Regulation of ubiquitination and antiviral activity of Cactin by deubiquitinase Usp14 in Drosophila.

Cactin, a highly conserved protein, plays a crucial role in various physiological processes in eukaryotes, including innate immunity. Recently, the function of Cactin in the innate immunity of Drosophila has been explored, revealing that Cactin regulates a non-canonical signaling pathway associated with the Toll and Imd pathways via the Cactin-Deaf1 axis. In addition, Cactin exhibits specific antiviral activity against the Drosophila C virus (DCV) in Drosophila, with an unknown mechanism. During DCV infection, it has been confirmed that the protein level and antiviral activity of Cactin are regulated by ubiquitination. However, the precise ubiquitination and deubiquitination mechanisms of Cactin in Drosophila remain unexplored. In this study, we identified ubiquitin-specific protease 14 (Usp14) as a major deubiquitinase for Cactin through comprehensive deubiquitinase screening. Our results demonstrate that Usp14 interacts with the C_Cactus domain of Cactin via its USP domain. Usp14 efficiently removes K48- and K63-linked polyubiquitin chains from Cactin, thereby preventing its degradation through the ubiquitin-proteasome pathway. Usp14 significantly inhibits DCV replication in Drosophila cells by stabilizing Cactin. Moreover, Usp14-deficient fruit flies exhibit increased susceptibility to DCV infection compared to wild-type flies. Collectively, our findings reveal the regulation of ubiquitination and antiviral activity of Cactin by the deubiquitinase Usp14, providing valuable insights into the modulation of Cactin-mediated antiviral activity in Drosophila.IMPORTANCEViral infections pose a severe threat to human health, marked by high pathogenicity and mortality rates. Innate antiviral pathways, such as Toll, Imd, and JAK-STAT, are generally conserved across insects and mammals. Recently, the multi-functionality of Cactin in innate immunity has been identified in Drosophila. In addition to regulating a non-canonical signaling pathway through the Cactin-Deaf1 axis, Cactin exhibits specialized antiviral activity against the Drosophila C virus (DCV) with an unknown mechanism. A previous study emphasized the significance of the Cactin level, regulated by the ubiquitin-proteasome pathway, in modulating antiviral signaling. However, the regulatory mechanisms governing Cactin remain unexplored. In this study, we demonstrate that Usp14 stabilizes Cactin by preventing its ubiquitination and subsequent degradation. Furthermore, Usp14 plays a crucial role in regulating the antiviral function mediated by Cactin. Therefore, our findings elucidate the regulatory mechanism of Cactin in Drosophila, offering a potential target for the prevention and treatment of viral infections.

Induced Self-Assembly of Vitamin E-Spermine/siRNA Nanocomplexes via Spermine/Helix Groove-Specific Interaction for Efficient siRNA Delivery and Antitumor Therapy.

Gene therapy has been one of potential strategies for the treatment of different diseases, where efficient and safe gene delivery systems are also extremely in need. Current lipid nanoparticles (LNP) technology highly depends on the packing and condensation of nucleic acids with amine moieties. Here, an attempt to covalently link two natural compounds, spermine and vitamin E, is made to develop self-assembled nucleic acid delivery systems. Among them, the spermine moieties specifically interact with the major groove of siRNA helix through salt bridge interaction, while vitamin E moieties are located around siRNA duplex. Such amphiphilic vitamin E-spermine/siRNA complexes can further self-assemble into nanocomplexes like multiblade wheels. Further studies indicate that these siRNA nanocomplexes with the neutrally charged surface of vitamin E can enter cells via caveolin/lipid raft mediated endocytosis pathway and bypass lysosome trapping. With these self-assembled delivery systems, efficient siRNA delivery is successfully achieved for Eg5 and Survivin gene silencing as well as DNA plasmid delivery. Further in vivo study indicates that VE-Su-Sper/DSPE-PEG2000/siSurvivin self-assembled nanocomplexes can accumulate in cancer cells and gradually release siRNA in tumor tissues and show significant antitumor effect in vivo. The self-assembled delivery system provides a novel strategy for highly efficient siRNA delivery.

Chromosome-level genome assembly and population genomic analysis provide novel insights into the immunity and evolution of Sogatella furcifera.

