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Annexin-1 (ANXA1) has shown neuroprotective effects and microglia play significant roles during central nervous system injury, yet the underlying mechanisms remain unclear. This study sought to determine whether ANXA1 regulates microglial response to oxygen–glucose deprivation/reperfusion (OGD/R) treatment and to clarify the downstream molecular mechanism. In rat hippocampal slices, OGD/R treatment enhanced the ANXA1 expression in neuron, the formyl peptide receptor (FPRs) expression in microglia, and the microglial activation in the CA1 region (cornu ammonis 1). These effects were reversed by the FPRs antagonist Boc1. The cell membrane currents amplitude of BV-2 microglia (the microglial like cell-line) was increased when treated with Ac2-26, the N-terminal peptide of ANXA1. Ac2-26 treatment enhanced BV-2 microglial migration whereas Boc1 treatment inhibited the migration. In BV-2 microglia, both the expression of the CK2 target phosphorylated α-E-catenin and the binding of casein kinase II (CK2) with α-E-catenin were elevated by Ac2-26, these effects were counteracted by the CK2 inhibitor TBB and small interfering (si) RNA directed against transcripts of CK2 and FPRs. Moreover, both TBB and siRNA-mediated inhibition of CK2 blocked Ac2-26-mediated BV-2 microglia migration. Our findings indicate that ANXA1 promotes microglial activation and migration during OGD/R via FPRs, and CK2 target α-E-catenin phosphorylation is involved in this process.

Bone homeostasis is tightly orchestrated and maintained by the balance between osteoblasts and osteoclasts. Recent studies have greatly expanded our understanding of the molecular mechanisms of cellular differentiation. However, the functional roles of non-coding RNAs particularly lncRNAs in remodeling bone architecture remain elusive. In our study, lncRNA H19 was found to be upregulated during osteogenesis in hMSCs. Stable expression of H19 significantly accelerated in vivo and in vitro osteoblast differentiation. Meanwhile, by using bioinformatic investigations and RIP assays combined with luciferase reporter assays, we demonstrated that H19 functioned as an miRNA sponge for miR-141 and miR-22, both of which were negative regulators of osteogenesis and Wnt/β-catenin pathway. Further investigations revealed that H19 antagonized the functions of these two miRNAs and led to de-repression of their shared target gene β-catenin, which eventually activated Wnt/β-catenin pathway and hence potentiated osteogenesis. In addition, we also identified a novel regulatory feedback loop between H19 and its encoded miR-675-5p. And miR-675-5p was found to directly target H19 and counteracted osteoblast differentiation. To sum up, these observations indicate that the lncRNA H19 modulates Wnt/β-catenin pathway by acting as a competing endogenous RNA, which may shed light on the functional role of lncRNAs in coordinating osteogenesis.

The rational design of α-helix–mimicking peptidomimetics provides a streamlined approach to discover potent inhibitors for protein− protein interactions (PPIs). However, designing cell-penetrating long peptidomimetic scaffolds equipped with various functional groups necessary for interacting with large protein-binding interfaces remains challenging. This is particularly true for targeting β-catenin/BCL9 PPIs. Here we designed a series of unprecedented helical sulfono-γ-AApeptides that mimic the binding mode of the α-helical HD2 domain of B Cell Lymphoma 9 (BCL9). Our studies show that sulfono-γ-AApeptides can structurally and functionally mimic the α-helical domain of BCL9 and selectively disrupt β-catenin/BCL9 PPIs with even higher potency. More intriguingly, these sulfono-γ-AApeptides can enter cancer cells, bind with β-catenin and disrupt β-catenin/BCL9 PPIs, and exhibit excellent cellular activity, which is much more potent than the BCL9 peptide. Furthermore, our enzymatic stability studies demonstrate the remarkable stability of the helical sulfono-γ-AApeptides, with no degradation in the presence of pronase for 24 h, augmenting their biological potential. This work represents not only an example of helical sulfono-γ-AApeptides that mimic α-helix and disrupt protein–protein interactions, but also an excellent example of potent, selective, and cell-permeable unnatural foldameric peptidomimetics that disrupt the β-catenin/BCL9 PPI. The design of helical sulfono-γ-AApeptides may lead to a new strategy to modulate a myriad of protein–protein interactions.

