Reduced Expression Levels Research Articles

The Rice Qa-SNAREs in SYP13 Subfamily Are Involved in Regulating Arbuscular Mycorrhizal Symbiosis and Seed Fertility.

Qa-SNARE gene SYP132 (isoform α) was previously reported to affect arbuscular mycorrhizal (AM) symbiosis in the legume species Medicago truncatula. In non-legumes especially monocots, it remains unknown whether certain SNARE genes are also involved in AM symbiosis. In this work, we studied a rice orthologous gene OsSYP132, which showed induced expression in mycorrhizal roots and two paralogous genes OsSYP131a and OsSYP131b, which were not induced by the AM fungus Rhizophagus irregularis. After employing CRISPR/Cas9 technique to generate their mutants, the Ossyp131a homozygous mutant T0 plants exhibited a dwarf phenotype and produced no fertile seeds, indicating a required role of this gene in seed fertility. Unlike the case in legume, the Ossyp132 mutants exhibited normal mycorrhizal phenotype, so did the Ossyp131b mutants. In the Ossyp131b Ossyp132 double mutants, however, the colonization rate and arbuscule abundance level decreased markedly, indicating an impaired fungal proliferation ability in rice roots. Such a defect was further confirmed by the reduced expression levels of AM marker genes. Our results in rice therefore demonstrated that while SYP13II members showed evolutionary and induction patterns specific to symbiosis, AM symbiosis is in fact controlled by the combined action of both SYP13I and SYP13II clades, revealing a functional redundancy among SYNTAXIN genes in mutualism.

Dickkopf-1 exerts protective effects by inhibiting PANoptosis and retinal neovascularization in diabetic retinopathy

Diabetic retinopathy (DR) is a key reason for legal blindness worldwide. Currently, it is urgently necessary to determine the etiology and pathological molecular mechanism of DR to search for resultful therapies. Dickkopf-1 (DKK1) is inhibitive for canonical Wnt signaling via negative feedback, and has been reported as a biomarker for DR. However, the related mechanisms are still unclear. In this work, our data showed that DKK1 was decreased in the vitreous tissues at an early stage of diabetes triggered by streptozotocin (STZ) injection in rats. We subsequently found that DKK1 intravitreal injection significantly ameliorated the physiological function of retina in STZ-challenged rats, accompanied by improved retinal structure. Surprisingly, our results indicated that DKK1 injection remarkably suppressed PANoptosis in retinal tissues of STZ-challenged rats with DR, as proved by ameliorated pyroptosis, apoptosis and necroptosis, which were mainly through the blockage of cleaved Gasdermin-D (GSDMD), Caspase-3 and receptor-interacting protein kinase-3 (RIPK3). Additionally, Wnt signaling including the expression of Wnt, β-catenin and LDL receptor-related protein 5/6 (LRP5/6) was also highly prohibited in retina of DKK1-injected rats with DR. Furthermore, retinal neovascularization and acellular vessel in DR rats were also considerably abolished after DKK1 injection, accompanied by reduced expression levels of retinal vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9). More in vitro experiments showed that DKK1 treatment markedly repressed the proliferative and migratory ability of endothelial cells via inhibiting angiogenesis-related molecules. Together, all our results broaden the knowledge of the correlation between DKK1 and DR, and then provide a novel therapeutic strategy for the suppression of management of DR.

An in‐Depth Analysis of the PAK1 Autophagy Signaling Pathway in H9C2 Cardiomyoblasts

