Single-Cell RNA-Sequencing Atlas Reveals the Tumor Microenvironment of Metastatic High-Grade Serous Ovarian Carcinoma.

Ovarian cancer is the most common and lethal gynecological tumor in women worldwide. High-grade serous ovarian carcinoma (HGSOC) is one of the histological subtypes of epithelial ovarian cancer, accounting for 70%. It often occurs at later stages associated with a more fatal prognosis than endometrioid carcinomas (EC), another subtype of epithelial ovarian cancer. However, the molecular mechanism and biology underlying the metastatic HGSOC (HG_M) immunophenotype remain poorly elusive. Here, we performed single-cell RNA sequencing analyses of primary HGSOC (HG_P) samples, metastatic HGSOC (HG_M) samples, and endometrioid carcinomas (EC) samples. We found that ERBB2 and HOXB-AS3 genes were more amplified in metastasis tumors than in primary tumors. Notably, high-grade serous ovarian cancer metastases are accompanied by dysregulation of multiple pathways. Malignant cells with features of epithelial-mesenchymal transition (EMT) affiliated with poor overall survival were identified. In addition, cancer-associated fibroblasts with EMT-program were enriched in HG_M, participating in angiogenesis and immune regulation, such as IL6/STAT3 pathway activity. Compared with ECs, HGSOCs exhibited higher T cell infiltration. PRDM1 regulators may be involved in T cell exhaustion in ovarian cancer. The CX3CR1_macro subpopulation may play a role in promoting tumor progression in ovarian cancer with high expression of BAG3, IL1B, and VEGFA. The new targets we discovered in this study will be useful in the future, providing guidance on the treatment of ovarian cancer.

Bnip3 mediates doxorubicin-induced cardiomyocyte pyroptosis via caspase-3/GSDME.

AIMS: This study was aimed to investigate the role of GSDME-mediated pyroptosis in cardiac injury induced by Doxorubicin (DOX), and to evaluate the role of BH3-only protein Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) in regulation of DOX-induced pyroptosis. MAIN METHODS: HL-1 cardiomyocytes and C57BL/6J mice were treated by DOX to establish DOX-induced cardiotoxicity in vitro and in vivo models, respectively. Cell transfection was applied to regulate the expression of caspase-3, GSDME and Bnip3. Western blot was used for measuring expression of protein level. LDH-cytotoxicity assay was used to detect the LDH release. The Flow cytometry analysis was used to detect the cell death. Echocardiography was used to determine the cardiac function. HE staining was used for observing pathological feature of heart tissues. KEY FINDINGS: Our results showed that GSDME-mediated pyroptosis was involved in DOX-induced cardiotoxicity in vivo. We showed that HL-1 cardiomyocytes exposed to DOX exhibited morphological features of pyroptosis in vitro. We also showed that DOX induced activation of caspase-3 and eventually triggered GSDME-dependent pyroptosis, which was reduced by the silence or inhibitor of caspase-3. We further showed that knockdown of GSDME inhibited DOX-induced cardiomyocyte pyroptosis in vitro. Finally, DOX increased the expression of Bnip3, whereas silencing of Bnip3 blunted cardiomyocyte pyroptosis induced by DOX, which was regulated through caspase-3 activation and GSDME cleavage. SIGNIFICANCE: Our findings revealed a novel pathway that cardiomyocyte pyroptosis is regulated through Bnip3-caspase-3-GSDME pathway following DOX treatment, suggesting that Bnip3-dependent pyroptosis may offer a novel therapeutic strategy to reduce cardiotoxicity induced by DOX.

Inhibition of microRNA-23b prevents polymicrobial sepsis-induced cardiac dysfunction by modulating TGIF1 and PTEN.

