels in mice plasma will be analyzed on 33 cytokines. The relative mass of the heart of the preeclamptic mice was increased by 11% (p = 0.017), with an important fibrosis. Ultrasonography revealed a lesser morphological adaptation to stress. Under dobutamin aortic peak velocity, blood pressure, and right ventricular outflow are significantly increased. The endothelial cells microarray analysis demonstrate that ∼3073 transcripts (p Our results demonstrate massive quasi-invisible long term effects of preeclampsia, affecting strongly the endothelial cells. Dilatation of hearts and fibrosis indicate a noxious cardiac tissue remodeling in preeclamptic mice. To the best of our knowledge, our study is one of the first to address the molecular effects of preeclampsia in the long term.

Preeclampsia induced by STOX1 overexpression in mice induces intrauterine growth restriction, abnormal ultrasonography and BOLD MRI signatures.

Preeclampsia is a major hypertensive disease caused by pregnancy, inducing proteinuria and increased blood pressure starting from the second half of pregnancy (early preeclampsia) or near the end of pregnancy (late preeclampsia). Pre-symptomatic diagnosis would allow for therapeutic interventions, such as with low-dose aspirin. Among non-invasive methods to explore organ physiology, Doppler ultrasonography (US) and functional blood oxygenation level-dependent (BOLD) MRI (which do not need radioactive contrast agents such as gadolinium) can be used in pregnant women. In this study, we used US and BOLD MRI to finely characterize the phenotype of preeclampsia induced by the foeto-placental overexpression of the transcription factor storkhead box 1A (STOX1A) in female mice. We could observe late fetal growth restriction consistent with the placental dysfunction revealed by US and the known association between preeclampsia and intra-uterine growth restriction. On US, uterine and umbilical artery as well as heart and kidney parameters were modified in preeclamptic mice. On BOLD MRI, mean T2* values revealed considerable differences between control and preeclamptic placentas, which suggests altered dynamics of oxygen release and ratio of oxyhemoglobin to deoxyhemoglobin in the model. These preliminary pre-clinical results suggest that BOLD MRI could be evaluated as a prognostic/diagnostic tool for preeclampsia.

Stox1 as a novel transcriptional suppressor of Math1 during cerebellar granule neurogenesis and medulloblastoma formation

Cerebellar granule neuronal progenitors (GNPs) are the precursors of cerebellar granule cells (CGCs) and are believed to be the cell of origin for medulloblastoma (MB), yet the molecular mechanisms governing GNP neurogenesis are poorly elucidated. Here, we demonstrate that storkhead box 1 (Stox1), a forkhead transcriptional factor, has a pivotal role in cerebellar granule neurogenesis and MB suppression. Expression of Stox1 is upregulated along with GNP differentiation and repressed by activation of sonic hedgehog (SHH) signaling. Stox1 exerts its neurogenic and oncosuppressing effect via direct transcriptional repression of Math1, a basic helix-loop-helix transcription activator essential for CGC genesis. This study illustrates a SHH-Stox1-Math1 regulatory axis in normal cerebellar development and MB formation.

Storkhead box 2 and melanoma inhibitory activity promote oral squamous cell carcinoma progression.

BackgroundStorkhead box protein 2 (STOX2) is a transcriptional factor associated with pre-eclampsia with fetal growth restriction. We recently reported that melanoma inhibitory activity (MIA) promotes oral squamous cell carcinoma (OSCC) progression. However, the relationship between STOX2 and MIA remains unknown in malignancies.MethodsWe used immunohistochemistry and PCR to investigate MIA and STOX2 expression in OSCC. We also performed functional analysis in human OSCC cells.ResultsMIA and STOX2 mRNA levels were higher in OSCCs than in normal oral epithelial cells, and upregulation of STOX2 was significantly correlated with overexpression of MIA. Immunostaining for STOX2 was associated with nodal metastasis (P = 0.0002) and MIA expression (P < 0.0001). Furthermore, MIA expression (P = 0.0035) and STOX2 expression (P = 0.0061) were associated with poor outcome in OSCCs. In vitro analysis using OSCC cells revealed that MIA increased expression of STOX2 by paracrine manner. Moreover, STOX2 accelerated OSCC cell growth, invasion, suppressed apoptosis, and enhanced resistance to paclitaxel, cisplatin, and 5-FU.ConclusionsOur results suggest that MIA-STOX2 signaling may be a useful diagnostic and therapeutic target in OSCCs.

