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 RCC 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 RCC proliferation in vitro and RCC progression in vivo. Importantly, elevated expression of FOXA2 in patients with RCC 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.

Effect of maternal body mass index on the steroid profile in women with gestational diabetes mellitus.

Objective: To explore the effect of maternal body mass index (BMI) on steroid hormone profiles in women with gestational diabetes mellitus (GDM) and those with normal glucose tolerance (NGT). Methods: We enrolled 79 women with NGT and 80 women with GDM who had a gestational age of 24-28 weeks. The participants were grouped according to their BMI. We quantified 11 steroid hormones profiles by liquid chromatography-tandem mass spectrometry and calculated the product-to-precursor ratios in the steroidogenic pathway. Results: Women with GDM and BMI<25kg/m2 showed higher concentrations of dehydroepiandrosterone (DHEA) (p<0.001), testosterone (T) (p=0.020), estrone (E1) (p=0.010) and estradiol (E2) (p=0.040) and lower Matsuda index and HOMA-ß than women with NGT and BMI<25kg/m2. In women with GDM, concentrations of E1 (p=0.006) and E2 (p=0.009) declined, accompanied by reduced E2/T (p=0.008) and E1/androstenedione (A4) (p=0.010) in the BMI>25 kg/m2 group, when compared to that in the BMI<25 kg/m2 group. The values of E2/T and E1/A4 were used to evaluate the cytochrome P450 aromatase enzyme activity in the steroidogenic pathway. Both aromatase activities negatively correlated with the maternal BMI and positively correlated with the Matsuda index in women with GDM. Conclusions: NGT women and GDM women with normal weight presented with different steroid hormone profiles. Steroidogenic pathway profiling of sex hormones synthesis showed a significant increase in the production of DHEA, T, E1, and E2 in GDM women with normal weight. Additionally, the alteration of steroid hormone metabolism was related to maternal BMI in women with GDM, and GDM women with overweight showed reduced estrogen production and decreased insulin sensitivity compared with GDM women with normal weight.

USF1/CD90 signaling in maintaining glioblastoma stem cells and tumor-associated macrophages adhesion.

BACKGROUND: Glioblastoma stem cells (GSCs) and their interplay with tumor-associated macrophages (TAMs) are responsible for malignant growth and tumor recurrence of glioblastoma multiforme (GBM), but the underlying mechanisms are largely unknown. METHODS: Cell viability, stemness, migration, and invasion were measured in GSCs after the knockdown of upstream stimulating factor 1 (USF1). Luciferase assay and chromatin immunoprecipitation qPCR were performed to determine the regulation of CD90 by USF1. Immunohistochemistry and immunofluorescent staining were used to examine the expression of USF1 and GSC markers, as well as the crosstalk between GSCs and TAMs. In addition, the interaction between GSCs and TAMs was confirmed using in vivo GBM models. RESULTS: We show that USF1 promotes malignant glioblastoma phenotypes and GSCs-TAMs physical interaction by inducing CD90 expression. USF1 predicts a poor prognosis for glioma patients and is upregulated in patient-derived GSCs and glioblastoma cell lines. USF1 overexpression increases the proliferation, invasion, and neurosphere formation of GSCs and glioblastoma cell lines, while USF1 knockdown exerts an opposite effect. Further mechanistic studies reveal that USF1 promotes GSC stemness by directly regulating CD90 expression. Importantly, CD90 of GSCs functions as an anchor for physical interaction with macrophages. Additionally, the USF1/CD90 signaling axis supports the GSCs and TAMs adhesion and immunosuppressive feature of TAMs, which in turn enhance the stemness of GSCs. Moreover, the overexpression of CD90 restores the stemness property in USF1 knockdown GSCs and its immunosuppressive microenvironment. CONCLUSIONS: Our findings indicate that the USF1/CD90 axis might be a potential therapeutic target for the treatment of glioblastoma.

MiR-30a-5p Regulates GLT-1 Function via a PKCα-Mediated Ubiquitin Degradation Pathway in a Mouse Model of Parkinson's Disease.