Cardiovascular dysfunction is a major complication associated with sepsis induced mortality. Cardiac fibrosis plays a critical role in sepsis induced cardiac dysfunction. The mechanisms of the activation of cardiac fibrosis is unclarified. In this study, we found that microRNA-23b (miR-23b) was up-regulated in heart tissue during cecal ligation and puncture (CLP)-induced sepsis and transfection of miR-23b inhibitor improved survival in late sepsis. Inhibition of miR-23b in the myocardium protected against cardiac output and enhanced left ventricular systolic function. miR-23b inhibitor also alleviated cardiac fibrosis in late sepsis. MiR-23b mediates the activation of TGF-ß1/Smad2/3 signaling to promote the differentiation of cardiac fibroblasts through suppression of 5'TG3'-interacting factor 1 (TGIF1). MiR-23b also induces AKT/N-Cadherin signaling to contribute to the deposition of extracellular matrix by inhibiting phosphatase and tensin homologue (PTEN). TGIF1 and PTEN were confirmed as the targets of miR-23b in vitro by Dual-Glo Luciferase assay. miR-23b inhibitor blocked the activation of adhesive molecules and restored the imbalance of pro-fibrotic and anti-fibrotic factors. These data provide direct evidence that miR-23b is a critical contributor to the activation of cardiac fibrosis to mediate the development of myocardial dysfunction in late sepsis. Blockade of miR-23b expression may be an effective approach for prevention sepsis-induced cardiac dysfunction.

Inhibition of MicroRNA-23b Attenuates Immunosuppression During Late Sepsis Through NIK, TRAF1, and XIAP.

Background: microRNA-23b (miR-23b) is a multiple functional miRNA. We hypothesize that miR-23b plays a role in the pathogenesis of sepsis. Our study investigated the effect of miR-23b on sepsis-induced immunosuppression. Methods: Mice were treated with miR-23b inhibitors by tail vein injection 2 days after cecal ligation puncture (CLP)-induced sepsis. Apoptosis in spleens and apoptotic signals were investigated, and survival was monitored. T-cell immunoreactivities were examined during late sepsis. Nuclear factor κB (NF-κB)-inducing kinase (NIK), tumor necrosis factor (TNF)-receptor associated factor 1 (TRAF1), and X-linked inhibitor of apoptosis protein (XIAP), the putative targets of miR-23b, were identified by a dual-luciferase reporter assay. Results: miR-23b expression is upregulated and sustained during sepsis. The activation of the TLR4/TLR9/p38 MAPK/STAT3 signal pathway contributes to the production of miR-23b in CLP-induced sepsis. miR-23b inhibitor decreased the number of spleen cells positive by terminal deoxynucleotidyl transferase dUTP nick-end labeling and improved survival. miR-23b inhibitor restored the immunoreactivity by alleviating the development of T-cell exhaustion and producing smaller amounts of immunosuppressive interleukin 10 and interleukin 4 during late sepsis. We demonstrated that miR-23b mediated immunosuppression during late sepsis by inhibiting the noncanonical NF-κB signal and promoting the proapoptotic signal pathway by targeting NIK, TRAF1, and XIAP. Conclusions: Inhibition of miR-23b reduces late-sepsis-induced immunosuppression and improves survival. miR-23b might be a target for immunosuppression.

The ROS/NF-κB/NR4A2 pathway is involved in H2O2 induced apoptosis of resident cardiac stem cells via autophagy.

Cardiac stem cells (CSCs)-based therapy provides a promising avenue for the management of ischemic heart diseases. However, engrafted CSCs are subjected to acute cell apoptosis in the ischemic microenvironment. Here, stem cell antigen 1 positive (Sca-1+) CSCs proved to own therapy potential were cultured and treated with H2O2 to mimic the ischemia situation. As autophagy inhibitor, 3-methyladenine (3MA), inhibited H2O2-induced CSCs apoptosis, thus we demonstrated that H2O2 induced autophagy-dependent apoptosis in CSCs, and continued to find key proteins responsible for the crosstalk between autophagy and apoptosis. Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), increased upon cardiomyocyte injury with unknown functions in CSCs, was increased by H2O2. NR4A2 siRNA attenuated H2O2 induced autophagy and apoptosis in CSCs, which suggested an important role of NR4A2 in CSCs survival in ischemia conditions. Reactive oxygen species (ROS) and NF-κB (P65) subunit were both increased by H2O2. Either the ROS scavenger, N-acetyl-l-cysteine (NAC) or NF-κB signaling inhibitor, bay11-7082 could attenuate H2O2-induced autophagy and apoptosis in CSCs, which suggested they were involved in this process. Furthermore, NAC inhibited NF-κB activities, while bay11-7082 inhibited NR4A2 expression, which revealed a ROS/NF-κB/NR4A2 pathway responsible for H2O2-induced autophagy and apoptosis in CSCs. Our study supports a new clue enhancing the survival rate of CSCs in the infarcted myocardium for cell therapy in ischemic cardiomyopathy.