P21‐activated serine/threonine Kinase 1 (PAK1) plays a critical role in cardiomyocyte survival under numerous stressful conditions. Autophagy is a cellular process that promotes homeostasis by removing and replacing antiquated or damaged cellular components. Mitophagy is a form of selective autophagy that eliminates damaged mitochondria to maintain a pool of healthy mitochondria. We have previously demonstrated that PAK1 is essential for maintaining autophagy and mitophagy activities. However, the downstream mediators have not yet been discovered. In this study, we explored the mechanisms of PAK1‐dependent autophagy and mitophagy by determining the protein expression levels of the major regulators of autophagy and mitophagy. H9c2 cardiac myoblasts were treated with siRNA to knockdown the expression of PAK1. Western blot analysis showed that PAK1 knockdown substantially reduced the protein expression levels of ATG5‐12 complex, an essential promotor of autophagosome formation, TFEB, a master regulator of lysosome biogenesis and autophagy, and p62, an autophagy receptor for ubiquitinated cargos. All these changes are expected to reduce autophagy activity. In addition, PAK1 knockdown also reduced the expression of mitophagy receptor FUNDC1 and diminished the association of p62 with mitochondria, which coincided with reduced mitophagy activity. Collectively, these results suggest that the ability of PAK1 knockdown to inhibit autophagy and mitophagy is mediated by reduced expression levels of several important regulators of autophagy and/or mitophagy pathways. Future research is warranted to determine whether restoring the expression levels of these target genes can overcome the inhibition of autophagy and mitophagy by PAK1 deficiency.

Rice transcription factor WRKY68 plays a positive role in <italic>Xa21</italic>-mediated resistance to <italic>Xanthomonas oryzae</italic> pv. <italic>oryzae</italic>

<p id="C3">Leaf blight is a bacterial disease of rice which leads to heavy yield losses. <italic>Xa21</italic> which harbors broad spectrum resistance to leaf blight is the first resistant gene cloned in rice and is widely used in rice breeding program. The identification of transcription factor involved in <italic>Xa21</italic>-mediated resistance to bacterial leaf blight is of great significance. In this study, transgenic <italic>WRKY68-RNAi</italic> lines with reduced expression level of WRKY68 protein in the background of <italic>Xa21 </italic>were generated. The <italic>WRKY68-RNAi</italic> transgenic lines showed reduced resistance to <italic>Xanthomonas oryzae</italic> pv. <italic>oryzae </italic>(<italic>Xoo</italic>), indicating that <italic>WRKY68</italic> played a positive role in <italic>Xa21</italic>-mediated resistance in rice. No detectable changes of WRKY68 protein abundance in leaves at different stages and positions after inoculation were identified by Western blot, indicating that the expression of WRKY68 protein were independent of <italic>Xa21</italic> and <italic>Xoo</italic>, and thus <italic>WRKY68</italic> gene may function as a transcription factor to regulate downstream genes. It was further found that the abundance of a number of pathogenesis-related proteins, including PR1A, PR5, PR10A, PR-pha, and PAL1 were dramatically changed in <italic>WRKY68-RNAi</italic> transgenic lines when inoculated with <italic>Xoo</italic>, suggested that these pathogenesis-related proteins were regulated by <italic>WRKY68 </italic>and the corresponding genes were involved in the downstream defenses responses.

Delivery of miRNA-126 through folic acid-targeted biocompatible polymeric nanoparticles for effective lung cancer therapy

Objective: Nanoparticle-based drug delivery systems (DDSs) have been playing a considerable role in the eradication of cancer. In this experimental study, we designed and synthesized folic acid (FA)-decorated chitosan (CS) nanocarrier for targeted delivery of miR-126 (as a therapeutic agent) to lung cancer A549 cells. Materials and methods: Therefore, the FA-CS-miR-126 nano-complex was perfectly developed and characterized by various analytical devices such as Fourier transform infrared (FT-IR) and dynamic light scattering (DLS) spectroscopies and as well as transmission electron microscopy (TEM). The size was determined lower than 100 nm for synthetics. Then, a gel retardation assay was performed to investigate the entrapment efficiency of nano-complex. Afterward, the sort of in vitro assays was implemented on A549 (FA receptor-positive lung cancer cell line) and MRC5 (normal human diploid cell line) to evaluate the therapeutic efficiency of FA-CS-miR-126. Results: As the cell viability (40.7 ± 2.98% cell viability after 72 h treatment with 500 nM), migration assay (weaker migration after 24 h and 48 h), apoptotic and autophagy genes expression level (Caspse9: sixfolds; BAX: 17 folds; ATG5: fourfolds; and BECLIN1: threefolds more than the control group), the reduced expression level of EGF-L7, as a target gene for miR-126 was evaluated by Real-Time PCR too, then, cell cycle arrest (8.66% of cells in sub-G1 phase), and cell apoptosis assay (21.0% of cancer cell in late apoptosis phase) were scrutinized. Conclusion: These results are remarkably approved the biocompatible and efficient performance of FA-CS-miR-126 as a promising DDS.

