Small molecule activators of TAK1 promotes its activity-dependent ubiquitination and TRAIL-mediated tumor cell death.

TAK1 is a key modulator of both NF-κB signaling and RIPK1. In TNF signaling pathway, activation of TAK1 directly mediates the phosphorylation of IKK complex and RIPK1. In a search for small molecule activators of RIPK1-mediated necroptosis, we found R406/R788, two small molecule analogs that could promote sustained activation of TAK1. Treatment with R406 sensitized cells to TNF-mediated necroptosis and RIPK1-dependent apoptosis by promoting sustained RIPK1 activation. Using click chemistry and multiple biochemical binding assays, we showed that treatment with R406 promotes the activation of TAK1 by directly binding to TAK1, independent of its original target Syk kinase. Treatment with R406 promoted the ubiquitination of TAK1 and the interaction of activated TAK1 with ubiquitinated RIPK1. Finally, we showed that R406/R788 could promote the cancer-killing activities of TRAIL in vitro and in mouse models. Our studies demonstrate the possibility of developing small molecule TAK1 activators to potentiate the effect of TRAIL as anticancer therapies.

MKRN3-mediated ubiquitination of Poly(A)-binding proteins modulates the stability and translation of GNRH1 mRNA in mammalian puberty.

The family of Poly(A)-binding proteins (PABPs) regulates the stability and translation of messenger RNAs (mRNAs). Here we reported that the three members of PABPs, including PABPC1, PABPC3 and PABPC4, were identified as novel substrates for MKRN3, whose deletion or loss-of-function mutations were genetically associated with human central precocious puberty (CPP). MKRN3-mediated ubiquitination was found to attenuate the binding of PABPs to the poly(A) tails of mRNA, which led to shortened poly(A) tail-length of GNRH1 mRNA and compromised the formation of translation initiation complex (TIC). Recently, we have shown that MKRN3 epigenetically regulates the transcription of GNRH1 through conjugating poly-Ub chains onto methyl-DNA bind protein 3 (MBD3). Therefore, MKRN3-mediated ubiquitin signalling could control both transcriptional and post-transcriptional switches of mammalian puberty initiation. While identifying MKRN3 as a novel tissue-specific translational regulator, our work also provided new mechanistic insights into the etiology of MKRN3 dysfunction-associated human CPP.

Ligand-Directed Caging Enables the Control of Endogenous DNA Alkyltransferase Activity with Light inside Live Cells.

The control of endogenous protein activity with light inside live cells is helpful for the high spatiotemporal probing of their dynamic roles. Herein, we report the first small-molecule-ligand-directed caging approach to control the endogenous human O6 -alkylguanine-DNA alkyltransferase (AGT) activity with light, and the caged AGT is constructed from the native intracellular AGT. The photo-responsive O6 -benzylguanine derivative O6 -NBG3 is developed to site-specifically cage the AGT's catalytic cysteine residue, and the light irradiation on-demand restores AGT's activity in vitro, in bacteria, and in mammalian cells. With O6 -NBG3, the alkylated AGT is dealkylated for the first time to recover the DNA repair activity in breast cancer MCF-7 cells by the dose-dependent light irradiation. This decaging strategy enables the localized modulation of endogenous AGT activity with high temporal precision without genetic engineering, which holds great potential for therapeutic applications.

Runt-related transcription factor 1 (Runx1) aggravates pathological cardiac hypertrophy by promoting p53 expression.