Hematopoietic stem progenitor cells prevent chronic stress-induced lymphocyte apoptosis.

Physical or psychological chronic stress can suppress the immune system. However, the mechanisms remain to be elucidated. We investigated the effect of hematopoietic stem-progenitor cells (HSPCs) on chronic stress-induced the alterations of immune responses. We demonstrate that HSPCs prevents stress-induced lymphocyte apoptosis. Moreover, we also demonstrate that the protective effect of HSPCs on stress-induced lymphocyte reduction exerts by steroid hormones. Furthermore, we reveal that chronic stress-induced T cell-mediated immune responses contributes to the protective effect of HSPCs. These results indicate that HPSCs might offer a novel therapeutic strategy against the deleterious effects of chronic stress on the immune system.

MicroRNA-155 attenuates late sepsis-induced cardiac dysfunction through JNK and ß-arrestin 2.

Cardiac dysfunction is correlated with detrimental prognosis of sepsis and contributes to a high risk of mortality. After an initial hyperinflammatory reaction, most patients enter a protracted state of immunosuppression (late sepsis) that alters both innate and adaptive immunity. The changes of cardiac function in late sepsis are not yet known. MicroRNA-155 (miR-155) is previously found to play important roles in both regulations of immune activation and cardiac function. In this study, C57BL/6 mice were operated to develop into early and late sepsis phases, and miR-155 mimic was injected through the tail vein 48 h after cecal ligation and puncture (CLP). The effect of miR-155 on CLP-induced cardiac dysfunction was explored in late sepsis. We found that increased expression of miR-155 in the myocardium protected against cardiac dysfunction in late sepsis evidenced by attenuating sepsis-reduced cardiac output and enhancing left ventricular systolic function. We also observed that miR-155 markedly reduced the infiltration of macrophages and neutrophils into the myocardium and attenuated the inflammatory response via suppression of JNK signaling pathway. Moreover, overexpression of ß-arrestin 2 (Arrb2) exacerbated the mice mortality and immunosuppression in late sepsis. Furthermore, transfection of miR-155 mimic reduced Arrb2 expression, and then restored immunocompetence and improved survival in late septic mice. We conclude that increased miR-155 expression through systemic administration of miR-155 mimic attenuates cardiac dysfunction and improves late sepsis survival by targeting JNK associated inflammatory signaling and Arrb2 mediated immunosuppression.

ß-Arrestin 2 Promotes Hepatocyte Apoptosis by Inhibiting Akt Protein.

Recent studies reveal that multifunctional protein ß-arrestin 2 (Arrb2) modulates cell apoptosis. Survival and various aspects of liver injury were investigated in WT and Arrb2 KO mice after bile duct ligation (BDL). We found that deficiency of Arrb2 enhances survival and attenuates hepatic injury and fibrosis. Following BDL, Arrb2-deficient mice as compared with WT controls displayed a significant reduction of hepatocyte apoptosis as demonstrated by the TUNEL assay. Following BDL, the levels of phospho-Akt and phospho-glycogen synthase kinase 3ß (GSK3ß) in the livers were significantly increased in Arrb2 KO compared with WT mice, although p-p38 increased in WT but not in Arrb2-deficient mice. Inhibition of GSK3ß following BDL decreases hepatic apoptosis and decreased p-p38 in WT mice but not in Arrb2 KO mice. Activation of Fas receptor with Jo2 reduces phospho-Akt and increases apoptosis in WT cells and WT mice but not in Arrb2-deficient cells and Arrb2-deficient mice. Consistent with direct interaction of Arrb2 with and regulating Akt phosphorylation, the expression of a full-length or N terminus but not the C terminus of Arrb2 reduces Akt phosphorylation and coimmunoprecipates with Akt. These results reveal that the protective effect of deficiency of Arrb2 is due to loss of negative regulation of Akt due to BDL and decreased downstream GSK3ß and p38 MAPK signaling pathways.