Sogatella furcifera is a destructive agricultural pest causing large threats to rice production in China and Southeast Asian countries. Despite recent breakthroughs in long-read sequencing, high quality genomic data are very limited in S. furcifera. In present study, a chromosome-level assembly of the S. furcifera genome was completed (0.64 GB), comprising 15 chromosomes covered 95.04% of the estimated genome size, along with other 624 small scaffolds making up the remaining 4.96% of the genome of S. furcifera. A total of 24,669 protein-coding genes, 1211 long noncoding RNA and 7595 circular RNA transcripts were predicted in this study. Comparative genomic analysis revealed rapidly evolved genes were associated with multiple immune-related pathways in S. furcifera. Genome resequencing of 44 individuals from 12 geographic populations revealed frequent gene flow among populations. The systemic genomic analysis will provide more insights into the understanding of the immunity and evolutionary adaptation of S. furcifera.

Praeruptorin A inhibits the activation of NF-κB pathway and the expressions of inflammatory factors in poly (I:C)-induced RAW264.7 cells.

Praeruptorin A (PA), a natural coumarin compound, has significant anti-inflammatory effects. In this study, we evaluate the anti-inflammatory effect of PA on RAW 264.7 mouse macrophages induced by Polyinosinic acid-polycytidylic acid (poly (I:C)). RAW 264.7 mouse macrophages induced by poly (I:C) were treated with or without PA, the viability of which was determined to screen working solution of PA. RNA-sequencing was applied to analyze the differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were carried out. The expressions of interleukin (IL)-1ß, heme oxygenase 1 (HMOX1), prostaglandin-endoperoxide synthase 2 (PTGS2), ATP binding cassette subfamily A member 1 (Abca1) and NF-κB-related proteins were measured by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. As a result, PA at 1, 2, 3, 4 and 5 µM slightly affected cell viability, while PA at 6 and 7 µM significantly inhibited cell viability. GO and KEGG analysis results revealed that DEGs were mainly enriched in the pathways related to inflammatory signaling. Through further analysis, we obtained five possible targets of PA, and verified that PA inhibited the expressions of IL-1ß, HMOX1, PTGS2 and Abca1 as well as the activation of NF-κB pathway in poly (I:C)-induced RAW264.7 cells. To summarize, PA may inhibit expressions of the inflammation-related genes in poly (I:C)-induced RAW264.7 cells, which demonstrates its potential as a drug against virus related diseases.

MAPK4 silencing in gastric cancer drives liver metastasis by positive feedback between cancer cells and macrophages.

Liver metastasis is a major cause of death in gastric cancer patients, but the underlying mechanisms are poorly understood. Through a combination of in vivo screening and transcriptome profiling followed by quantitative RT-PCR and tissue array analyses, we found that mitogen-activated protein kinase 4 (MAPK4) downregulation in gastric cancer tissues from patients is significantly associated with liver metastasis and poor prognosis. The knockdown of MAPK4 in gastric cancer cells promotes liver metastasis in orthotopic mouse models. MAPK4 depletion in gastric cancer cells induces the secretion of macrophage migration inhibitory factor (MIF) to polarize tumor-associated macrophages (TAMs) in orthotopic xenograft tumors. Moreover, TAMs activate epithelial-mesenchymal transition of gastric cancer cells to suppress MAPK4 expression, which further increases MIF secretion to polarize TAMs. Taken together, our results suggest a previously undescribed positive feedback loop between cancer cells and macrophages mediated by MAPK4 silencing that facilitates gastric cancer liver metastasis.

Deciphering the Genetic Basis of Silkworm Cocoon Colors Provides New Insights into Biological Coloration and Phenotypic Diversification.

The genetic basis of phenotypic variation is a long-standing concern of evolutionary biology. Coloration has proven to be a visual, easily quantifiable, and highly tractable system for genetic analysis and is an ever-evolving focus of biological research. Compared with the homogenized brown-yellow cocoons of wild silkworms, the cocoons of domestic silkworms are spectacularly diverse in color, such as white, green, and yellow-red; this provides an outstanding model for exploring the phenotypic diversification and biological coloration. Herein, the molecular mechanism underlying silkworm green cocoon formation was investigated, which was not fully understood. We demonstrated that five of the seven members of a sugar transporter gene cluster were specifically duplicated in the Bombycidae and evolved new spatial expression patterns predominantly expressed in silk glands, accompanying complementary temporal expression; they synergistically facilitate the uptake of flavonoids, thus determining the green cocoon. Subsequently, polymorphic cocoon coloring landscape involving multiple loci and the evolution of cocoon color from wild to domestic silkworms were analyzed based on the pan-genome sequencing data. It was found that cocoon coloration involved epistatic interaction between loci; all the identified cocoon color-related loci existed in wild silkworms; the genetic segregation, recombination, and variation of these loci shaped the multicolored cocoons of domestic silkworms. This study revealed a new mechanism for flavonoids-based biological coloration that highlights the crucial role of gene duplication followed by functional diversification in acquiring new genetic functions; furthermore, the results in this work provide insight into phenotypic innovation during domestication.