Cardiac hypertrophy and the resultant heart failure are among the most common causes of morbidity and mortality worldwide; thus, identifying the key factor mediating pathological cardiac hypertrophy is critically important for developing the strategy to protect against heart failure. Runx1 (Runt-related transcription factor 1) acts as an essential transcription factor that functions in a variety of cellular processes including differentiation, proliferation, tissue growth and DNA damage response. However, relatively little is known about the role of Runx1 in heart, especially cardiac hypertrophy and heart failure. In the present study, we investigated the role of Runx1 in experimentally pathological cardiac hypertrophy. The in vitro model was induced by Ang II exposure to cultured neonatal rat cardiomyocytes, and the in vivo pathological cardiac hypertrophy models were induced by chronic pressure overload in mice. Runx1 expression is increased in heart tissues from mice with pressure overload-induced cardiac hypertrophy and in neonatal rat cardiomyocytes in response to Ang II stimulation. Moreover, knockdown of cardiac Runx1 alleviates the pressure overload-induced cardiac hypertrophy. Mechanistically, Runx1 activates the p53 signalling by binding to the p53 gene and promotes its transcription. Rescue experiments indicate that Runx1 promotes cardiac hypertrophy in a p53-dependent manner. Remarkably, we demonstrated that Ro5-3335 (a Runx1 inhibitor) acts as a potential therapeutic drug for treating pathological cardiac hypertrophy. In summary, we conclude that Runx1 is a novel mediator and therapeutic target for pathological cardiac hypertrophy.

Identification of 22 N-glycosites on spike glycoprotein of SARS-CoV-2 and accessible surface glycopeptide motifs: Implications for vaccination and antibody therapeutics.

Coronaviruses hijack human enzymes to assemble the sugar coat on their spike glycoproteins. The mechanisms by which human antibodies may recognize the antigenic viral peptide epitopes hidden by the sugar coat are unknown. Glycosylation by insect cells differs from the native form produced in human cells, but insect cell-derived influenza vaccines have been approved by the US Food and Drug Administration. In this study, we analyzed recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein secreted from BTI-Tn-5B1-4 insect cells, by trypsin and chymotrypsin digestion followed by mass spectrometry analysis. We acquired tandem mass spectrometry (MS/MS) spectrums for glycopeptides of all 22 predicted N-glycosylated sites. We further analyzed the surface accessibility of spike proteins according to cryogenic electron microscopy and homolog-modeled structures and available antibodies that bind to SARS-CoV-1. All 22 N-glycosylated sites of SARS-CoV-2 are modified by high-mannose N-glycans. MS/MS fragmentation clearly established the glycopeptide identities. Electron densities of glycans cover most of the spike receptor-binding domain of SARS-CoV-2, except YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ, similar to a region FSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQ in SARS-CoV-1. Other surface-exposed domains include those located on central helix, connecting region, heptad repeats and N-terminal domain. Because the majority of antibody paratopes bind to the peptide portion with or without sugar modification, we propose a snake-catching model for predicted paratopes: a minimal length of peptide is first clamped by a paratope and sugar modifications close to the peptide either strengthen or do not hinder the binding.

The Crc protein participates in down-regulation of the Lon gene to promote rhamnolipid production and rhl quorum sensing in Pseudomonas aeruginosa.

Rhamnolipid acts as a virulence factor during Pseudomonas aeruginosa infection. Here, we show that deletion of the catabolite repression control (crc) gene in P. aeruginosa leads to a rhamnolipid-negative phenotype. This effect is mediated by the down-regulation of rhl quorum sensing (QS). We discover that a disruption of the gene encoding the Lon protease entirely offsets the effect of crc deletion on the production of both rhamnolipid and rhl QS signal C4-HSL. Crc is unable to bind lon mRNA in vitro in the absence of the RNA chaperon Hfq, while Crc contributes to Hfq-mediated repression of the lon gene expression at a posttranscriptional level. Deletion of crc, which results in up-regulation of lon, significantly reduces the in vivo stability and abundance of the RhlI protein that synthesizes C4-HSL, causing the attenuation of rhl QS. Lon is also capable of degrading the RhlI protein in vitro. In addition, constitutive expression of rhlI suppresses the defects of the crc deletion mutant in rhamnolipid, C4-HSL and virulence on lettuce leaves. This study therefore uncovers a novel posttranscriptional regulatory cascade, Crc-Hfq/Lon/RhlI, for the regulation of rhamnolipid production and rhl QS in P. aeruginosa.

Study on progressive failure mode of surrounding rock of shallow buried bias tunnel considering strain-softening characteristics.