Palmitate promotes autophagy and apoptosis through ROS-dependent JNK and p38 MAPK.

Palmitate (PA), one of the most prevalent saturated fatty acids, causes myocardial dysfunction. However, the mechanisms by which PA induces cell apoptosis and autophagy remain to be elucidated. We showed that autophagy was induced in an mTORC1-dependent way and played a protective role against PA-induced apoptosis, which was verified by pretreatment with 3-methyladenine (3MA) and rapamycin. However, p62 began to accumulate after 18 h treatment with PA, suggesting prolonged exposure to PA lead to an impairment of autophagic flux. PA enhanced ROS production as well as activated p38-mitogen-activated protein kinase (p38 MAPK) and c-jun NH2 terminal kinases (JNKs). The antioxidant N-Acety-l-Cysteine (NAC) was found to attenuate the JNK and p38 MAPK activation with a concomitant reduction of PA-induced autophagy and apoptosis. Furthermore, both JNK and p38 MAPK inhibitors were shown to directly abrogate caspase 7 cleavage as well as the conversion of LC3BI to LC3BII. Thus, we demonstrate that PA stimulates autophagy and apoptosis via ROS-dependent JNK and p38 MAPK pathways.

Inhibition of Toll-like receptor 9 attenuates sepsis-induced mortality through suppressing excessive inflammatory response.

Sepsis, a major clinical problem with high morbidity and mortality, is caused by overwhelming systemic host-inflammatory response. Toll-like receptors (TLRs) play a fundamental role in induction of hyperinflammation and tissue damage in sepsis. In this study, we demonstrate a protective role of TLR9 inhibition against the dysregulated inflammatory response and tissue injury in sepsis. TLR9 deficiency decreased the mortality of mice following cecal ligation and puncture (CLP)-induced sepsis. TLR9 knockout mice showed dampened p38 activation and augmented Akt phosphorylation in the spleen, lung and liver. In addition, TLR9 deficiency decreased the levels of inflammatory cytokines and attenuated splenic apoptosis after CLP. These results indicate that TLR9 inhibition might offer a novel therapeutic strategy for the management of sepsis.

The role of toll-like receptor 9 in chronic stress-induced apoptosis in macrophage.

Emerging evidence implied that chronic stress has been exerting detrimental impact on immune system functions in both humans and animals. Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival. We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress. However, the exact role of TLR9 in stress-mediated change of macrophage function remains unclear. The results of the current study showed that when BALB/c mice were treated with restraint stress (12 h daily for 2 days), the number of macrophages recruited to the peritoneal cavity was obviously increased. Results also demonstrated that the sustained effects of stress elevated cytokine IL-1ß, TNF-α and IL-10 production yet diminished IFN-γ production from macrophage, which led to apoptotic cell death. However, TLR9 deficiency prevented the chronic stress-mediated accumulation of macrophages. In addition, knocking out TLR9 significantly abolished the chronic stress-induced imbalance of cytokine levels and apoptosis in macrophage. TLR9 deficiency was also found to reverse elevation of plasma IL-1ß, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress. These results indicated that TLR9-mediated macrophage responses were required for chronic stress-induced immunosuppression. Further exploration showed that TLR9 deficiency prevented the increment of p38 MAPK phosphorylation and reduction of Akt/Gsk-3ß phosphorylation; TLR9 deficiency also attenuated the release of mitochondrial cytochrome c into cytoplasm, caused upregulation of Bcl-2/Bax protein ratio, downregulation of cleavage of caspase-3 and PARP, as well as decreased TUNEL-positive cells in macrophage of stressed mice. Collectively, our studies demonstrated that deficiency of TLR9 maintained macrophage function by modulating macrophage accumulation and attenuating macrophage apoptosis, thus preventing immunosuppression in restraint-stressed mice.

TLR2 Involved in Naive CD4+ T Cells Rescues Stress-Induced Immune Suppression by Regulating Th1/Th2 and Th17.