Paraquat (PQ) poisoning‐induced pulmonary fibrosis is one of the primary causes of death in patients with PQ poisoning. Hypoxia‐inducible factor‐1α (HIF‐1α) and epithelial‐mesenchymal transition (EMT) are involved in the progression of pulmonary fibrosis. Snail and β‐catenin are two other factors involved in promoting EMT. However, the relationship among HIF‐1α, Snail and β‐catenin in PQ poisoning‐induced pulmonary fibrosis is not clear. Our research aimed to determine whether the regulation of HIF‐1α in EMT occurs via the Snail and β‐catenin pathways in PQ poisoning‐induced pulmonary fibrosis. Sixty‐six Sprague–Dawley rats were randomly and evenly divided into a control group and a PQ group. The PQ group was treated with an intragastric infusion of a 20% PQ solution (50 mg/kg) for 2, 6, 12, 24, 48 and 72 hrs. A549 and RLE‐6TN cell lines were transfected with HIF‐1α siRNA for 48 hrs before being exposed to PQ. Western blotting, real‐time quantitative PCR, immunofluorescence, immunohistochemistry and other assays were used in our research. In vivo, the protein levels of HIF‐1α and α‐SMA were increased at 2 hrs and the level of ZO‐1 (Zonula Occluden‐1) was reduced at 12 hrs. In vitro, the transient transfection of HIF‐1α siRNA resulted in a decrease in the degree of EMT. The expression levels of Snail and β‐catenin were significantly reduced when HIF‐α was silenced. These data demonstrate that EMT may be involved in PQ poisoning‐induced pulmonary fibrosis and regulated by HIF‐1α via the Snail and β‐catenin pathways. Hypoxia‐inducible factor‐1α may be a therapeutic target for the treatment of PQ poisoning‐induced pulmonary fibrosis.

Only a limited number of human cells can fuse to form a multinucleated syncytium. Cell fusion occurs as part of the differentiation of some cell types, including myotubes in muscle and osteoclasts in remodeling bone. In the differentiation of the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form endocrinologically active, non-proliferative, multinucleated syncytia. These syncytia allow the exchange of nutrients and gases between the maternal and fetal circulation. Alteration of syncytial formation during pregnancy affects fetal growth and the outcome of the pregnancy. Here, we demonstrate the role of annexin A5 (AnxA5) in syncytial formation by cellular delivery of recombinant AnxA5 and RNA interference. By a variety of co-immunoprecipitation, immunolocalization and proximity experiments, we show that a pool of AnxA5 organizes at the inner-leaflet of the plasma membrane in the vicinity of a molecular complex that includes E-Cadherin, α-Catenin and β-Catenin, three proteins previously shown to form adherens junctions implicated in cell fusion. A combination of knockdown and reconstitution experiments with AnxA5, with or without the ability to self-assemble in 2D-arrays, demonstrate that this AnxA5 2D-network mediates E-Cadherin mobility in the plasmalemma that triggers human trophoblasts aggregation and thereby cell fusion.

