NDRG2 acts as a negative regulator of the progression of small-cell lung cancer through the modulation of the PTEN-AKT-mTOR signalling cascade.

N-myc downstream-regulated gene 2 (NDRG2) has been recognised as a negative regulator of the progression of numerous tumours, yet its specific role in small-cell lung carcinoma (SCLC) is not fully understood. The purpose of the current study was to investigate the biological role and mechanism of NDRG2 in SCLC. Initial investigation using the Gene Expression Omnibus (GEO) dataset revealed marked downregulation of NDRG2 transcripts in SCLC. The decreased abundance of NDRG2 in SCLC was verified by examining clinical specimens. Increasing NDRG2 expression in SCLC cell lines caused significant changes in cell proliferation, cell cycle progression, colony formation, and chemosensitivity. NDRG2 overexpression decreased the levels of phosphorylated PTEN, AKT and mTOR. In PTEN-depleted SCLC cells, the upregulation of NDRG2 did not result in any noticeable impact on AKT or mTOR activation. Additionally, the reactivation of AKT reversed the antitumour effects of NDRG2 in SCLC cells. Notably, increasing NDRG2 expression retarded the growth of SCLC cell-derived xenografts in vivo. In conclusion, NDRG2 serves as an inhibitor of SCLC, and its cancer-inhibiting effects are achieved through the suppression of AKT/mTOR via the activation of PTEN. This work suggests that NDRG2 is a potential druggable target for SCLC treatment.

Divergent sequences of tetraspanins enable plants to specifically recognize microbe-derived extracellular vesicles.

Extracellular vesicles (EVs) are important for cell-to-cell communication in animals. EVs also play important roles in plant-microbe interactions, but the underlying mechanisms remain elusive. Here, proteomic analyses of EVs from the soybean (Glycine max) root rot pathogen Phytophthora sojae identify the tetraspanin family proteins PsTET1 and PsTET3, which are recognized by Nicotiana benthamiana to trigger plant immune responses. Both proteins are required for the full virulence of P. sojae. The large extracellular loop (EC2) of PsTET3 is the key region recognized by N. benthamiana and soybean cells in a plant receptor-like kinase NbSERK3a/b dependent manner. TET proteins from oomycete and fungal plant pathogens are recognized by N. benthamiana thus inducing immune responses, whereas plant-derived TET proteins are not due to the sequence divergence of sixteen amino acids at the C-terminal of EC2. This feature allows plants to distinguish self and non-self EVs to trigger active defense responses against pathogenic eukaryotes.

The CAP superfamily protein PsCAP1 secreted by Phytophthora triggers immune responses in Nicotiana benthamiana through a leucine-rich repeat receptor-like protein.

The role of cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 (CAP) superfamily proteins in the innate immune responses of mammals is well characterized. However, the biological function of CAP superfamily proteins in plant-microbe interactions is poorly understood. We used proteomics and transcriptome analyses to dissect the apoplastic effectors secreted by the oomycete Phytophthora sojae during early infection of soybean leaves. By transiently expressing these effectors in Nicotiana benthamiana, we identified PsCAP1, a novel type of secreted CAP protein that triggers immune responses in multiple solanaceous plants including N. benthamiana. This secreted CAP protein is conserved among oomycetes, and multiple PsCAP1 homologs can be recognized by N. benthamiana. PsCAP1-triggered immune responses depend on the N-terminal immunogenic fragment (aa 27-151). Pretreatment of N. benthamiana with PsCAP1 or the immunogenic fragment increases plant resistance against Phytophthora. The recognition of PsCAP1 and different homologs requires the leucine-rich repeat receptor-like protein RCAP1, which associates with two central receptor-like kinases BRI1-associated receptor kinase 1 (BAK1) and suppressor of BIR1-1 (SOBIR1) in planta. These findings suggest that the CAP-type apoplastic effectors act as an important player in plant-microbe interactions that can be perceived by plant membrane-localized receptor to activate plant resistance.

NLR surveillance of pathogen interference with hormone receptors induces immunity.