Layout Comparison and Parameter Optimization of Supercritical Carbon Dioxide Coal-Fired Power Generation Systems under Environmental and Economic Objectives.

In the current studies, the supercritical carbon dioxide coal-fired power generation systems show efficiency and cost advantages over the traditional steam-based power systems. However, few studies have considered simultaneously environmental and economic objectives in the multi-objective analysis process. This study conducts a layout comparison and parameter optimization of the systems under the above two objectives. Initially, the thermodynamic, environmental, and economic models of the systems are established. Subsequently, the optimal layout is determined by the two-stage layout comparison. Further, multi-objective optimization is performed for the selected layout, and the optimal design parameters are determined by the decision process. Finally, the sensitivities of three selected parameters to the optimization results are analyzed. The results show that the basic layout coupled with overlap and intercooling schemes is optimal. Its ultimate environmental impact (UEI) and levelized cost of electricity (LCOE) are 219.8 kp-eq and 56.9 USD/MWh, respectively. The two objectives UEI and LCOE are conflicting. Based on a trade-off between them, the maximum temperature/pressure of the system is determined to be 635.3 °C/30.1 MPa. The coal price per unit of heat shows the highest sensitivity, and the pinch temperature difference of the recuperator shows opposite sensitivities at the UEI below 218 kp-eq and above 223 kp-eq.

Dimethyl itaconate alleviates the pyroptosis of macrophages through oxidative stress.

Macrophages are involved in the pathophysiology of many diseases as critical cells of the innate immune system. Pyroptosis is a form of macrophage death that induces cytokinesis of phagocytic substances in the macrophages, thereby defending against infection. Dimethyl itaconate (DI) is an analog of itaconic acid with anti-inflammatory effects. However, the effect of dimethyl itaconate on macrophage pyroptosis has not been elucidated clearly. Thus, the present study aimed to analyze the effect of DI treatment on a macrophage pyroptosis model (Lipopolysaccharide, LPS + Adenosine Triphosphate, ATP). The results showed that 0.25 mM DI ameliorated macrophage pyroptosis and downregulated interleukin (IL)-1ß expression. Then, real-time quantitative polymerase chain reaction (RT-qPCR) was used to confirm the result of RNA-sequencing of the upregulated oxidative stress-related genes (Gclc and Gss) and downregulated inflammation-related genes (IL-12ß and IL-1ß). In addition, Gene Ontology (GO) enrichment analysis showed that differential genes were associated with transcript levels and DNA replication. Kyoto encyclopedia of genes and genomes (KEGG) enrichment showed that signaling pathways, such as tumor necrosis factor (TNF), Jak, Toll-like receptor and IL-17, were altered after DI treatment. N-acetyl-L-cysteine (NAC) reversed the DI effect on the LPS + ATP-induced macrophage pyroptosis and upregulated the IL-1ß expression. Oxidative stress-related protein Nrf2 is involved in the DI regulation of macrophage pyroptosis. Taken together, these findings suggested that DI alleviates the pyroptosis of macrophages through oxidative stress.

Prediction of Mental Health in Medical Workers During COVID-19 Based on Machine Learning.

Mental health prediction is one of the most essential parts of reducing the probability of serious mental illness. Meanwhile, mental health prediction can provide a theoretical basis for public health department to work out psychological intervention plans for medical workers. The purpose of this paper is to predict mental health of medical workers based on machine learning by 32 factors. We collected the 32 factors of 5,108 Chinese medical workers through questionnaire survey, and the results of Self-reporting Inventory was applied to characterize mental health. In this study, we propose a novel prediction model based on optimization algorithm and neural network, which can select and rank the most important factors that affect mental health of medical workers. Besides, we use stepwise logistic regression, binary bat algorithm, hybrid improved dragonfly algorithm and the proposed prediction model to predict mental health of medical workers. The results show that the prediction accuracy of the proposed model is 92.55%, which is better than the existing algorithms. This method can be used to predict mental health of global medical worker. In addition, the method proposed in this paper can also play a role in the appropriate work plan for medical worker.