Stress, either physical or psychological, can have a dramatic impact on our immune system. There has been little progress, however, in understanding chronic stress-induced immunosuppression. Naive CD4+ T cells could modulate immune responses via differentiation to T helper (Th) cells. In this study, we showed that stress promotes the release of the Th1 cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α, the Th2 cytokines interleukin (IL)-4 and IL-10 and the Th17 cytokine IL-17 of splenic naive CD4+ T cells. This suggests that stress promotes the differentiation of naive CD4+ T cells to Th1, Th2 and Th17 cells. Knockout strategies verified that TLR2 might modulate the differentiation of Th1/Th2 cells by inhibiting p38 mitogen-activated protein kinase (MAPK). Taken together, our data suggest that chronic stress induces immune suppression by targeting TLR2 and p38 MAPK in naive CD4+ T cells.

Chronic morphine-induced microRNA-124 promotes microglial immunosuppression by modulating P65 and TRAF6.

Opioids have been widely applied in clinics as one of the most potent pain relievers for centuries, but their abuse has deleterious physiological effects including immunosuppression. However, the mechanisms are unclear. TLRs and acetylcholine are widely expressed in the immune and nervous systems, and play critical roles in immune responses. In this article, we show that morphine suppresses the innate immunity in microglia and bone marrow-derived macrophages through differential regulation of TLRs and acetylcholinesterase. Either morphine or inhibition of acetylcholine significantly promotes upregulation of microRNA-124 (miR-124) in microglia, bone marrow-derived macrophages, and the mouse brain, where miR-124 mediates morphine inhibition of the innate immunity by directly targeting a subunit of NF-κB p65 and TNFR-associated factor 6 (TRAF6). Furthermore, transcription factors AP-1 and CREB inhibited miR-124, whereas p65 bound directly to promoters of miR-124, thereby enhancing miR-124 transcription. Moreover, acute morphine treatment transiently upregulated the expression of p65 and phospho-p65 in both nucleus and cytoplasm priming the expression of miR-124, whereas long exposure of morphine maintained miR-124 expression, which inhibited p65- and TRAF6-dependent TLR signaling. These data suggest that modulation of miRs is capable of preventing opioid-induced damage to microglia.

Valsartan blocked alcohol-induced, Toll-like receptor 2 signaling-mediated inflammation in human vascular endothelial cells.

BACKGROUND: Alcohol consumption induces inflammatory damage in vessels, and the underlying mechanism is unclear. Valsartan, as one of the angiotensin receptor blockers (ARBs), plays a role in the inhibition of inflammatory reactions in vascular dysfunction. This study is to investigate the role of Toll-like receptor 2 (TLR2) in alcohol-induced inflammatory damage in vascular endothelial cells in vitro and to explore the protective effect of valsartan on alcohol-induced and TLR2-mediated inflammatory damage. METHODS: The human umbilical vein cell line (EA.hy926) were exposed to alcohol at 0 to 80 mM for 0 to 48 hours with or without valsartan pretreatment. The expression of TLR2 signaling, including TLR2, tumor necrosis factor receptor associated factor 6 (TRAF-6) and nuclear factor kappa B (NF-κB) p65 were detected by Western blot. The levels of proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were determined by ELISA. To confirm the role of TLR2, we functionally up-regulated or down-regulated TLR2 by using TLR2 agonist or TLR2 small interfering RNA (siRNA), respectively. To further investigate the mechanism of alcohol on renin-angiotensin system, we detected the expression of angiotensin II receptor type 1 (AGTR1) in protein levels. RESULTS: The expression of TLR2, TRAF-6, NF-κB p65, and the proinflammatory cytokines, TNF-α and IL-6, were significantly increased after alcohol exposure in EA.hy926 endothelial cells. This was enhanced by TLR2 agonist, and was inhibited by TLR2 siRNA transfection. The pretreatment of valsartan resulted in an inhibition of TLR2 signaling and proinflammatory cytokines. The expression of AGTR1 was up-regulated after alcohol exposure, and was blocked by valsartan pretreatment. CONCLUSIONS: TLR2 signaling-mediated alcohol induced inflammatory response in human vascular epithelial cells in vitro, which was inhibited by valsartan.