Fulvestrant is a selective estrogen receptor downregulator (SERD) and highly effective antagonist to hormone-sensitive breast cancers following failure of previous tamoxifen or aromatase inhibitor therapies. However, after prolonged fulvestrant therapy, acquired resistance eventually occurs in the majority of breast cancer patients, due to poorly understood mechanisms. To examine a possible role(s) of aberrantly expressed microRNAs (miRNAs) in acquired fulvestrant resistance, we compared antiestrogen-resistant and -sensitive breast cancer cells, revealing the overexpression of miR-221/222 in the SERD-resistant cell lines. Fulvestrant treatment of estradiol (E2)- and fulvestrant-sensitive MCF7 cells resulted in increased expression of endogenous miR-221/222. Ectopic upregulation of miR-221/222 in estrogen receptor-α (ERα)-positive cell lines counteracted the effects of E2 depletion or fulvestrant-induced cell death, thus also conferring hormone-independent growth and fulvestrant resistance. In cells with acquired resistance to fulvestrant, miR-221/222 expression was essential for cell growth and cell cycle progression. To identify possible miR-221/222 targets, miR-221- or miR-222- induced alterations in global gene expression profiles and target gene expression at distinct time points were determined, revealing that miR-221/222 overexpression resulted in deregulation of multiple oncogenic signaling pathways previously associated with drug resistance. Activation of β-catenin by miR-221/222 contributed to estrogen-independent growth and fulvestrant resistance, whereas TGF-β-mediated growth inhibition was repressed by the two miRNAs. This first in-depth investigation into the role of miR-221/222 in acquired fulvestrant resistance, a clinically important problem, demonstrates that these two ‘oncomirs’ may represent promising therapeutic targets for treating hormone-independent, SERD-resistant breast cancer.

Polycystic ovary syndrome (PCOS) is an endocrine disorder in women of reproductive age. In addition to anovulation, endometrial dysfunction can reduce fertility in PCOS. The cyclical changes of endometrium are controlled by estrogen and progesterone via modulating the Wnt/B-catenin pathway. Clomiphene citrate (CC) and letrozole are used to induce ovulation; unlike letrozole, there is a discrepancy between ovulation and pregnancy rates in CC-treated cycles. Because of the antiestrogenic effects of CC on endometrium, we compared the expression of the key molecules of the Wnt/B-catenin pathway in the endometrium of women taking CC and letrozole. This study included PCOS and healthy women divided into the groups stimulated with letrozole (5 mg) or CC (100 mg) as well as NO-treatment groups. The endometrial thickness and hormonal profile were measured on day 12 of the menses. Using real-time polymerase chain reaction and western blot, we evaluated mRNA and protein expression of B-catenin, glycogen synthase kinase 3 beta (GSK3B), dickkopf Wnt signaling pathway inhibitor 1 (DKK1), and estrogen receptor 1 (ESR1) in the endometrial samples. Significantly, the mean serum estrogen and progesterone were lower and higher, respectively, in letrozole than CC groups. The endometrial thickness was significantly reduced in CC. The proteins expression of active B-catenin, inactive GSK3B, and ESR1 were significantly decreased in CC-treated groups. The mRNA and protein assessment of DKK1 showed significantly higher expression in CC. Our results indicate that letrozole can provide an acceptable activation of the Wnt/B-catenin pathway, resulting in adequate proliferation of endometrium in the women receiving letrozole compared to CC. Summary Sentence The expression of B-catenin, GSK3B, DKK1, and ESR1 were adversely affected in the endometrium of women induced with clomiphene citrate compared to letrozole, resulting in inefficacity of endometrium.

In a new study, Yingzi Yang and her colleagues show that a careful balance between Wnt and Hedgehog signaling is required to maintain proper differentiation of osteogenic precursor cells. Upon mutation of GNAS , this balance is disturbed and severe bone disease develops, including either heterotopic ossification or fibrous dysplasia. Heterotopic ossification, the pathologic formation of extraskeletal bone, occurs as a common complication of trauma or in genetic disorders and can be disabling and lethal. However, the underlying molecular mechanisms are largely unknown. Here we demonstrate that Gα s restricts bone formation to the skeleton by inhibiting Hedgehog signaling in mesenchymal progenitor cells. In progressive osseous heteroplasia, a human disease caused by null mutations in GNAS , which encodes Gα s , Hedgehog signaling is upregulated in ectopic osteoblasts and progenitor cells. In animal models, we show that genetically-mediated ectopic Hedgehog signaling is sufficient to induce heterotopic ossification, whereas inhibition of this signaling pathway by genetic or pharmacological means strongly reduces the severity of this condition. As our previous work has shown that GNAS gain-of-function mutations upregulate WNT–β-catenin signaling in osteoblast progenitor cells, resulting in their defective differentiation and fibrous dysplasia, we identify Gα s as a key regulator of proper osteoblast differentiation through its maintenance of a balance between the Wnt–β-catenin and Hedgehog pathways. Also, given the results here of the pharmacological studies in our mouse model, we propose that Hedgehog inhibitors currently used in the clinic for other conditions, such as cancer, may possibly be repurposed for treating heterotopic ossification and other diseases caused by GNAS inactivation.