Combining gene expression signature with clinical features for survival stratification of gastric cancer.

The AJCC staging system is considered as the golden standard in clinical practice. However, it remains some pitfalls in assessing the prognosis of gastric cancer (GC) patients with similar clinicopathological characteristics. We aim to develop a new clinic and genetic risk score (CGRS) to improve the prognosis prediction of GC patients. We established genetic risk score (GRS) based on nine-gene signature including APOD, CCDC92, CYS1, GSDME, ST8SIA5, STARD3NL, TIMEM245, TSPYL5, and VAT1 based on the gene expression profiles of the training set from the Asian Cancer Research Group (ACRG) cohort by LASSO-Cox regression algorithms. CGRS was established by integrating GRS with clinical risk score (CRS) derived from Surveillance, Epidemiology, and End Results (SEER) database. GRS and CGRS dichotomized GC patients into high and low risk groups with significantly different prognosis in four independent cohorts with different data types, such as microarray, RNA sequencing and qRT-PCR (all HR > 1, all P < 0.001). Both GRS and CGRS were prognostic signatures independent of the AJCC staging system. Receiver operating characteristic (ROC) analysis showed that area under ROC curve of CGRS was larger than that of the AJCC staging system in most cohorts we studied. Nomogram and web tool (http://39.100.117.92/CGRS/) based on CGRS were developed for clinicians to conveniently assess GC prognosis in clinical practice. CGRS integrating genetic signature with clinical features shows strong robustness in predicting GC prognosis, and can be easily applied in clinical practice through the web application.

Macrophages enhance lipopolysaccharide induced apoptosis via Ang1 and NF-κB pathways in human umbilical vein endothelial cells.

Lipopolysaccharide (LPS) could induce apoptosis and dysfunction of endothelial cells. We aimed to reveal the effects of macrophages on cell proliferation and apoptosis in LPS induced human umbilical vein endothelial cells (HUVECs). THP-1 derived macrophages and HUVECs were co-cultured in the presence of LPS. Cell viability was measured by Cell Counting Kit-8 and apoptosis was analyzed by flow cytometry. Expression of Ang1, the NF-κB component p65 was evaluated by western blot and quantitative PCR. Small interfering RNAs (siRNAs) were used to knockdown the expression of proinflammatory cytokines and p65 in HUVECs. Plasmid transfection-mediated overexpression of Ang1 was employed to see its effects on cell proliferation and apoptosis in HUVECs. Macrophages enhanced LPS-induced proliferation impairments and apoptosis in HUVECs, which could be attenuated by siRNA-mediated knockdown of cytokines TNF-α, IL-1ß, IL-6 and IL-12p70 in macrophages. The dysfunction of HUVECs was tightly associated with reduced Ang1 expression and increased phosphorylated p65 (p-65). Overexpression of Ang1 in HUVECs significantly decreased p-p65, suggesting negatively regulation of p-p65 by Ang1. Overexpression of Ang1, adding recombinant Ang1 or silencing of p65 substantially attenuated the dysfunction of HUVECs in terms of cell proliferation and apoptosis. In conclusions, THP-1-derived macrophages enhance LPS induced dysfunction of HUVECs via Ang1 and NF-κB pathways, suggesting new therapeutic targets for sepsis.

[Paeoniflorin improves the permeability of cardiac microvascular endothelial cells by regulating Src/vascular endothelial-cadherin pathway].