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

FTO Alleviates CdCl2-Induced Apoptosis and Oxidative Stress via the AKT/Nrf2 Pathway in Bovine Granulosa Cells.

Cadmium (Cd) is a common environmental heavy metal contaminant of reproduction toxicity. Cd accumulation in animals leads to the damage of granulosa cells. However, its mechanism needs to be elucidated. This research found that treating granulosa cells with Cd resulted in reduced cell viability. The flow cytometry results showed that Cd increased the degree of apoptosis and level of superoxide anion (O2−) in granulosa cells. Further analysis showed that Cd treatment resulted in reduced expression levels of nuclear factor erythroid 2-related factor-2 (Nrf2), superoxide dismutase (SOD), catalase (CAT) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and an increased expression level of malondialdehyde (MDA); the expression levels of Bcl-2 associated X (Bax) and caspase-3 increased, whereas that of B-cell lymphoma 2 (Bcl-2) decreased. Changes in m6A methylation-related enzymes were noted with Cd-induced damage to granulosa cells. The results of transcriptome and MeRIP sequencing revealed that the AKT pathway participated in Cd-induced damage in granulosa cells, and the MAX network transcriptional repressor (MNT) may be a potential target gene of fat mass and obesity-associated protein (FTO). FTO and YTH domain family member 2 (YTHDF2) regulated MNT expression through m6A modification. FTO overexpression alleviated Cd-induced apoptosis and oxidative stress through the activation of the AKT/Nrf2 pathway; this process could be reversed using siMNT. Overall, these findings associated m6A with Cd-induced damage to granulosa cells and provided insights into Cd-induced granulosa cell cytotoxicity from a new perspective centered on m6A modification.

An EGFP Knock-in Zebrafish Experimental Model Used in Evaluation of the Amantadine Drug Safety During Early Cardiogenesis.

BackgroundDrug exposure during gestation or in prematurely born children represents a significant risk to congenital heart disease (CHD). Amantadine is an antiviral agent also effective in the treatment of Parkinson’s disease. However, while its potential side effects associated with tetralogy of fallot (ToF) and birth defects were implicated, its underlying etiologic mechanisms of action remain unknown. Here, we report teratogenic effects of amantadine drug during early cardiogenesis through developing a novel zebrafish (Danio rerio) knock-in (KI) animal model and explore the underlying mechanisms.MethodsHomologous recombination (HR) pathway triggered by CRISPR/Cas9 system was utilized to generate an enhanced green fluorescent protein (EGFP) KI zebrafish animal model. Dynamic fluorescence imaging coupled with a whole-mount in-situ hybridization (WISH) assay was employed to compare the spatial and temporal expression patterns of the EGFP reporter in the KI animal model with the KI-targeted endogenous gene. Heart morphology and EGFP expression dynamics in the KI animal models were monitored to assess cardiac side effects of different doses of amantadine hydrochloride. Expression of key genes required for myocardium differentiation and left–right (LR) asymmetry was analyzed using WISH and quantitative reverse transcription-PCR (RT-PCR).ResultsA novel EGFP KI line targeted at the ventricular myosin heavy chain (vmhc) gene locus was successfully generated, in which EGFP reporter could faithfully recapitulate the endogenous expression dynamics of the ventricle chamber-specific expression of the vmhc gene. Amantadine drug treatment-induced ectopic expression of vmhc gene in the atrium and caused cardiac-looping or LR asymmetry defects to dose-dependently during early cardiogenesis, concomitant with dramatically reduced expression levels of key genes required for myocardium differentiation and LR asymmetry.ConclusionWe generated a novel zebrafish KI animal model in which EGFP reports the ventricle chamber-specific expression of vmhc gene dynamics that is useful to effectively assess drug safety on the cardiac morphology in vivo. Specifically, this study identified teratogenic effects of amantadine drug during early cardiogenesis dose dependent, which could be likely conveyed by inhibiting expression of key genes required for cardiac myocardium differentiation and LR asymmetry.