Mountain tunnels portal often have to pass through slope terrain unavoidably, thus forming a shallow buried bias tunnel. During the construction of shallow buried bias tunnel, disasters such as slope sliding and tunnel collapse frequently occur. The failure mode of surrounding rock obtained by current research is based on the limit equilibrium theory, which cannot reflect the progressive failure characteristics of the surrounding rock of shallow buried bias tunnel. In order to reveal the failure mechanism of the gradual instability of surrounding rock of shallow buried bias tunnel, the problem of gradual failure of the surrounding rock is reduced to an elastic-plastic analysis problem for surrounding rock considering the strain-softening characteristics. Based on the elastic-plastic analysis of the failure process of shallow buried bias tunnel, MATLAB was used to compile a program to read the finite-difference calculation result file, extract the effective information such as shear strain and tensile strain at the center point of each unit, and establish the analysis method of the progressive failure mode of shallow buried bias tunnel. The reliability of the method proposed was verified by comparing the failure process of the model test with the development process of shear strain increment. Under the condition of no support, the formation mechanism of failure plane of surrounding rock on both sides of shallow buried bias tunnel is different. The shallow buried side is the shear failure plane formed by the collapse of surrounding rock, while the deep buried side of the tunnel is the shear failure plane formed by the collapse of surrounding rock and slope sliding. Under the conditions of excavation and support, the failure plane of the shallow buried bias tunnel can be divided into three parts according to the formation sequence and reasons. The part I is the failure plane, which is formed by active shear under the influence of tunnel excavation. The part II is the failure plane formed by tensile crack of slope top. The part III is the failure plane formed by passive shear under the push of the soil in the upper part of the slope.

Melanoma differentiation-associated protein 5 prevents cardiac hypertrophy via apoptosis signal-regulating kinase 1-c-Jun N-terminal kinase/p38 signaling.

Pathological cardiac hypertrophy (CH) is driven by maladaptive changes in myocardial cells in response to pressure overload or other stimuli. CH has been identified as a significant risk factor for the development of various cardiovascular diseases, ultimately resulting in heart failure. Melanoma differentiation-associated protein 5 (MDA5), encoded by interferon-induced with helicase C domain 1 (IFIH1), is a cytoplasmic sensor that primarily functions as a detector of double-stranded ribonucleic acid (dsRNA) viruses in innate immune responses; however, its role in CH pathogenesis remains unclear. Thus, the aim of this study was to examine the relationship between MDA5 and CH using cellular and animal models generated by stimulating neonatal rat cardiomyocytes with phenylephrine and by performing transverse aortic constriction on mice, respectively. MDA5 expression was upregulated in all models. MDA5 deficiency exacerbated myocardial pachynsis, fibrosis, and inflammation in vivo, whereas its overexpression hindered CH development in vitro. In terms of the underlying molecular mechanism, MDA5 inhibited CH development by promoting apoptosis signal-regulating kinase 1 (ASK1) phosphorylation, thereby suppressing c-Jun N-terminal kinase/p38 signaling pathway activation. Rescue experiments using an ASK1 activation inhibitor confirmed that ASK1 phosphorylation was essential for MDA5-mediated cell death. Thus, MDA5 protects against CH and is a potential therapeutic target.

PLEKHM2 deficiency induces impaired mitochondrial clearance and elevated ROS levels in human iPSC-derived cardiomyocytes.