Phytohormone signalling pathways have an important role in defence against pathogens mediated by cell-surface pattern recognition receptors and intracellular nucleotide-binding leucine-rich repeat class immune receptors1,2 (NLR). Pathogens have evolved counter-defence strategies to manipulate phytohormone signalling pathways to dampen immunity and promote virulence3. However, little is known about the surveillance of pathogen interference of phytohormone signalling by the plant innate immune system. The pepper (Capsicum chinense) NLR Tsw, which recognizes the effector nonstructural protein NSs encoded by tomato spotted wilt orthotospovirus (TSWV), contains an unusually large leucine-rich repeat (LRR) domain. Structural modelling predicts similarity between the LRR domain of Tsw and those of the jasmonic acid receptor COI1, the auxin receptor TIR1 and the strigolactone receptor partner MAX2. This suggested that NSs could directly target hormone receptor signalling to promote infection, and that Tsw has evolved a LRR resembling those of phytohormone receptors LRR to induce immunity. Here we show that NSs associates with COI1, TIR1 and MAX2 through a common repressor-TCP21-which interacts directly with these phytohormone receptors. NSs enhances the interaction of COI1, TIR1 or MAX2 with TCP21 and blocks the degradation of corresponding transcriptional repressors to disable phytohormone-mediated host immunity to the virus. Tsw also interacts directly with TCP21 and this interaction is enhanced by viral NSs. Downregulation of TCP21 compromised Tsw-mediated defence against TSWV. Together, our findings reveal that a pathogen effector targets TCP21 to inhibit phytohormone receptor function, promoting virulence, and a plant NLR protein has evolved to recognize this interference as a counter-virulence strategy, thereby activating immunity.

CircLPAR3 knockdown suppresses esophageal squamous cell carcinoma cell oncogenic phenotypes and Warburg effect through miR-873-5p/LDHA axis.

BACKGROUND: Circular RNAs (circRNAs) have been identified to participate in regulating multiple malignancies. Herein, this study aimed to explore the clinical significance, biological function, and regulatory mechanisms of circRNA lysophosphatidic acid receptor 3 (circLPAR3) in esophageal squamous cell carcinoma (ESCC) cell malignant phenotypes and Warburg effect. METHODS: The qRT-PCR and Western blot were used to detect the levels of genes and proteins. Glucose uptake and lactate production were detected to determine the Warburg effect. The effects of circLPAR3 on ESCC cell proliferation, apoptosis, and metastasis were evaluated by MTT, 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, wound healing, and transwell assays. The binding interaction between miR-873-5p and circLPAR3 or lactate dehydrogenase A (LDHA) was verified using dual-luciferase reporter and RIP assays. Xenograft mice models were established to conduct in vivo analysis. RESULTS: CircLPAR3 is a stable circRNA and was increased in ESCC tissues and cells. Functionally, circLPAR3 knockdown suppressed ESCC cell Warburg effect, proliferation, metastasis, and induced apoptosis in vitro, and impeded xenograft tumor growth and Warburg effect in ESCC mice models. Mechanistically, circLPAR3 served as a sponge for miR-873-5p, which targeted LDHA. Moreover, circLPAR3 could regulate LDHA expression by sponging miR-873-5p. Thereafter, rescue experiments suggested that miR-873-5p inhibition reversed the anticancer effects of circLPAR3 silencing on ESCC cells. Furthermore, miR-873-5p overexpression restrained ESCC cell Warburg effect and oncogenic phenotypes, which were abolished by LDHA up-regulation. CONCLUSION: CircLPAR3 knockdown suppressed ESCC cell growth, metastasis, and Warburg effect by miR-873-5p/LDHA axis, implying a promising molecular target for ESCC therapy.

The N-terminus of an Ustilaginoidea virens Ser-Thr-rich glycosylphosphatidylinositol-anchored protein elicits plant immunity as a MAMP.

Many pathogens infect hosts through specific organs, such as Ustilaginoidea virens, which infects rice panicles. Here, we show that a microbe-associated molecular pattern (MAMP), Ser-Thr-rich Glycosyl-phosphatidyl-inositol-anchored protein (SGP1) from U. virens, induces immune responses in rice leaves but not panicles. SGP1 is widely distributed among fungi and acts as a proteinaceous, thermostable elicitor of BAK1-dependent defense responses in N. benthamiana. Plants specifically recognize a 22 amino acid peptide (SGP1 N terminus peptide 22, SNP22) in its N-terminus that induces cell death, oxidative burst, and defense-related gene expression. Exposure to SNP22 enhances rice immunity signaling and resistance to infection by multiple fungal and bacterial pathogens. Interestingly, while SGP1 can activate immune responses in leaves, SGP1 is required for U. virens infection of rice panicles in vivo, showing it contributes to the virulence of a panicle adapted pathogen.