Glutamate excitotoxicity is caused by dysfunctional glutamate transporters and plays an important role in the pathogenesis of Parkinson's disease (PD); however, the mechanisms that underlie the regulation of glutamate transporters in PD are still not fully elucidated. MicroRNAs(miRNA), which are abundant in astrocytes and neurons, have been reported to play key roles in regulating the translation of glutamate-transporter mRNA. In this study, we hypothesized that the miR-30a-5p contributes to the pathogenesis of PD by regulating the ubiquitin-mediated degradation of glutamate transporter 1 (GLT-1). We demonstrated that short-hairpin RNA-mediated knockdown of miR-30a-5p ameliorated motor deficits and pathological changes like astrogliosis and reactive microgliosis in a mouse model of PD (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice). Western blotting and immunofluorescent labeling revealed that miR-30a-5p suppressed the expression and function of GLT-1 in MPTP-treated mice and specifically in astrocytes treated with 1-methyl-4-phenylpyridinium (MPP+) (cell model of PD). Both in vitro and in vivo, we found that miR-30a-5p knockdown promoted glutamate uptake and increased GLT-1 expression by hindering GLT-1 ubiquitination and subsequent degradation in a PKCα-dependent manner. Therefore, we conclude that miR-30a-5p represents a potential therapeutic target for the treatment of PD.

Combination of metformin and cold atmospheric plasma induces glioma cell death to associate with c-Fos.

Glioma is the most common type of brain cancer. Chemotherapy combination with surgery and radiotherapy is a standard treatment for patients. Although there are many advances in glioma therapy, the prognosis of glioma patients has not significantly been improved over the past decades. Hence, there is still an urgent need to develop a new therapy to treat glioma. Cell viability was assessed by CellTiter Blue assay; flow cytometry (FCM) was used for detecting cell apoptosis; ROS detection was detected by ROS Assay; H2O2 detection was performed by hydrogen peroxide detection kits; real-time PCR and WB were used to determine gene expression. Using the glioma cell line U251 and U87, we investigated a possible combination inhibitory effect includes metformin and cold atmospheric plasma (CAP). The combination treatment showed a synergistic inhibitory effect on cell viability, significantly inducing cell apoptosis. Furthermore, we also found H2O2 produced by CAP has an important role in the synergistic inhibitory effect, eliminating H2O2 with catalase reversed the synergistic inhibitory effect. In addition, the transcript and protein levels of c-FOS were robustly increased after co-treated with metformin and CAP. Taken together, we propose that pre-treatment of glioma cells with metformin sensitize tumor cells to CAP, which may serve as a potential therapeutic strategy for glioma.

Ticagrelor prevents tumor metastasis via inhibiting cell proliferation and promoting platelet apoptosis.

Tumor cells can activate platelets, which in turn facilitate tumor cell survival and dissemination. Platelets inhibition or blocking platelet-tumor cell interactions has become a strategy to suppress tumor progression. In this study, we investigated the effect of ticagrelor, a new antiplatelet drug, on tumor cell proliferation and metastasis. Our results show that ticagrelor not only inhibits the proliferation, migration, and invasion of B16F10 and Lewis lung carcinoma cells but also induces platelet apoptosis. In addition, we find that apoptosis of the platelet cells is dose dependent. Further, the result of in-vivo experiments proved that ticagrelor treatment decreased the tumor metastasis. The results of this study demonstrate that ticagrelor may be a potential anti-tumor agent for tumor metastasis.

Generation of a Novel Mouse Model of Parkinson's Disease via Targeted Knockdown of Glutamate Transporter GLT-1 in the Substantia Nigra.