Pleckstrin homology domain-containing family M member 2 (PLEKHM2) is an essential adaptor for lysosomal trafficking and its homozygous truncation have been reported to cause early onset dilated cardiomyopathy (DCM). However, the molecular mechanism of PLEKHM2 deficiency in DCM pathogenesis and progression is poorly understood. Here, we generated an in vitro model of PLEKHM2 knockout (KO) induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to elucidate the potential pathogenic mechanism of PLEKHM2-deficient cardiomyopathy. PLEKHM2-KO hiPSC-CMs developed disease phenotypes with reduced contractility and impaired calcium handling. Subsequent RNA sequencing (RNA-seq) analysis revealed altered expression of genes involved in mitochondrial function, autophagy and apoptosis in PLEKHM2-KO hiPSC-CMs. Further molecular experiments confirmed PLEKHM2 deficiency impaired autophagy and resulted in accumulation of damaged mitochondria, which triggered increased reactive oxygen species (ROS) levels and decreased mitochondrial membrane potential (Δψm). Importantly, the elevated ROS levels caused oxidative stress-induced damage to nearby healthy mitochondria, resulting in extensive Δψm destabilization, and ultimately leading to impaired mitochondrial function and myocardial contractility. Moreover, ROS inhibition attenuated oxidative stress-induced mitochondrial damage, thereby partially rescued PLEKHM2 deficiency-induced disease phenotypes. Remarkably, PLEKHM2-WT overexpression restored autophagic flux and rescued mitochondrial function and myocardial contractility in PLEKHM2-KO hiPSC-CMs. Taken together, these results suggested that impaired mitochondrial clearance and increased ROS levels play important roles in PLEKHM2-deficient cardiomyopathy, and PLEKHM2-WT overexpression can improve mitochondrial function and rescue PLEKHM2-deficient cardiomyopathy.

Global metabolomic and network analysis of Escherichia coli responses to exogenous biofuels.

Although synthetic biology progress has made it possible to produce various biofuels in more user-friendly hosts, such as Escherichia coli, the large-scale biofuel production in these non-native systems is still challenging, mostly due to the very low tolerance of these non-native hosts to the biofuel toxicity. To address the issues, in this study we determined the metabolic responses of E. coli induced by three major biofuel products, ethanol, butanol, and isobutanol, using a gas chromatography-mass spectrometry (GC-MS) approach. A metabolomic data set of 65 metabolites identified in all samples was then subjected to principal component analysis (PCA) to compare their effects and a weighted correlation network analysis (WGCNA) to identify the metabolic modules specifically responsive to each of the biofuel stresses, respectively. The PCA analysis showed that cellular responses caused by the biofuel stress were in general similar to aging cells at stationary phase, inconsistent with early studies showing a high degree of dissimilarity between metabolite responses during growth cessation as induced through stationary phases or through various environmental stress applications. The WGCNA analysis allowed identification of 2, 4, and 2 metabolic modules specifically associated with ethanol, butanol, and isobutanol treatments, respectively. The biofuel-associated modules included amino acids and osmoprotectants, such as isoleucine, valine, glycine, glutamate, and trehalose, suggesting amino acid metabolism and osmoregulation are among the key protection mechanisms against biofuel stresses in E. coli. Interestingly, no module was found associated with all three biofuel products, suggesting differential effects of each biofuel on E. coli. The findings enhanced our understanding of E. coli responses to exogenous biofuels and also demonstrated the effectiveness of the metabolomic and network analysis in identifying key targets for biofuel tolerance.

Establishment of a human TLR4 compound heterozygous knockout hESC line (WAe009-A-N) to model toll-like receptor 4 deficiency by CRISPR/Cas9 system.

Toll-like receptor 4 (TLR4) is a pattern recognition receptor (PRRS) and an important protective immune receptor. TLR4 deficiency can lead to Inflammatory bowel disease. To explore the role of TLR4, we used CRISPR/Cas9 system to produce TLR4 compound heterozygous knockout embryonic stem cells in H9 cell line. The WAe009-A-N has a compound heterozygous 7 bp deletion/8 bp deletion in TLR4 exon 3, which resulted in a frameshift in the translation of TLR4, and TLR4 protein wasn't detectable in this cell line. In addition, the WAe009-A-N with normal karyotype can express pluripotent markers and differentiate into three germ layers in vitro.

Generation of a TRPM8 knockout hESC line (WAe009-A-A) derived from H9 using CRISPR/Cas9.