MicorRNA-195 links long non-coding RNA SEMA3B antisense RNA 1 (head to head) and cyclin D1 to regulate the proliferation of glioblastoma cells

ABSTRACT Long non-coding RNA (lncRNA) SEMA3B antisense RNA 1 (head to head) (SEMA3B-AS1) is a recently identified tumor suppressor in gastric cancer. However, its role in glioblastoma (GBM) is unclear. This study was conducted to explore the role of SEMA3B-AS1 in GBM. In this study, the expression of SEMA3B-AS1, cyclin D1 and miR-195 were determined by RT-qPCR. Gene interactions were evaluated by dual-luciferase assay and overexpression experiments. BrdU assay was performed to monitor cell proliferation. We observed downregulation of SEMA3B-AS1 in GBM. The expression of SEMA3B-AS1 was inversely correlated with the expression of cyclin D1 but positively correlated with the expression of miR-195. In GBM cells, overexpression of SEMA3B-AS1 and miR-195 caused reduced expression levels of cyclin D1. MiR-195 inhibitor reduced the effects of overexpression of SEMA3B-AS1 on the expression of cyclin D1. Moreover, overexpression of SEMA3B-AS1 increased the expression levels of miR-195. Cell proliferation data showed that, SEMA3B-AS1 and miR-195 decreased cell proliferation, while overexpression of cyclin D1 increased GBM cell proliferation. In addition, miR-195 inhibitor inhibited the role of overexpression of SEMA3B-AS1 in cancer cell proliferation. Moreover, miR-195 interacted with cyclin D1, but not SEMA3B-AS1. Furthermore, SEMA3B-AS1 decreased the methylation of the promoter region of miR-195. Therefore, we concluded that miR-195 links lncRNA SEMA3B-AS1 and cyclin D1 to regulate the proliferation of GBM cells.

Molecular Mechanism of Pancreatic β-Cell Failure in Type 2 Diabetes Mellitus.

Various important transcription factors in the pancreas are involved in the process of pancreas development, the differentiation of endocrine progenitor cells into mature insulin-producing pancreatic β-cells and the preservation of mature β-cell function. However, when β-cells are continuously exposed to a high glucose concentration for a long period of time, the expression levels of several insulin gene transcription factors are substantially suppressed, which finally leads to pancreatic β-cell failure found in type 2 diabetes mellitus. Here we show the possible underlying pathway for β-cell failure. It is likely that reduced expression levels of MafA and PDX-1 and/or incretin receptor in β-cells are closely associated with β-cell failure in type 2 diabetes mellitus. Additionally, since incretin receptor expression is reduced in the advanced stage of diabetes mellitus, incretin-based medicines show more favorable effects against β-cell failure, especially in the early stage of diabetes mellitus compared to the advanced stage. On the other hand, many subjects have recently suffered from life-threatening coronavirus infection, and coronavirus infection has brought about a new and persistent pandemic. Additionally, the spread of coronavirus infection has led to various limitations on the activities of daily life and has restricted economic development worldwide. It has been reported recently that SARS-CoV-2 directly infects β-cells through neuropilin-1, leading to apoptotic β-cell death and a reduction in insulin secretion. In this review article, we feature a possible molecular mechanism for pancreatic β-cell failure, which is often observed in type 2 diabetes mellitus. Finally, we are hopeful that coronavirus infection will decline and normal daily life will soon resume all over the world.

Amelioration of Ovalbumin-Induced Allergic Asthma by Juglans regia via Downregulation of Inflammatory Cytokines and Upregulation of Aquaporin-1 and Aquaporin-5 in Mice.