The Phytophthora effector Avh241 interacts with host NDR1-like proteins to manipulate plant immunity.

Plant pathogens rely on effector proteins to suppress host innate immune responses and facilitate colonization. Although the Phytophthora sojae RxLR effector Avh241 promotes Phytophthora infection, the molecular basis of Avh241 virulence remains poorly understood. Here we identified non-race specific disease resistance 1 (NDR1)-like proteins, the critical components in plant effector-triggered immunity (ETI) responses, as host targets of Avh241. Avh241 interacts with NDR1 in the plasma membrane and suppresses NDR1-participated ETI responses. Silencing of GmNDR1s increases the susceptibility of soybean to P. sojae infection, and overexpression of GmNDR1s reduces infection, which supports its positive role in plant immunity against P. sojae. Furthermore, we demonstrate that GmNDR1 interacts with itself, and Avh241 probably disrupts the self-association of GmNDR1. These data highlight an effective counter-defense mechanism by which a Phytophthora effector suppresses plant immune responses, likely by disturbing the function of NDR1 during infection.

N-glycosylation shields Phytophthora sojae apoplastic effector PsXEG1 from a specific host aspartic protease.

Hosts and pathogens are engaged in a continuous evolutionary struggle for physiological dominance. A major site of this struggle is the apoplast. In Phytophthora sojae-soybean interactions, PsXEG1, a pathogen-secreted apoplastic endoglucanase, is a key focal point of this struggle, and the subject of two layers of host defense and pathogen counterdefense. Here, we show that N-glycosylation of PsXEG1 represents an additional layer of this coevolutionary struggle, protecting PsXEG1 against a host apoplastic aspartic protease, GmAP5, that specifically targets PsXEG1. This posttranslational modification also attenuated binding by the previously described host inhibitor, GmGIP1. N-glycosylation of PsXEG1 at N174 and N190 inhibited binding and degradation by GmAP5 and was essential for PsXEG1's full virulence contribution, except in GmAP5-silenced soybeans. Silencing of GmAP5 reduced soybean resistance against WT P. sojae but not against PsXEG1 deletion strains of P. sojae. The crucial role of N-glycosylation within the three layers of defense and counterdefense centered on PsXEG1 highlight the critical importance of this conserved apoplastic effector and its posttranslational modification in Phytophthora-host coevolutionary conflict.

Circular RNA SMARCA5 suppressed non-small cell lung cancer progression by regulating miR-670-5p/RBM24 axis.

Circular RNAs (circRNAs) have good stability and long half-life in blood and other body fluid, and possess regulatory effects on various biological processes as miRNA/RNA-binding protein sponges, or by competing endogenous RNA, indicating their great potential as biomarkers or targets of cancer therapy. In this study, we mainly explored the role and mechanism of circular RNA SMARCA5 (circsSMARCA5) in non-small cell lung cancer (NSCLC). Quantitative RT-PCR was applied to measure the expression levels of genes, and then, the relationships among circsSMARCA5, microRNA-670-5p (miR-670-5p), and RBM24 were further analyzed. Animal and cell experiments were performed to explore the functions of circsSMARCA5 in NSCLC cells. The results showed that circsSMARCA5 was expressed at low level in NSCLC tissues and cells, while miR-670-5p had high level in NSCLC tissues. Dual luciferase reporter assay verified that miR-670-5p was the target of circsSMARCA5, and RBM24 has the binding site of miR-670-5p. Further analysis showed that circsSMARCA5 could negatively regulate miR-670-5p and had positive relationship with RBM24. Moreover, circsSMARCA5 obviously inhibited tumor growth in vivo, reduced cell proliferation and increased cell apoptosis in vitro, while miR-670-5p mimic or RBM24 knockdown could reverse these effects. Thus, circsSMARCA5 may serve as an NSCLC suppressor by regulating the miR-670-5p/RBM24 axis, and it may have the potential to be a biomarker or therapeutic target for NSCLC.