Parkinson's disease (PD) is a common neurodegenerative disease that is characterized by pathological dopaminergic (DA) neuronal death and α-synuclein aggregation. Glutamate excitotoxicity is a well-established pathogenesis of PD that involves dysfunctional expression of glutamate transporters. Glutamate transporter-1 (GLT-1) is mainly responsible for clearance of glutamate at synapses, including DA synapses. However, the role of GLT-1 in the aberrant synaptic transmission in PD remains elusive. In the present study, we generated small-interfering RNAs (siRNAs) to knockdown GLT-1 expression in primary astrocytes, and we report that siRNA knockdown of astrocytic GLT-1 decreased postsynaptic density-95 (PSD-95) expression in neuron-astrocyte cocultures in vitro. Using adeno-associated viruses (AAVs) targeting GLT-1 short-hairpin RNA (shRNA) sequences with a glial fibrillary acidic protein (GFAP) promoter, we abolished astrocytic GLT-1 expression in the substantia nigra pars compacta (SNpc) of mice. We found that GLT-1 deficiency in the SNpc induced parkinsonian phenotypes in terms of progressive motor deficits and nigral DA neuronal death in mice. We also found that there were reactive astrocytes and microglia in the SNpc upon GLT-1 knockdown. Furthermore, we used RNA sequencing to determine altered gene expression patterns upon GLT-1 knockdown in the SNpc, which revealed that disrupted calcium signaling pathways may be responsible for GLT-1 deficiency-mediated DA neuronal death in the SNpc. Taken together, our findings provide evidence for a novel role of GLT-1 in parkinsonian phenotypes in mice, which may contribute to further elucidation of the mechanisms of PD pathogenesis.

Regulatory Mechanism of miR-543-3p on GLT-1 in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) features the degeneration and death of dopamine neurons in the substantia nigra pars compacta and the formation of Lewy bodies that contain α-synuclein. Among the numerous PD etiologies, glutamate excitotoxicity is a research hot spot, and glutamate transporters play key roles in this theory. It has been shown that the expression of the glutamate transporter is regulated by microRNAs. In this study, we found that the levels of expression and function of glutamate transporter type 1 (GLT-1) were significantly reduced and miR-543-3p was upregulated during the development of PD. Furthermore, our results indicated that GLT-1 plays an important role in the pathomechanism of PD. We found that miR-543-3p can suppress the expression and function of GLT-1 in MPP+-treated astrocytes and MPTP-treated mice. Inhibition of miR-543-3p can rescue the expression and function of GLT-1 and relieve dyskinesia in the PD model, which suggests that inhibition of miR-543-3p could serve as a potential therapeutic target for PD.

Akt-mediated platelet apoptosis and its therapeutic implications in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count which can cause fatal hemorrhage. ITP patients with antiplatelet glycoprotein (GP) Ib-IX autoantibodies appear refractory to conventional treatments, and the mechanism remains elusive. Here we show that the platelets undergo apoptosis in ITP patients with anti-GPIbα autoantibodies. Consistent with these findings, the anti-GPIbα monoclonal antibodies AN51 and SZ2 induce platelet apoptosis in vitro. We demonstrate that anti-GPIbα antibody binding activates Akt, which elicits platelet apoptosis through activation of phosphodiesterase (PDE3A) and PDE3A-mediated PKA inhibition. Genetic ablation or chemical inhibition of Akt or blocking of Akt signaling abolishes anti-GPIbα antibody-induced platelet apoptosis. We further demonstrate that the antibody-bound platelets are removed in vivo through an apoptosis-dependent manner. Phosphatidylserine (PS) exposure on apoptotic platelets results in phagocytosis of platelets by macrophages in the liver. Notably, inhibition or genetic ablation of Akt or Akt-regulated apoptotic signaling or blockage of PS exposure protects the platelets from clearance. Therefore, our findings reveal pathogenic mechanisms of ITP with anti-GPIbα autoantibodies and, more importantly, suggest therapeutic strategies for thrombocytopenia caused by autoantibodies or other pathogenic factors.

Simvastatin Inhibits Activation of NADPH Oxidase/p38 MAPK Pathway and Enhances Expression of Antioxidant Protein in Parkinson Disease Models.