Juglans regia (J. regia) has been used traditionally to treat cough and asthma. The present study evaluates the immunomodulatory and anti-inflammatory potential of J. regia against ovalbumin (OVA)-induced allergic asthma. Intraperitoneal sensitization proceeded by intranasal challenge with OVA was used to induce allergic asthma. BALB/c mice were treated with methanol, n-hexane, and ethyl acetate extracts of J. regia and methylprednisolone one week after 2nd sensitization with OVA and continued for 7 days. mRNA expression levels of IL-4, IL-5, IL-13, AQP-1, AQP-5 TNF-α, TGF-β, and NF-kB were determined using reverse transcription polymerase chain reaction. Hematoxylin and eosin, and periodic acidic-Schiff stains were used for histopathological studies of lung tissues. The data presented all three extracts of J. regia significantly ameliorated airway inflammation by reducing expression levels of IL-4, IL-5, and IL-13 and TNF-α in OVA-treated mice. The suppression of goblet cells hyperplasia and inflammatory cells infiltration by J. regia involved low TGF-β and NF-kB levels. Pretreatment with J. regia also increased the AQP-1 and AQP-5 expression levels in mice treated with OVA. This study supported the traditional use of J. regia and proposed that J. regia ameliorated allergic asthma by suppression of proinflammatory cytokines and elevation of AQP-1 and AQP-5 expression levels.

MicroRNA-582-3p regulates osteoporosis through regulating homeobox A10 and osteoblast differentiation

Objective Recent studies have demonstrated that micro RNAs (miRNAs) are involved in bone formation and bone cell differentiation, but the role of miR-582-3p in osteoporosis is unclear. We want to study the mechanism of miR-582-3p on osteogenic differentiation. Method The expression of miR-582-3p and homeobox (Hox) A10 were analyzed by quantitative RT-PCR. The expression levels of HOXA10 protein were determined by Western blot. The target of HOXA10 was identified by bioinformatics and luciferase reporter gene assay. Results The results showed that miR-582-3p was up-regulated in OP tissues and down-regulated in osteogenic differentiated C2C12 cells compared with that in the control group. Overexpression of miR-582-3p resulted in reduced expression levels of osteocalcin (OC), alkaline phosphatase (ALP), and collagen, type I, α1 (COL1A1). miR-582-3p had a potential binding site with HOXA10. Moreover, miR-582-3p inhibited the expression of HOXA10, and overexpression of HOXA10 reduced the effect of miR-582-3p on osteoblast markers. HOXA10 was the target gene of miR-582-3p, which could inhibit the expression of HOXA10. Furthermore, HOXA10 reduced the role of miR-582-3p in osteoblast markers. miR-582-3p inhibited the development of osteoporosis by regulating HOXA10 and osteoblast differentiation. Conclusions miR-582-3p may be a therapeutic target of osteoporosis treatment.

Inherited variants in CHD3 show variable expressivity in Snijders Blok-Campeau syndrome

PurposeCommon diagnostic next-generation sequencing strategies are not optimized to identify inherited variants in genes associated with dominant neurodevelopmental disorders as causal when the transmitting parent is clinically unaffected, leaving a significant number of cases with neurodevelopmental disorders undiagnosed. MethodsWe characterized 21 families with inherited heterozygous missense or protein-truncating variants in CHD3, a gene in which de novo variants cause Snijders Blok-Campeau syndrome. ResultsComputational facial and Human Phenotype Ontology–based comparisons showed that the phenotype of probands with inherited CHD3 variants overlaps with the phenotype previously associated with de novo CHD3 variants, whereas heterozygote parents are mildly or not affected, suggesting variable expressivity. In addition, similarly reduced expression levels of CHD3 protein in cells of an affected proband and of healthy family members with a CHD3 protein-truncating variant suggested that compensation of expression from the wild-type allele is unlikely to be an underlying mechanism. Notably, most inherited CHD3 variants were maternally transmitted. ConclusionOur results point to a significant role of inherited variation in Snijders Blok-Campeau syndrome, a finding that is critical for correct variant interpretation and genetic counseling and warrants further investigation toward understanding the broader contributions of such variation to the landscape of human disease.

MiR-217-5p suppresses epithelial-mesenchymal transition and the NF-κB signaling pathway in breast cancer via targeting of metadherin.