Extraction of acetanilide herbicides in naked oat (Avena nuda L.) by using ionic-liquid-based matrix solid-phase dispersion-foam flotation solid-phase extraction.

The herbicides in naked oat (Avena nuda L.) samples were extracted, separated, and determined by using ionic-liquid-based matrix solid-phase dispersion-solvent flotation coupled with high-performance liquid chromatography. The experimental parameters were optimized and evaluated by a univariate method and orthogonal experiment. A good linear relationship was obtained in the range of 5-5000 µg/kg, and the linear correlation coefficient are between 0.9989∼0.9993. The quantification limits for alachlor, metazachlor, propanil, acetochlor, pretilachlor, metolachlor, and butachlor are 5.03, 2.62, 2.73, 4.58, 7.28, 5.05, 5.78 µg/kg, respectively. The average recoveries of the acetanilide herbicides at spiked concentrations of 10, 100, and 500 µg/kg ranged from 92.1 to 104.7%, and relative standard deviations were equal to or lower than 2.9%.

Homeobox D10, a tumor suppressor, inhibits the proliferation and migration of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common types of esophageal cancer, which is the sixth leading cause of cancer death globally. Homeobox D10 (HOXD10) is a member of the homeobox (HOX) gene family and has been reported to act as a tumor suppressor. However, the potential role of HOXD10 in ESCC has not been reported. Thus, the aim of this study was to examine the expression and function of HOXD10 in ESCC. The expressions of HOXD10 in human ESCC tissues and cell lines were detected by quantitative reverse transcription polymerase chain reaction and Western blot. The HOXD10 overexpressing cell lines were established, then CCK-8 and Transwell assays were performed to examine cell proliferation, migration, and invasion, respectively. The expression of EMT-related proteins and signaling pathway-related proteins were detected by Western blot. Our results showed that HOXD10 is lowly expressed in ESCC tissues as well as in ESCC cell lines. Ectopic overexpression of HOXD10 inhibited cell proliferation, migration, and invasion of ESCC cells (P < 0.05). HOXD10 overexpression repressed the epithelial-mesenchymal transition (EMT) process in ESCC cells. Besides, HOXD10 overexpression suppressed the activation of PI3K/AKT/mTOR signaling pathway. PI3K/Akt agonist, insulin-like growth factor-1, reversed the inhibitory effects of HOXD10 on cell proliferation and migration in ESCC cells. Additional in vivo study proved that ectopic expression of HOXD10 caused an obvious inhibitory effect on the tumor growth. These findings indicated that overexpression of HOXD10 suppressed the proliferation, migration, and invasion via regulating the PI3K/AKT/mTOR signaling pathway in ESCC cells. Thus, targeting HOXD10 may be considered as a therapeutic strategy for ESCC treatment.

Casticin inhibits esophageal cancer cell proliferation and promotes apoptosis by regulating mitochondrial apoptotic and JNK signaling pathways.

Casticin, a flavonoid isolated from Vitex species, has been found to have anti-tumor property in multiple human cancers. The present study aimed to investigate the effect of casticin on the proliferation and apoptosis of esophageal cancer (EC) cells, and further illustrate the underlying mechanisms. In in vitro studies, human EC cell lines TE-1 and ECA-109 were treated with various concentrations of casticin (low-, middle-, and high-dose groups). The results showed that casticin dose-dependently inhibited the proliferation and clonogenicity of EC cells and induced cell cycle arrest in sub-G1 and G2 phases. Furthermore, casticin markedly enhanced EC cell apoptosis as detected by flow cytometry and Hoechst 33342 staining. The level of anti-apoptotic Bcl-2 protein was decreased, while the levels of pro-apoptotic Bax, cleaved-caspase-3, cleaved-caspase-9, and cleaved-PARP were conversely increased in casticin-treated TE-1 and ECA-109 cells. Moreover, casticin decreased the mitochondrial membrane potential and increased the release of mitochondrial cytochrome C into cytoplasm. In addition, the JNK signaling pathway was involved in casticin-medicated anti-proliferation and pro-apoptosis. Cells pretreated with SP600125, a JNK pathway inhibitor, partially abolished the effect of casticin. Finally, the anti-tumor property of casticin was confirmed in in vivo xenograft models. Overall, we provided both in vitro and in vivo evidences that casticin inhibited the proliferation and induced apoptosis of EC cells, and the anti-tumor action of casticin was mediated, in part, by the mitochondrial-dependent apoptosis and the activation of JNK signaling pathway.