The loss of microRNA-122 (miR-122) expression is strongly associated with increased invasion and metastasis, and poor prognosis of hepatocellular carcinoma (HCC), however, the underlying mechanisms remain poorly understood. In the present study, we observed that miR-122 over-expression in HCC cell lines Sk-hep-1 and Bel-7402 triggered the mesenchymal-epithelial transition (MET), as demonstrated by epithelial-like morphological changes, up-regulated epithelial proteins (E-cadherin, ZO-1, α-catenin, occludin, BVES, and MST4), and down-regulated mesenchymal proteins (vimentin and fibronectin). The over-expression of miRNA-122 also caused cytoskeleton disruption, RhoA/Rock pathway inactivation, enhanced cell adhesion, and suppression of migration and invasion of Sk-hep-1 and Bel-7402 cells, whereas, these effects could be reversed through miR-122 inhibition. Additional studies demonstrated that the inhibition of wild-type RhoA function induced MET and inhibited cell migration and invasion, while RhoA over-expression reversed miR-122-induced MET and inhibition of migration and invasion of HCC cells, suggesting that miR-122 induced MET and suppressed the migration and invasion of HCC cells by targeting RhoA. Moreover, our results demonstrated that HNF4α up-regulated its target gene miR-122 that subsequently induced MET and inhibited cell migration and invasion, whereas miR-122 inhibition reversed these HNF4α-induced phenotypes. These results revealed functional and mechanistic links among the tumor suppressors HNF4α, miR-122, and RhoA in EMT and invasive and metastatic phenotypes of HCC. Taken together, our study provides the first evidence that the HNF4α/miR-122/RhoA axis negatively regulates EMT and the migration and invasion of HCC cells.

AML1‐ETO, the most common fusion oncoprotein by t (8;21) in acute myeloid leukaemia (AML), enhances hematopoietic self‐renewal and leukemogenesis. However, currently no specific therapies have been reported for t (8;21) AML patients as AML1‐ETO is still intractable as a pharmacological target. For this purpose, leukaemia cells and AML1‐ETO‐induced murine leukaemia model were used to investigate the degradation of AML1‐ETO by melatonin (MLT), synthesized and secreted by the pineal gland. MLT remarkedly decreased AML1‐ETO protein in leukemic cells. Meanwhile, MLT induced apoptosis, decreased proliferation and reduced colony formation. Furthermore, MLT reduced the expansion of human leukemic cells and extended the overall survival in U937T‐AML1‐ETO‐xenografted NSG mice. Most importantly, MLT reduced the infiltration of leukaemia blasts, decreased the frequency of leukaemia stem cells (LSCs) and prolonged the overall survival in AML1‐ETO‐induced murine leukaemia. Mechanistically, MLT increased the expression of miR‐193a, which inhibited AML1‐ETO expression via targeting its putative binding sites. Furthermore, MLT decreased the expression of β‐catenin, which is required for the self‐renewal of LSC and is the downstream of AML1‐ETO. Thus, MLT presents anti‐self‐renewal of LSC through miR‐193a‐AML1‐ETO‐β‐catenin axis. In conclusion, MLT might be a potential treatment for t (8;21) leukaemia by targeting AML1‐ETO oncoprotein.