OBJECTIVE: To investigate the effect and mechanism of paeoniflorin on the permeability of cardiac microvascular endothelial cells (CMECs) in sepsis. METHODS: Primary rat CMECs were isolated and cultured in vitro, and the cells in the logarithmic growth phase were used for experiments. Tetramethylazozolium colorimetry (MTT) was used to screen the safe and effective concentrations of paeoniflorin at 10, 20, and 40 µmol/L. The cells were divided into blank control group, lipopolysaccharide (LPS) group and low, medium and high concentration paeoniflorin pretreatment group. The cells in the blank control group were cultured in complete medium; the cells in the LPS group were challenged with LPS (1 mg/L) in complete medium; and the cells in the paeoniflorin pretreatment groups were pretreated with 10, 20, and 40 µmol/L paeoniflorin at 4 hours before LPS stimulation. The cells in each group were further cultured for 24 hours after LPS stimulation. The horseradish peroxidase (HRP) method was used to detect the permeability of rat CMECs. The enzyme-linked immunosorbent assay (ELISA) was used to detect the CXC chemokine ligand (CXCL1, CXCL2) levels in the cell supernatant. The real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of CXCL1 and CXCL2 in the cells. Western Blot was used to detect phosphorylated Src (p-Src), vascular endothelial-cadherin (VE-cadherin) and phosphorylated mitogen activated protein kinase (p-MAPK). RESULTS: Compared with the blank control group, the permeability of rat CMECs in the LPS group was significantly increased. The cell permeability was improved to some extent after paeoniflorin pretreatment at different concentrations, and the improvement was most obvious in the 40 µmol/L paeoniflorin group, with statistically significant difference as compared with the LPS group (A value: 1.61±0.07 vs. 2.13±0.06, P < 0.01). ELISA results showed that there were moderate amounts of CXCL1 and CXCL2 in the cell supernatant of rat CMECs in the blank control group. However, the secretion of CXCL1 and CXCL2 in the cell supernatant was increased significantly under the induction of LPS. After pretreatment with paeoniflorin at different concentrations, the secretion of CXCL1 and CXCL2 in the cell supernatant was significantly reduced. The most obvious inhibitory effect on CXCL1 was 40 µmol/L paeoniflorin, and the most obvious inhibition on CXCL2 was 20 µmol/L paeoniflorin, the differences were statistically significant as compared with the LPS group [CXCL1 (ng/L): 337.51±68.04 vs. 829.86±65.06, CXCL2 (ng/L): 4.48±0.11 vs. 9.41±0.70, both P < 0.01]. RT-qPCR results showed that the mRNA expressions of CXCL1 and CXCL2 in the rat CMECs were consistent with the ELISA results. LPS could increase mRNA expressions of CXCL1 and CXCL2 in the rat CMECs, and pretreatment with different concentrations of paeoniflorin could significantly reduce the mRNA expressions of CXCL1 and CXCL2. The 40 µmol/L paeoniflorin had the best inhibitory effect on CXCL1 mRNA expression, and the 20 µmol/L paeoniflorin had the best inhibitory effect on CXCL2 mRNA expression, the differences were statistically significant as compared with the LPS group [CXCL1 mRNA (2-ΔΔCt): 0.543±0.004 vs. 0.812±0.089, CXCL2 mRNA (2-ΔΔCt): 10.52±0.71 vs. 17.68±1.09, both P < 0.01]. Western Blot results showed that moderate amounts of p-Src, VE-cadherin and p-MAPK proteins were expressed in the rat CMECs in the blank control group. After LPS stimulation, the expressions of p-Src and p-MAPK proteins were increased significantly, while the expression of VE-cadherin protein was decreased significantly. After pretreatment with different concentrations of paeoniflorin, the expressions of p-Src and p-MAPK proteins in the cells were decreased to varying degrees, while the expression of VE-cadherin protein was increased, and 40 µmol/L paeoniflorin had the most obvious effect, the differences were statistically significant as compared with the LPS group [p-Src protein (p-Src/GAPDH): 1.02±0.09 vs. 1.29±0.05, p-MAPK proteins (p-MAPK/GAPDH): 0.24±0.02 vs. 0.62±0.02, VE-cadherin protein (VE-cadherin/GAPDH): 0.64±0.03 vs. 0.31±0.02, all P < 0.01]. CONCLUSIONS: Paeoniflorin can regulate the Src/VE-cadherin pathway in CMECs, inhibit the expression and secretion of inflammation-related proteins and chemokines, and improve the cell permeability of CMECs induced by LPS.

Upregulation of BCAM and its sense lncRNA BAN are associated with gastric cancer metastasis and poor prognosis.