Targeting of the ß6 gene to suppress degradation of ECM via inactivation of the MAPK pathway in breast adenocarcinoma cells.

Integrin ανß6 has emerged as a potential novel target for anticancer and plays a major role in promoting malignant tumor progression. Recent studies indicate that integrin ανß6 occurs in many cancers. However, whether and how ανß6 is regulated by genetic and epigenetic mechanisms in breast cancer remain unknown. In the present study, two different short hairpin RNAs (shRNAs) targeting the ß6 gene were designed and constructed into pSUPER, respectively, which were transfected into the MCF-7 human breast adenocarcinoma cell line. The ß6-shRNA stably transfected cells were successfully established, and significant lower levels of ανß6 mRNA and protein expression were confirmed. Furthermore, inhibition of integrin ανß6 markedly downregulated the expression of matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-3 (MMP-3) and urokinase plasminogen activator (uPA) in tumor conditioned medium. Furthermore, ß6-shRNA-mediated silencing of the ανß6 gene obviously decreased the expression of ERK1/2. In particular, supression of integrin ανß6 caused significant downregulation of the degradation of basement membrane type IV collagen secretion via modulation of the plasminogen activation cascade. Our results thus indicate that ανß6 plays a fundamental role in promoting invasion and growth of breast adenocarcinoma cells. Taken together, this study revealed that targeting of the ß6 gene by RNA interference (RNAi) could efficiently downregulate ανß6 expression and suppress the ERK1/2-dependent extracellular matrix degradation in vitro, which is dependent upon inactivation of the mitogen-activated protein kinase (MAPK) pathway. These findings may offer a useful therapeutic approach to block invasion and migration of breast cancer cells.

Hepatitis B virus X protein promotes hepatocellular carcinoma transformation through interleukin-6 activation of microRNA-21 expression.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and chronic hepatitis B virus (HBV) infection is the major risk factor of HCC. The virus encodes HBV X (HBx) protein that plays a critical role in the development of HCC. Studies have revealed numerous HBx-altered genes and signalling pathways that heavily contribute to tumourigenesis of non-tumour hepatocytes. However, the role of HBx in regulating other critical gene regulators such as microRNAs is poorly understood, which impedes the exploration of a complete HBx-associated carcinogenic network. Besides, critical microRNAs that drive the transformation of non-tumour hepatocytes are yet to be identified. Here, we overexpressed C-terminal truncated HBx protein in a non-tumour hepatocyte cell line MIHA, and measured a panel of cancer-associated miRNAs. We observed that oncogenic miR-21 was upregulated upon ectopic expression of this viral protein variant. HBx-miR-21 pathway was prevalent in HCC cells as inhibition of HBx in Hep3B and PLC/PRF/5 cells significantly suppressed miR-21 expression. Subsequently, we showed that the upregulation of miR-21 was mediated by HBx-induced interleukin-6 pathway followed by activation of STAT3 transcriptional factor. The high dependency of miR-21 expression to HBx protein suggested a unique viral oncogenic pathway that could aberrantly affect a network of gene expression. Importantly, miR-21 was essential in the HBx-induced transformation of non-tumour hepatocytes. Inhibition of miR-21 effectively attenuated anchorage-independent colony formation and subcutaneous tumour growth of MIHA cells. Our study suggested that overexpression of miR-21 was critical to promote early carcinogenesis of hepatocytes upon HBV infection.

ß-arrestin2/miR-155/GSK3ß regulates transition of 5'-azacytizine-induced Sca-1-positive cells to cardiomyocytes.