Evidence suggests that oxidative stress is involved in the pathogenesis of Parkinson disease (PD). Simvastatin has been suggested to protect against oxidative stress in several diseases. However, the molecular mechanisms by which simvastatin protects against neuropathology and oxidative damage in PD are poorly elucidated. In this study, we aimed to investigate the potential neuroprotective effects of simvastatin owing to its anti-oxidative properties in 6-hydroxydopamine (6-OHDA)-treated SH-SY5Y cells and mice. The results of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence and CCK-8 assay demonstrated that simvastatin reduced intracellular reactive oxygen species (ROS) levels and reversed apoptosis in 6-OHDA-treated SH-SY5Y cells. Mechanistic studies revealed that 6-OHDA-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/p38 mitogen-activated protein kinase (MAPK) pathway was inhibited and nuclear factor-κB (NF-κB) nuclear transcription decreased in SH-SY5Y cells after simvastatin treatment. Enhanced expression levels of superoxide dismutase (SOD), heme oxygenase-1 (HO-1), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and glutamate-cysteine ligase modifier subunit (GCLM) were observed after simvastatin treatment in 6-OHDA-treated SH-SY5Y cells. In vivo studies revealed that administration of simvastatin by gavage decreased limb-use asymmetry and apomorphine-induced rotations in 6-OHDA-lesioned mice. Simvastatin increased dopaminergic neurons and reduced protein tyrosine nitration and gliosis in the midbrain of PD mice. An inhibitory effect on activation of the NADPH oxidase/p38 MAPK was observed, and increased antioxidant protein expression in the midbrain were seen in the simvastatin plus 6-OHDA group compared with the 6-OHDA-lesioned group. Taken together, these results demonstrate that simvastatin might inhibit the activation of NADPH oxidase/p38 MAPK pathway, enhance antioxidant protein expression and protect against oxidative stress, thereby providing a novel antioxidant mechanism that has therapeutic validity.

Protein kinase A determines platelet life span and survival by regulating apoptosis.

Apoptosis delimits platelet life span in the circulation and leads to storage lesion, which severely limits the shelf life of stored platelets. Moreover, accumulating evidence indicates that platelet apoptosis provoked by various pathological stimuli results in thrombocytopenia in many common diseases. However, little is known about how platelet apoptosis is initiated or regulated. Here, we show that PKA activity is markedly reduced in platelets aged in vitro, stored platelets, and platelets from patients with immune thrombocytopenia (ITP), diabetes, and bacterial infections. Inhibition or genetic ablation of PKA provoked intrinsic programmed platelet apoptosis in vitro and rapid platelet clearance in vivo. PKA inhibition resulted in dephosphorylation of the proapoptotic protein BAD at Ser155, resulting in sequestration of prosurvival protein BCL-XL in mitochondria and subsequent apoptosis. Notably, PKA activation protected platelets from apoptosis induced by storage or pathological stimuli and elevated peripheral platelet levels in normal mice and in a murine model of ITP. Therefore, these findings identify PKA as a homeostatic regulator of platelet apoptosis that determines platelet life span and survival. Furthermore, these results suggest that regulation of PKA activity represents a promising strategy for extending platelet shelf life and has profound implications for the treatment of platelet number-related diseases and disorders.

Microarray analysis of anti-cancer effects of docosahexaenoic acid on human colon cancer model in nude mice.

Docosahexaenoic acid (DHA), a derivative of ω3- polyunsaturated fatty acids present in fish oil, is well known to have anticancer activity on colon cancer cells, but the molecular and cellular mechanisms remain to be further clarified. In this study, anti-cancer effects of DHA on colon cancer cells were observed in a nude mouse HCT-15 xenograft model. And then, the different genes expression and signal pathways involved in this process were screened and identified using cDNA microarray analysis. Results of genes expression profiles indicated a reprogramming pattern of previously known and unknown genes and transcription factors associated with the action of DHA on colon cancer cells. And several genes related to tumor growth and metastasis including COX2, HIF-1α, VEGF-A, COMP, MMP-1, MMP-9, SCP2, SDC3, which were down-regulated by DHA, were further confirmed in HCT-15 cell line using RT-PCR method. In summary, our data might provide novel information for anti-cancer mechanism of DHA in colon cancer model.

Elevated DNA polymerase iota (Poli) is involved in the acquisition of aggressive phenotypes of human esophageal squamous cell cancer.

DNA polymerase iota (Polι) can repair several types of DNA damage but has extremely low fidelity. Previous studies have shown an aberrantly elevated Polι expression in human esophageal squamous cell cancer tissues. However, there were few reports describing the role of Polι in esophageal cancer progression. Based on Real-time PCR assay, we found Polι expression was up-regulated in esophageal cancer tissues compared to adjacent normal tissues and overexpression of Polι was correlated to lymph node metastasis. Clonogenic assay and transwell chamber assay showed that overexpression of Polι had higher clongenic capability and invasive tendency in human esophageal squamous cell cancer cells. Expression of cyclin D1, an important cell cycle regulator, was found to be associated with that of Polι in tissue samples and cancer cells as analyzed by real-time PCR, immunohistochemistry, Western blotting and immunofluorescence assay. Flow cytometry analysis further showed that cell cycle distribution was altered in Polι overexpressing cells. These results indicated that expression of Polι correlates significantly with tumor proliferation and invasion. We conclude that Polι is involved in the degree of aggressiveness of human esophageal squamous cell cancer.