The transient receptor potential cation channel subfamily M member 8 (TRPM8) is a kind of non-selective cation channel which controls Ca2+ homeostasis. Mutations in TRPM8 were related to dry eye diseases (DED). Here we constructed a TRPM8 knockout cell line WAe009-A-A from the original embryonic stem cell line H9 using CRISPR/Cas9 technology, which maybe helpful for exploring the pathogenesis of DED. WAe009-A-A cells possess stem cell morphology and pluripotency as well as normal karyotype, and have the ability of differentiating into three germ layers in vitro.

Severe fever with thrombocytopenia syndrome virus replicates in platelets and enhances platelet activation.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) infection causes an emerging hemorrhagic fever in East Asia with a high mortality rate. Thrombocytopenia is a consistent feature of SFTS illness, but the mechanism remains elusive. OBJECTIVES: We aimed to better understand the role of platelets in the pathophysiology of SFTSV infection, including the development of thrombocytopenia. METHODS: Using platelets from healthy volunteers and patients with SFTS, we evaluated the functional changes in platelets against SFTSV infection. We investigated the direct effect of glycoprotein VI on platelet-SFTSV interaction by quantitative real-time PCR, molecular docking, surface plasmon resonance spectrometry, flow cytometry, western blot, and platelet functional studies in vitro. Interactions of SFTSV and platelet-SFTSV complexes with macrophages were also determined by scanning electron microscope, quantitative real-time PCR, and flow cytometry. RESULTS: This study is the first to demonstrate that platelets are capable of harboring and producing SFTSV particles. Structural and functional studies found that SFTSVs bind platelet glycoprotein VI to potentiate platelet activation, including platelet aggregation, adenosine triphosphate release, spreading, clot retraction, coagulation, phosphatidylserine exposure, thrombus formation, and adherence. In vitro mechanistic studies highlighted that the interaction of platelets with human THP-1 cells promoted SFTSV clearance and suppressed cytokine production in macrophages. However, unwanted SFTSV replication in macrophages reciprocally aggravated SFTSV persistence in the circulation, which may contribute to thrombocytopenia and other complications during SFTSV infection. CONCLUSION: These findings together highlighted the pathophysiological role of platelets in initial intrinsic defense against SFTSV infections, as well as intertwined processes with host immunity, which can also lead to thrombocytopenia and poor prognosis.

Selective Covalent Targeting of Pyruvate Kinase M2 Using Arsenous Warheads.

There is growing interest in covalent targeted inhibitors in drug discovery against previously "undruggable" sites and targets. These molecules typically feature an electrophilic warhead that reacts with nucleophilic groups of protein residues, most notably the thiol group of cysteines. One main challenge in the field is to develop versatile utilizable warheads. Here, we characterize the unique features of novel arsenous warheads for reaction with thiol species in a reversible manner and further demonstrate that organoarsenic probes can be chemically tuned toward specific molecular targets by developing selective and potent inhibitors of pyruvate kinase M2 (PKM2). We show that compound 24 is a covalent and allosteric inhibitor of PKM2 and its orally bioavailable prodrug 25 exerts efficacious inhibition of PKM2-dependent tumor growth in vitro and in vivo. Our results introduce 25 and its derivatives as useful pharmacological tools and provide a general road map for targeting the protein cysteinome using arsenous warheads.

Quantitative iTRAQ LC-MS/MS proteomics reveals metabolic responses to biofuel ethanol in cyanobacterial Synechocystis sp. PCC 6803.