MicroRNAs (miRNAs) have been associated with a number of human malignancies, including breast cancer (BC). However, the expression, biological function and fundamental underlying mechanism of miR-217-5p in BC remain unclear. Therefore, in the present study, the expression levels of miR-217-5p and metadherin (MTDH) were examined in BC tissues and BC cell lines using reverse transcription-quantitative PCR. Cell Counting Kit-8 assays, cell proliferation, wound healing assays, Transwell assays and western blotting were used to examine the effects of miR-217-5p on cell proliferation, migration, the epithelial-mesenchymal transition (EMT) and NF-κB signaling pathway expression. The direct relationship between miR-217-5p and MTDH was assessed using a dual-luciferase reporter assay. The results demonstrated that significantly reduced expression levels of miR-217-5p but significantly increased mRNA expression levels of MTDH were observed in BC tissues from 35 patients with BC compared with non-tumor breast tissues. Furthermore, BC cell lines SK-BR3 and BT549 expressed miR-217-5p at markedly lower levels and MTDH at markedly higher levels compared with the breast epithelial MCF10A cell line. miR-217-5p overexpression significantly inhibited cell proliferation, invasion and migration and suppressed the EMT in BC cells. miR-217-5p overexpression also inhibited the NF-κB signaling pathway by markedly decreasing p65 mRNA and protein expression levels but significantly increasing IκBα expression levels. Furthermore, miR-217-5p knockdown markedly increased MTDH mRNA and protein expression levels. The expression levels of miR-217-5p were negatively correlated with those of MTDH in BC tissues. These results suggested that restoration of MTDH expression levels could potentially attenuate the inhibitory effects of miR-217-5p overexpression on BC cell proliferation. Therefore, in conclusion miR-217-5p overexpression may inhibit cell migration, invasion, the EMT and NF-κB signaling pathway in BC via targeting of MTDH. miR-217-5p may serve as an important potential target in BC therapy.

Telomere shortening impairs alveolar regeneration.

ObjectivesShort telomeres in alveolar type 2 (AT2) cells have been associated with many lung diseases. The study aimed to investigate the regeneration capacity of AT2 cells with short telomeres by knocking out Tert in mice (G4 Tert −/− ) from the whole to the cellular level.Materials and MethodsThe lung injury model of mice was established by left pneumonectomy (PNX). The proliferation and differentiation of AT2 cells were observed by immunofluorescence staining in vivo and in vitro. The difference of the gene expression between control and G4 Tert −/− group during the regeneration of AT2 cells was compared by RNA sequencing. The expression of tubulin polymerization promoting protein 3 (TPPP3) was reduced by adeno‐associated virus delivery.ResultsThe alveolar regeneration in G4 Tert −/− mice was impaired after PNX‐induced lung injury. The regulation of cytoskeleton remodelling was defective in G4 Tert −/− AT2 cells. The expression of TPPP3 was gradually increased during AT2 cell differentiation. The expression level of TPPP3 was reduced in G4 Tert −/− AT2 cells. Reducing TPPP3 expression in AT2 cells limits the microtubule remodelling and differentiation of AT2 cells.ConclusionShort telomeres in AT2 cells result in the reduced expression level of TPPP3, leading to impaired regeneration capacity of AT2 cells.

Overexpression of the Salix matsudana SmAP2-17 gene improves Arabidopsis salinity tolerance by enhancing the expression of SOS3 and ABI5