A Phytophthora effector recruits a host cytoplasmic transacetylase into nuclear speckles to enhance plant susceptibility.

Oomycete pathogens secrete host cell-entering effector proteins to manipulate host immunity during infection. We previously showed that PsAvh52, an early-induced RxLR effector secreted from the soybean root rot pathogen, Phytophthora sojae, could suppress plant immunity. Here, we found that PsAvh52 is required for full virulence on soybean and binds to a novel soybean transacetylase, GmTAP1, in vivo and in vitro. PsAvh52 could cause GmTAP1 to relocate into the nucleus where GmTAP1 could acetylate histones H2A and H3 during early infection, thereby promoting susceptibility to P. sojae. In the absence of PsAvh52, GmTAP1 remained confined to the cytoplasm and did not modify plant susceptibility. These results demonstrate that GmTAP1 is a susceptibility factor that is hijacked by PsAvh52 in order to promote epigenetic modifications that enhance the susceptibility of soybean to P. sojae infection.

Knockdown of DDX5 Inhibits the Proliferation and Tumorigenesis in Esophageal Cancer.

DEAD (Asp-Glu-Ala-Asp) box protein 5 (DDX5), a prototypical member of the DEAD/H-box protein family, has been involved in several human malignancies. However, the expression and biological role of DDX5 in esophageal cancer (EC) remain largely unknown. In this study, we examined the role of DDX5 in regulating EC cell proliferation and tumorigenesis and explored its possible molecular mechanism. We found that DDX5 was overexpressed in human EC cell lines. In addition, knockdown of DDX5 significantly inhibited the proliferation of EC cells in vitro and the growth of EC xenografts in vivo. Knockdown of DDX5 also suppressed the migration/invasion and epithelial-to-mesenchymal transition (EMT) phenotype in EC cells. Furthermore, we observed that knockdown of DDX5 inhibited the expression of ß-catenin, c-Myc, and cyclin D1 in EC cells. In conclusion, our findings provide the first evidence that siRNA-DDX5 inhibited the proliferation and invasion of EC cells through suppressing the Wnt/ß-catenin signaling pathway. Therefore, DDX5 may be a novel potential therapeutic target for the prevention and treatment of EC.

A Phytophthora Effector Manipulates Host Histone Acetylation and Reprograms Defense Gene Expression to Promote Infection.

Immune response during pathogen infection requires extensive transcription reprogramming. A fundamental mechanism of transcriptional regulation is histone acetylation. However, how pathogens interfere with this process to promote disease remains largely unknown. Here we demonstrate that the cytoplasmic effector PsAvh23 produced by the soybean pathogen Phytophthora sojae acts as a modulator of histone acetyltransferase (HAT) in plants. PsAvh23 binds to the ADA2 subunit of the HAT complex SAGA and disrupts its assembly by interfering with the association of ADA2 with the catalytic subunit GCN5. As such, PsAvh23 suppresses H3K9 acetylation mediated by the ADA2/GCN5 module and increases plant susceptibility. Expression of PsAvh23 or silencing of GmADA2/GmGCN5 resulted in misregulation of defense-related genes, most likely due to decreased H3K9 acetylation levels at the corresponding loci. This study highlights an effective counter-defense mechanism by which a pathogen effector suppresses the activation of defense genes by interfering with the function of the HAT complex during infection.

A paralogous decoy protects Phytophthora sojae apoplastic effector PsXEG1 from a host inhibitor.

The extracellular space (apoplast) of plant tissue represents a critical battleground between plants and attacking microbes. Here we show that a pathogen-secreted apoplastic xyloglucan-specific endoglucanase, PsXEG1, is a focus of this struggle in the Phytophthora sojae-soybean interaction. We show that soybean produces an apoplastic glucanase inhibitor protein, GmGIP1, that binds to PsXEG1 to block its contribution to virulence. P. sojae, however, secretes a paralogous PsXEG1-like protein, PsXLP1, that has lost enzyme activity but binds to GmGIP1 more tightly than does PsXEG1, thus freeing PsXEG1 to support P. sojae infection. The gene pair encoding PsXEG1 and PsXLP1 is conserved in many Phytophthora species, and the P. parasitica orthologs PpXEG1 and PpXLP1 have similar functions. Thus, this apoplastic decoy strategy may be widely used in Phytophthora pathosystems.

miR-1908 Overexpression Inhibits Proliferation, Changing Akt Activity and p53 Expression in Hypoxic NSCLC Cells.