Recent progress in metabolic engineering has led to autotrophic production of ethanol in various cyanobacterial hosts. However, cyanobacteria are known to be sensitive to ethanol, which restricts further efforts to increase ethanol production levels in these renewable host systems. To understand the mechanisms of ethanol tolerance so that engineering more robust cyanobacterial hosts can be possible, in this study, the responses of model cyanobacterial Synechocystis sp. PCC 6803 to ethanol were determined using a quantitative proteomics approach with iTRAQ LC-MS/MS technologies. The resulting high-quality proteomic data set consisted of 24,887 unique peptides corresponding to 1509 identified proteins, a coverage of approximately 42% of the predicted proteins in the Synechocystis genome. Using a cutoff of 1.5-fold change and a p-value less than 0.05, 135 and 293 unique proteins with differential abundance levels were identified between control and ethanol-treated samples at 24 and 48 h, respectively. Functional analysis showed that the Synechocystis cells employed a combination of induced common stress response, modifications of cell membrane and envelope, and induction of multiple transporters and cell mobility-related proteins as protection mechanisms against ethanol toxicity. Interestingly, our proteomic analysis revealed that proteins related to multiple aspects of photosynthesis were up-regulated in the ethanol-treated Synechocystis cells, consistent with increased chlorophyll a concentration in the cells upon ethanol exposure. The study provided the first comprehensive view of the complicated molecular mechanisms against ethanol stress and also provided a list of potential gene targets for further engineering ethanol tolerance in Synechocystis PCC 6803.

Generation of an iPSC line (ZZUNEUi021-A) from a hypertrophic cardiomyopathy patient with TNNT2 mutation.

A 25-years-old hypertrophic cardiomyopathy male patient donated his peripheral blood mononuclear cells (PBMCs) with heterozygote mutation in theTNNT2 gene. We generated induced pluripotent stem cell (iPSC) with normal karyotypic and expressing NANOG, Lin28, GDF3 and DNMT3. The iPSC line has demonstrated pluripotency by differentiating into three germ layers in vitro. The ZZUNEUi021-A would serve as an in vitro model for loss of TNNT2 function.

Identification of oleoylethanolamide as an endogenous ligand for HIF-3α.

Hypoxia-inducible factors (HIFs) are α/ß heterodimeric transcription factors modulating cellular responses to the low oxygen condition. Among three HIF-α isoforms, HIF-3α is the least studied to date. Here we show that oleoylethanolamide (OEA), a physiological lipid known to regulate food intake and metabolism, binds selectively to HIF-3α. Through crystallographic analysis of HIF-3 α/ß heterodimer in both apo and OEA-bound forms, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics (MD) simulations, and biochemical and cell-based assays, we unveil the molecular mechanism of OEA entry and binding to the PAS-B pocket of HIF-3α, and show that it leads to enhanced heterodimer stability and functional modulation of HIF-3. The identification of HIF-3α as a selective lipid sensor is consistent with recent human genetic findings linking HIF-3α with obesity, and demonstrates that endogenous metabolites can directly interact with HIF-α proteins to modulate their activities, potentially as a regulatory mechanism supplementary to the well-known oxygen-dependent HIF-α hydroxylation.

Myotubularin-Related Protein14 Prevents Neointima Formation and Vascular Smooth Muscle Cell Proliferation by Inhibiting Polo-Like Kinase1.

Background Restenosis is one of the main bottlenecks in restricting the further development of cardiovascular interventional therapy. New signaling molecules involved in the progress have continuously been discovered; however, the specific molecular mechanisms remain unclear. MTMR14 (myotubularin-related protein 14) is a novel phosphoinositide phosphatase that has a variety of biological functions and is involved in diverse biological processes. However, the role of MTMR14 in vascular biology remains unclear. Herein, we addressed the role of MTMR14 in neointima formation and vascular smooth muscle cell (VSMC) proliferation after vessel injury. Methods and Results Vessel injury models were established using SMC-specific conditional MTMR14-knockout and -transgenic mice. Neointima formation was assessed by histopathological methods, and VSMC proliferation and migration were assessed using fluorescence ubiquitination-based cell cycle indicator, transwell, and scratch wound assay. Neointima formation and the expression of MTMR14 was increased after injury. MTMR14 deficiency accelerated neointima formation and promoted VSMC proliferation after injury, whereas MTMR14 overexpression remarkably attenuated this process. Mechanistically, we demonstrated that MTMR14 suppressed the activation of PLK1 (polo-like kinase 1) by interacting with it, which further leads to the inhibition of the activation of MEK/ERK/AKT (mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase/protein kinase B), thereby inhibiting the proliferation of VSMC from the medial to the intima and thus preventing neointima formation. Conclusions MTMR14 prevents neointima formation and VSMC proliferation by inhibiting PLK1. Our findings reveal that MTMR14 serves as an inhibitor of VSMC proliferation and establish a link between MTMR14 and PLK1 in regulating VSMC proliferation. MTMR14 may become a novel potential therapeutic target in the treatment of restenosis.