BackgroundSalix matsudana (Koidz.) is a widely planted ornamental allotetraploid tree species. Genetic engineering can be used to enhance the tolerance of this species to soil salinization, endowing varieties with the ability to grow along coastlines, thereby mitigating afforestation and protecting the environment. The AP2/ERF family of transcription factors (TFs) plays multidimensional roles in plant biotic/abiotic stress tolerance and plant development. In this study, we cloned the SmAP2-17 gene and performed functional analysis of its role in salt tolerance. This study aims to identify key genes for future breeding of stress-resistant varieties of Salix matsudana.ResultsSmAP2-17 was predicted to be a homolog of AP2-like ethylene-responsive transcription factor ANT isoform X2 from Arabidopsis, with a predicted ORF of 2058 bp encoding an estimated protein of 685 amino acids containing two conserved AP2 domains (PF00847.20). SmAP2-17 had a constitutive expression pattern and was localized to the nucleus. The overexpression of the native SmAP2-17 CDS sequence in Arabidopsis did not increase salt tolerance because of the reduced expression level of ectopic SmAP2-17, potentially caused by salt-induced RNAi. Transgenic lines with high expression of optimized SmAP2-17 CDS under salt stress showed enhanced tolerance to salt. Moreover, the expression of general stress marker genes and important salt stress signaling genes, including RD29A, ABI5, SOS3, AtHKT1, and RBohF, were upregulated in SmAP2-17-overexpressed lines, with expression levels consistent with that of SmAP2-17 or optimized SmAP2-17. Promoter activity analysis using dual luciferase analysis showed that SmAP2-17 could bind the promoters of SOS3 and ABI5 to activate their expression, which plays a key role in regulating salt tolerance.ConclusionsThe SmAP2-17 gene isolated from Salix matsudana (Koidz.) is a positive regulator that improves the resistance of transgenic plants to salt stress by upregulating SOS3 and ABI5 genes. This study provides a potential functional gene resource for future generation of salt-resistant Salix lines by genetic engineering.

Mechanism of polyphyllin A in inhibition of proliferation and induction of apoptosis of gastric cancer cells by directly targeting ETS-1

The present study investigated the mechanism of polyphyllin A(PPA) in inhibiting gastric cancer(GC) cells. GC cells(SGC7901 and MGC803 cell lines) were treated with PPA at different concentrations. The effect of PPA on the proliferation of GC cells was detected by MTT assay, real-time cell analysis(RTCA) assay, and clone-forming assay, respectively. Reactive oxygen species(ROS) of GC cells was detected by flow cytometry. The change of mitochondrial membrane potential was detected by JC-1 assay. The expression and phosphorylation levels of apoptosis-related proteins(caspase-9, caspase-3, and PARP) and proteins related to the signaling pathway(ETS-1, CIP2 A, and Akt) were detected by Western blot. The binding sites of PPA to ETS-1 were analyzed by molecular docking. The affinity of PPA and ETS-1 was detected by drug affinity responsive target stability(DARTS) assay. PPA had a significant inhibitory effect on the proliferation and colony formation of GC cells at a low concentration. The PPA groups showed increased ROS and decreased mitochondrial membrane potential. PPA down-regulated the precursor expression of caspase-9 and caspase-3 and promoted the cleavage of PARP, suggesting that PPA induced the apoptosis of GC cells through the mitochondrial pathway. PPA significantly reduced expression levels of CIP2 A and the phosphorylation of downstream Akt. Molecular docking showed that PPA bound to the ETS domain of ETS-1, the transcription factor of CIP2 A, and formed hydrogen bonds with Pro319 and Asp317. DARTS assay further confirmed that PPA significantly prevented the hydrolysis of ETS-1 by pronase, which was inductive of the direct binding effect of PPA and ETS-1. PPA inhibits the proliferation and induces the apoptosis of GC cells by directly targeting ETS-1 to down-regulate the ETS-1/CIP2 A/Akt signaling pathway.

Factor Xa inhibitor, edoxaban ameliorates renal injury after subtotal nephrectomy by reducing epithelial-mesenchymal transition and inflammatory response.

Chronic kidney disease (CKD) is an increasing and life‐threatening disease worldwide. Recent evidence indicates that blood coagulation factors promote renal dysfunction in CKD patients. Activated factor X (FXa) inhibitors are safe and first‐line drugs for the prevention of thrombosis in patients with atrial fibrillation. Here, we investigated the therapeutic effects of edoxaban on CKD using the mouse 5/6 nephrectomy model. Eight‐week‐old wild‐type mice were subjected to 5/6 nephrectomy surgery and randomly assigned to two groups, edoxaban or vehicle admixture diet. Edoxaban treatment led to reduction of urinary albumin excretion and plasma UN levels compared with vehicle group, which was accompanied with reduced glomerular cross‐sectional area and cell number. Edoxaban treatment also attenuated fibrinogen positive area in the remnant kidneys after subtotal nephrectomy. Moreover, edoxaban treatment resulted in attenuated tubulointerstitial fibrosis after 5/6 nephrectomy, which was accompanied by reduced expression levels of epithelial‐mesenchymal transition (EMT) markers, inflammatory mediators, and oxidative stress markers in the remnant kidneys. Treatment of cultured proximal tubular cells, HK‐2 cells, with FXa protein led to increased expression levels of EMT markers, inflammatory mediators, and oxidative stress markers, which were abolished by pretreatment with edoxaban. Treatment of HK‐2 cells with edoxaban attenuated FXa‐stimulated phosphorylation levels of extracellular signal‐regulated kinase (ERK) and NF‐κB. Our findings indicate that edoxaban can improve renal injury after subtotal nephrectomy by reducing EMT and inflammatory response, suggesting that FXa inhibition could be a novel therapeutic target for CKD patients with atrial fibrillation.