The ribosomal protein (RP)-p53 pathway has been shown to play a key role in apoptosis and senescence of cancer cells. miR-1908 is a newly found miRNA that was reported to have prognostic potential in melanoma. However, its role and mechanism in the progression of non-small cell lung cancer (NSCLC) are largely unknown. In this study, we found that expression of miR-1908 was significantly downregulated in human NSCLC cell lines, including SK-MES-1, A549, and NCI-H460. Then the role of miR-1908 in NSCLC cell proliferation was explored. The miR-1908 mimic was transfected into NSCLC cell lines, and their proliferation was detected. MTT and Cell Titer-Blue H analyses showed that the cell proliferation was notably reduced by the miR-1908 mimic transfection. Moreover, we found the RP-p53 pathway was activated by miR-1908 mimic. Moreover, the miR-1908 inhibitor transfection had a completely opposite effect on the NSCLC cell proliferation than that of miR-1908 mimic. To explore the underlying mechanism of that, TargetScan bioinformatics server and 3'-UTR luciferase reporter assay were applied to identify the targets of miR-1908. Our results showed that AKT1 substrate 1 (AKT1S1), a newly proven suppressor of the RP-p53 pathway, was a target of miR-1908, suggesting a probable mechanism for miR-191 suppressing NSCLC cell proliferation. Our findings provide a novel molecular target for the regulation of NSCLC cell proliferation.

Downregulation of O-linked N-acetylglucosamine transferase by RNA interference decreases MMP9 expression in human esophageal cancer cells.

O-linked N-acetylglucosamine transferase (OGT) catalyzes O-linked glycosylation (O-GlcNAcylation). O-GlcNAcylation is a post-translational carbohydrate modification of diverse nuclear and cytosolic proteins by the addition of O-linked ß-N-acetylglucosamine. It was recently demonstrated that OGT and the level of O-GlcNAcylation are upregulated in esophageal cancer; however, the physiological consequences of this upregulation remain unknown. The current study reports that OGT knockdown by short hairpin RNA (shRNA) did not affect cell viability; however, cell migration in esophageal cancer Eca-109 cells was significantly reduced. OGT-specific shRNA vectors efficiently decreased the protein and mRNA levels of OGT and the RL2 level (a marker of O-GlcNAcylation levels) in Eca-109 esophageal cancer cells. In addition, colony formation and cell proliferation assays demonstrated that OGT-specific shRNA decreased the proliferation of Eca-109 cells; however, there was no significant statistical difference between OGT-specific shRNA and control shRNA. Notably, transwell assays demonstrated that the migratory ability of Eca-109 cells was significantly suppressed following knockdown of the OGT gene. Correspondingly, western blot analyses demonstrated that OGT knockdown significantly downregulated the expression of matrix metalloproteinase 9 (MMP9) in Eca-109 cells. These results suggest that OGT may promote the migration, invasion and metastasis of esophageal cancer cells by enhancing the stability or expression of MMP9.

Sequencing study on familial lung squamous cancer.

Lung cancer is the leading cause of cancer-related mortality worldwide. The majority of lung cancers are sporadic, and familial cases are extremely rare. Previous studies have mainly focused on sporadic lung cancer and identified a large quantity of driver genes. However, familial lung cancers are rarer and studied less. The present study recruited a Chinese family in which multiple members had developed lung squamous carcinoma. To find the causative mutations, whole exome sequencing was conducted using a peripheral blood sample of one lung squamous carcinoma patient, and certain variants were validated in more samples. Whole exome sequencing analysis obtained ~2.0 Gb of data (an average of 60x depth for each targeted base), and further validation experiments identified two functional variants in two cancer-related genes (c.1218delA:p.E406fs in PDE4DIP and C1342A:p.L448I in CLTCL1). This study therefore provides useful sources for the further study of hereditary lung cancer.