Integrating microRNA and mRNA expression profiles in response to radiation-induced injury in rat lung.

PURPOSE: Exposure to radiation provokes cellular responses, which are likely regulated by gene expression networks. MicroRNAs are small non-coding RNAs, which regulate gene expression by promoting mRNA degradation or inhibiting protein translation. The expression patterns of both mRNA and miRNA during the radiation-induced lung injury (RILI) remain less characterized and the role of miRNAs in the regulation of this process has not been studied. The present study sought to evaluate miRNA and mRNA expression profiles in the rat lung after irradiation. METHODS AND MATERIALS: Male Wistar rats were subjected to single dose irradiation with 20 Gy using 6 MV x-rays to the right lung. (A dose rate of 5 Gy/min was applied). Rats were sacrificed at 3, 12 and 26 weeks after irradiation, and morphological changes in the lung were examined by haematoxylin and eosin. The miRNA and mRNA expression profiles were evaluated by microarrays and followed by quantitative RT-PCR analysis. RESULTS: A cDNA microarray analysis found 2183 transcripts being up-regulated and 2917 transcripts down-regulated (P ≤ 0.05, ≥2.0 fold change) in the lung tissues after irradiation. Likewise, a miRNAs microarray analysis indicated 15 miRNA species being up-regulated and 8 down-regulated (P ≤ 0.05). Subsequent bioinformatics analyses of the differentially expressed mRNA and miRNAs revealed that alterations in mRNA expression following irradiation were negatively correlated with miRNAs expression. CONCLUSIONS: Our results provide evidence indicating that irradiation induces alterations of mRNA and miRNA expression in rat lung and that there is a negative correlation of mRNA and miRNA expression levels after irradiation. These findings significantly advance our understanding of the regulatory mechanisms underlying the pathophysiology of radiation-induced lung injury. In summary, RILI does not develop gradually in a linear process. In fact, different cell types interact via cytokines in a very complex network. Furthermore, this study suggests that microRNAs may serve an important role in the pathogenesis of RILI and that understanding their role in RILI may have a significant effect on patient management and diagnosis in the future.

Amelioration of radiation-induced skin injury by adenovirus-mediated heme oxygenase-1 (HO-1) overexpression in rats.

OBJECTIVE: Radiation-induced skin injury remains a serious concern for radiation therapy. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has been reported to have potential antioxidant and anti-apoptotic properties. However, the role of HO-1 in radiation-induced skin damage remains unclear. This study aims to elucidate the effects of HO-1 on radiation-induced skin injury in rats. METHODS: A control adenovirus (Ad-EGFP) and a recombinant adenovirus (Ad-HO1-EGFP) were constructed. Rats were irradiated to the buttock skin with a single dose of 45 Gy followed by a subcutaneous injection of PBS, 5 × 109 genomic copies of Ad-EGFP or Ad-HO1-EGFP (n = 8). After treatment, the skin MDA concentration, SOD activity and apoptosis were measured. The expression of antioxidant and pro-apoptotic genes was determined by RT-PCR and real-time PCR. Skin reactions were measured at regular intervals using the semi-quantitative skin injury score. RESULTS: Subcutaneous injection of Ad-HO1-EGFP infected both epidermal and dermal cells and could spread to the surrounding regions. Radiation exposure upregulated the transcription of the antioxidant enzyme genes, including SOD-1, GPx2 and endogenous HO-1. HO-1 overexpression decreased lipid peroxidation and inhibited the induction of ROS scavenging proteins. Moreover, HO-1 exerted an anti-apoptotic effect by suppressing FAS and FASL expression. Subcutaneous injection of Ad-HO1-EGFP demonstrated significant improvement in radiation-induced skin injury. CONCLUSIONS: The present study provides evidences for the protective role of HO-1 in alleviating radiation-induced skin damage in rats, which is helpful for the development of therapy for radiation-induced skin injury.