Transcription factor STOX1 regulates proliferation of inner ear epithelial cells via the AKT pathway

Storkhead box 1 (STOX1) belongs to the forkhead family of transcription factors, and is reported to be involved in apoptosis of Caenorhabditis elegans. However, up to now the precise role of STOX1 in mammalian epithelial development has not been established. Here, we report that it plays an important role in regulation of proliferation of inner ear epithelial cells. Immunohistochemistry and reverse transcription-PCR assays were used to determine expression pattern of STOX1 in the mouse inner ear. Furthermore, its overexpression and knockdown effects on mouse inner ear epithelial cells were studied using RT-PCR, immunofluorescence, MTT assay, BrdU labelling and western blotting. Storkhead box 1 was selectively expressed in epithelial cells, but not in stromal cells of the inner ear. Its over-expression enhanced cell proliferation and sphere formation, however, STOX1 knockdown inhibited cell proliferation and sphere formation in purified utricular epithelial cells in culture. Consistently, several cell cycle regulatory genes such as for PCNA, cyclin A and cyclin E, were up-regulated by STOX1 over-expression. Furthermore, biochemical analyses indicated that proliferation-promoting effects induced by STOX1 were mediated via phosphorylation of AKT in these cells. Taken together, we demonstrate that STOX1 is a novel stimulatory factor for inner ear epithelial cell proliferation and might be an important target to be considered in regeneration or repair of inner ear epithelium.

Nitroso-redox balance and mitochondrial homeostasis are regulated by STOX1, a pre-eclampsia-associated gene.

Storkhead box 1 (STOX1) is a winged-helix transcription factor that is implicated in the genetic forms of a high-prevalence human gestational disease, pre-eclampsia. STOX1 overexpression confers pre-eclampsia-like transcriptomic features to trophoblastic cell lines and pre-eclampsia symptoms to pregnant mice. The aim of this work was to evaluate the impact of STOX1 on free radical equilibrium and mitochondrial function, both in vitro and in vivo. Transcriptome analysis of STOX1-transgenic versus nontransgenic placentas at 16.5 days of gestation revealed alterations of mitochondria-related pathways. Placentas overexpressing STOX1 displayed altered mitochondrial mass and were severely biased toward protein nitration, indicating nitroso-redox imbalance in vivo. Trophoblast cells overexpressing STOX1 displayed an increased mitochondrial activity at 20% O2 and in hypoxia, despite reduction of the mitochondrial mass in the former. STOX1 overexpression is, therefore, associated with hyperactive mitochondria, resulting in increased free radical production. Moreover, nitric oxide (NO) production pathways were activated, resulting in peroxynitrite formation. At low oxygen pressure, STOX1 overexpression switched the free radical balance from reactive oxygen species (ROS) to reactive nitrogen species (RNS) in the placenta as well as in a trophoblast cell line. In pre-eclamptic placentas, NO interacts with ROS and generates peroxynitrite and nitrated proteins as end products. This process will deprive the maternal organism of NO, a crucial vasodilator molecule. Our data posit STOX1 as a genetic switch in the ROS/RNS balance and suggest an explanation for elevated blood pressure in pre-eclampsia.

STOX2 but not STOX1 is differentially expressed in decidua from pre-eclamptic women: data from the Second Nord-Trondelag Health Study.

Variation in the Storkhead box-1 (STOX1) gene has previously been associated with pre-eclampsia. In this study, we assess candidate single nucleotide polymorphisms (SNPs) in STOX1 in an independent population cohort of pre-eclamptic (n = 1.139) and non-pre-eclamptic (n = 2.269) women (the HUNT2 study). We also compare gene expression levels of STOX1 and its paralogue, Storkhead box-2 (STOX2) in decidual tissue from pregnancies complicated by pre-eclampsia and/or fetal growth restriction (FGR) (n = 40) to expression levels in decidual tissue from uncomplicated pregnancies (n = 59). We cannot confirm association of the candidate SNPs to pre-eclampsia (P > 0.05). For STOX1, no differential gene expression was observed in any of the case groups, whereas STOX2 showed significantly lower expression in deciduas from pregnancies complicated by both pre-eclampsia and FGR as compared with controls (P = 0.01). We further report a strong correlation between transcriptional alterations reported previously in choriocarcinoma cells over expressing STOX1A and alterations observed in decidual tissue of pre-eclamptic women with FGR.