Asiatic acid alleviates Ang-II induced cardiac hypertrophy and fibrosis via miR-126/PIK3R2 signaling.

BACKGROUND: Cardiac hypertrophy is an independent risk factor of many cardiovascular diseases. Studies have demonstrated that microRNA-126 (miR-126) was involved in angiogenesis during physiological and pathological process. However, its role in cardiac hypertrophy has not been known clearly. Our previous study demonstrated that asiatic acid (AA) has obvious protective effect on cardiac hypertrophy. Here, this study aimed to discover the regulatory role of miR-126 and its mechanism in cardiac hypertrophy, and to determine whether AA's anti-hypertrophy effect is partially miR-126 dependent. METHODS: Male Sprague Dawley rats were AngII infused via osmotic minipumps for 4 weeks and were treated with AA (20 mg/kg/day) by oral gavage. Cardiac hypertrophy was assessed using the echocardiography and histological analysis. In vitro studies,cardiomyocyte and cardiac fibroblasts (CF) were treted with AngII and AngII plus AA. And, the effect of AA on miR-126 and PI3K/AKT signaling pathway was investigated. RESULTS: Treatment of rats with AA decreased the ratio of heart weight to tibia length and hypertrophy markers. In vitro exprements demonstrated that AA significantly attenuated AngII-induced cardiac growth and cardiac fibroblast collagen expression. Moreover, our results found downregulation of miR-126 and activation of PI3K/AKT signaling pathway in AngII infusion induced cardiac hypertrophy model. It was also determined that miR-126 targets PIK3R2 directly. CONCLUSIONS: AA supplementation upregulated the expression of miR-126 and conferred cardio-protection effect against AngII induced cardiac hypertrophy.

MiRNA-223-3p Affects Mantle Cell Lymphoma Development by Regulating the CHUK/NF-ƘB2 Signaling Pathway.

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive malignancy that accounts for 5-10% of non-Hodgkin's lymphoma. MiRNA-223-3p has been demonstrated to be down-regulated in MCL and is a useful prognostic factor. However, little is known about underlying molecular mechanism of miRNA-233-3p in MCL. METHODS: The expression levels of miRNA-223-3p and CHUK mRNA in MCL cells were detected by real-time quantitative PCR (RT-qPCR). The effects of miRNA-223-3p/CHUK overexpression/knockdown on MCL cell proliferation and apoptosis were measured by CCK-8 assay and annexin V PE/7-AAD-based flow cytometry/TUNEL assay, respectively. A nude mouse subcutaneous xenograft model was used to further evaluate the potential effects in vivo. Dual-luciferase reporter assay was used to verify the inhibitory effect of miRNA-223-3p on CHUK. Furthermore, the regulatory function of miRNA-223-3p on the CHUK/NF-ƘB2 axis was assessed by RT-qPCR, western blot and immunofluorescence. RESULTS: In the present study, miRNA-223-3p overexpression inhibited proliferation and accelerated apoptosis of MCL cells in vitro and in vivo. The results of Luciferase reporter assay showed that CHUK was a direct target of miRNA-223-3p in HEK293T cells. Furthermore, the results of RT-qPCR, western blot confirmed that CHUK was targeted and negatively regulated by miRNA-223-3p for repressing NF-ƘB2 pathway activation in MCL cells. Importantly, CHUK overexpression promoted proliferation and suppressed apoptosis of MCL cells, whereas CHUK knockdown reversed down-regulated miRNA-223-3p -accelerated cell proliferation in vitro. CONCLUSION: In conclusion, miRNA-223-3p affects MCL development by regulating the CHUK/NF-ƘB2 signaling pathway, which is crucial to provide a novel therapeutic strategy.