Stem-cell antigen 1-positive (Sca-1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5'-azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. ß-arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of ß-arrestin2 in Sca-1+ CSC differentiation, we used ß-arrestin2-knockout mice and overexpression strategies. Real-time PCR revealed that ß-arrestin2 promoted 5'-azacytizine-induced Sca-1+ CSC differentiation in vitro. Because the microRNA 155 (miR-155) may regulate ß-arrestin2 expression, we detected its role and relationship with ß-arrestin2 and glycogen synthase kinase 3 (GSK3ß), another probable target of miR-155. Real-time PCR revealed that miR-155, inhibited by ß-arrestin2, impaired 5'-azacytizine-induced Sca-1+ CSC differentiation. On luciferase report assay, miR-155 could inhibit the activity of ß-arrestin2 and GSK3ß, which suggests a loop pathway between miR-155 and ß-arrestin2. Furthermore, ß-arrestin2-knockout inhibited the activity of GSK3ß. Akt, the upstream inhibitor of GSK3ß, was inhibited in ß-arrestin2-Knockout mice, so the activity of GSK3ß was regulated by ß-arrestin2 not Akt. We transplanted Sca-1+ CSCs from ß-arrestin2-knockout mice to mice with myocardial infarction and found similar protective functions as in wild-type mice but impaired arterial elastance. Furthermore, low level of ß-arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3ß, similar to in vitro findings. The ß-arrestin2/miR-155/GSK3ß pathway may be a new mechanism with implications for treatment of heart disease.

The protective effect of astaxanthin on fetal alcohol spectrum disorder in mice.

Astaxanthin is a strong antioxidant with the ability of reducing the markers of inflammation. To explore the protective effect of astaxanthin on maternal ethanol induced embryonic deficiency, and to investigate the underlying mechanisms, we detected the morphology, expression of neural marker genes, oxidative stress indexes, and inflammatory factors in mice model of fetal alcohol spectrum disorder with or without astaxanthin pretreatment. Our results showed that astaxanthin blocked maternal ethanol induced retardation of embryonic growth, and the down-regulation of neural marker genes, Otx1 and Sox2. Moreover, astaxanthin also reversed the increases of malondialdehyde (MDA), hydrogen peroxide (H2O2), and the decrease of glutathione peroxidase (GPx) in fetal alcohol spectrum disorder. In addition, maternal ethanol induced up-regulation of toll-like receptor 4 (TLR4), and the down-streaming myeloid differentiation factor 88 (MyD88), NF-κB, TNF-α, and IL-1ß in embryos, and this was inhibited by astaxanthin pretreatment. These results demonstrated a protective effect of astaxanthin on fetal alcohol spectrum disorder, and suggested that oxidative stress and TLR4 signaling associated inflammatory reaction are involved in this process.

Suppression of tumorigenicity by microRNA-138 through inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop in glioblastoma multiforme.

Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to identify the tumor suppressive miRNA not only down-regulated in glioblastoma multiforme (GBM) but also potent to inhibit the oncogene EZH2, and then investigate the biological function and pathophysiologic role of the candidate miRNA in GBM. In this study, we show that miRNA-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients from The Cancer Genome Atlas dataset (TCGA) data portal. Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138. Additionally, miRNA-138 negatively correlates to mRNA levels of EZH2 and CDK6 among GBM clinical samples from both TCGA and our small amount datasets. In conclusion, our data demonstrate a tumor suppressive role of miRNA-138 in GBM tumorigenicity, suggesting a potential application in GBM therapy.

ß-Arrestin prevents cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt pathways.

Our previous studies have shown that ß-arrestin 2 plays an anti-apoptotic effect. However, the mechanisms by which ß-arrestin contribute to anti-apoptotic role remain unclear. In this study, we show that a deficiency of either ß-arrestin 1 or ß-arrestin 2 significantly increases serum deprivation (SD)-induced percentage of apoptotic cells. ß-arrestin 2 deficient-induced apoptosis was inhibited by transfection with ß-arrestin 2 full-length plasmid, revealing that SD-induced apoptosis is dependent on ß-arrestin 2. Furthermore, in the absence of either ß-arrestin 1 or ß-arrestin 2 significantly enhances SD-induced the level of pro-apoptotic proteins, including cleaved caspase-3, extracellular-signal regulated kinase 1/2 (ERK1/2) and p38, members of mitogen-activated protein kinases (MAPKs). In addition, a deficiency of either ß-arrestin 1 or ß-arrestin 2 inhibits phosphorylation of Akt. The SD-induced changes in cleaved caspase-3, ERK1/2 and p38 MAPKs, Akt, and apoptotic cell numbers could be blocked by double knockout of ß-arrestin 1/2. Our study thus demonstrates that ß-arrestin inhibits cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt signaling pathways.