MiR-200c-3p Regulates DUSP1/MAPK Pathway in the Nonalcoholic Fatty Liver After Laparoscopic Sleeve Gastrectomy

AimNon-alcoholic fatty liver disease (NAFLD) is a health burden worldwide, which is closely related to obesity. The effect of sleeve gastrectomy (SG) on NAFLD is efficient, and the underlying mechanism remains unknown. Our study sought to investigate the mechanism of dual-specificity protein phosphatase 1 (DUSP1) expression regulation following the SG procedure in NAFLD patients and C57BL/6J mice via miR-200c-3p.MethodsThe serum was extracted from NAFLD patients who underwent laparoscopic sleeve gastrectomy (LSG) and volunteers. Next, the correlation between miR-200c-3p and DUSP1 was identified in vitro. NAFLD mice were modelled by high-fat diets (HFD). The hepatic tissue expression levels of miR-200c-3p, DUSP1, phospho-extracellular regulated protein kinases1/2 (p-ERK1/2), phospho -p38 mitogen-activated protein kinases (p-p38), and phospho-c-Jun N-terminal kinases (p-JNK) induced by SG procedure were evaluated.ResultsThe SG procedure contributed to significant weight loss, reduced lipids in NAFLD patients and mice. The increased expression level of miR-200c-3p and reduced expression of DUSP1 were observed in NAFLD patients and mice (p<0.05). The reduced expression levels of miR-200c-3p and increased expression of DUSP1 were observed in patients and mice with NAFLD who underwent SG procedure. DUSP1 is a potential target of miR-200c-3p.ConclusionsA novel mechanism was identified in which miR-200c-3p regulates the MAPK-dependent signals that are linked to the promotion of hepatosteatosis via DUSP1 after sleeve gastrectomy. The findings suggested that miR-200c-3p should be further explored as a potential target for the treatments of NAFLD.

The protective effects of butorphanol tartrate against homocysteine-induced blood-brain barrier dysfunction

ABSTRACT A high concentration of homocysteine (Hcy) has been recently reported to be closely associated with the development of stroke, which is related to the Hcy-induced blood-brain barrier (BBB) dysfunction. Butorphanol tartrate is a promising analgesic agent that targets the opiate receptor and shows promising protective effects on ischemia/reperfusion injury. The present research proposes to investigate the protective effect of butorphanol tartrate on Hcy-induced BBB disruption to explore the potential application of butorphanol tartrate in treating Hcy-induced stroke. Hcy was utilized to establish both an in vivo animal model and in vitro human brain vascular endothelial cells (HBVECs) injury model. We found that the increased diffusion of sodium fluorescein and Evan’s blue, declined expression of Claudin-5, and increased production of interleukin- 6 (IL-6) and tumor necrosis factor-α (TNF-α) were observed in Hcy-treated mice, which were all significantly reversed by butorphanol tartrate. In Hcy-stimulated HBVECs, increased endothelial permeability and reduced expression levels of Claudin-5 and Krüppel-like factor 5 (KLF5) were observed, all of which were dramatically rescued by 2 and 5 µM butorphanol tartrate. Lastly, the protective function of butorphanol tartrate in Hcy-stimulated HBVECs was dramatically abolished by the knockdown of KLF5. Collectively, butorphanol tartrate showed protective effects on Hcy-induced BBB disruption by upregulating the KLF5/Claudin-5 axis.