Patients with metastatic gastric cancer (GC) have a poor prognosis; however, the molecular mechanism of GC metastasis remains unclear. Here, we employed bioinformatics to systematically screen the metastasis-associated genes and found that the levels of basal cell adhesion molecule (BCAM) were significantly increased in GC tissues from patients with metastasis, as compared to those without metastasis. The upregulation of BCAM was also significantly associated with a shorter survival time. Depletion of BCAM inhibited GC cell migration and invasion. Knockout (KO) of BCAM by the CRISPR/Cas9 system reduced the invasion and metastasis of GC cells. To explore the mechanism of BCAM upregulation, we identified a previously uncharacterized BCAM sense lncRNA that spanned from exon 6 to intron 6 of BCAM, and named it as BCAM-associated long noncoding RNA (BAN). Knockdown of BAN inhibited BCAM expression at both mRNA and protein levels. Knockdown of BAN suppressed GC cell migration and invasion, which was effectively rescued by ectopic expression of BCAM. Further clinical data showed that BAN upregulation was associated with GC metastasis and poor prognosis. Importantly, BAN expression was also significantly associated with that of BCAM in GC tissues. Taken together, these results indicate that increased expression of BCAM and its sense lncRNA BAN promote GC cell invasion and metastasis, and are associated with poor prognosis of GC patients.

lncRNA Sensing of a Viral Suppressor of RNAi Activates Non-canonical Innate Immune Signaling in Drosophila.

Antiviral immunity in insects is mediated by the RNA interference (RNAi) pathway. Viruses evade antiviral RNAi by expressing virulence factors known as viral suppressors of RNAi (VSR). Here, we report the identification of VINR, a Drosophila VSR-interacting long non-coding (lnc) RNA that activates non-canonical innate immune signaling upon detection of the dsRNA-binding VSR of Drosophila C virus (DCV). VINR is required for the induction of antimicrobial peptide (AMP) genes but dispensable for antiviral RNAi. VINR functions by preventing the ubiquitin proteasome-dependent degradation of Cactin, a coiled-coil and arginine-serine-rich domain-containing protein that regulates a non-cannonical antimicrobial pathway for AMP induction. CRISPR-Cas9 knockout of VINR in Drosophila cells enhances DCV replication independently of antiviral RNAi, and VINR-knockout adult flies exhibit enhanced disease susceptibility to DCV and bacteria. Our findings reveal a counter counter-defense strategy activated by a lncRNA in response to the viral suppression of the primary antiviral RNAi immunity.

[Research progress of interleukin-33 and its receptor ST2 in sepsis].

Sepsis is a life-threatening organ dysfunction caused by an imbalance in the response to infection. Clinically the effects of anti-infection and fluid resuscitation are limited, and the morbidity and mortality of sepsis are still high. Interleukin-33 (IL-33), a member of the IL-1 family, binds to various cell types through the ST2-IL-1 receptor helper protein complex. IL-33 and its receptor ST2 play an important role as immune regulatory factors in sepsis. This article reviews the pathophysiological characteristics of sepsis, the biological characteristics of IL-33 and its receptor ST2, and the relationship between IL-33/ST2 and sepsis, so as to provide new ideas for the diagnosis and treatment of sepsis.

Genome-wide characterization and developmental expression profiling of long non-coding RNAs in Sogatella furcifera.

The genome-wide characterization of long non-coding RNA (lncRNA) in insects demonstrates their importance in fundamental biological processes. Essentially, an in-depth understanding of the functional repertoire of lncRNA in insects is pivotal to insect resources utilization and sustainable pest control. Using a custom bioinformatics pipeline, we identified 1861 lncRNAs encoded by 1852 loci in the Sogatella furcifera genome. We profiled lncRNA expression in different developmental stages and observed that the expression of lncRNAs is more highly temporally restricted compared to protein-coding genes. More up-regulated Sogatella furcifera lncRNA expressed in the embryo, 4th and 5th instars, suggesting that increased lncRNA levels may play a role in these developmental stages. We compared the relationship between the expression of Sogatella furcifera lncRNA and its nearest protein gene and found that lncRNAs were more correlated to their downstream coding neighbors on the opposite strand. Our genome-wide profiling of lncRNAs in Sogatella furcifera identifies exciting candidates for characterization of lncRNAs, and also provides information on lncRNA regulation during insect development.

Identification of genes underlying phenotypic plasticity of wing size via insulin signaling pathway by network-based analysis in Sogatella furcifera.

BACKGROUND: Phenotypic plasticity is a common and highly adaptive phenomenon where the same genotype produces different phenotypes in response to environmental cues. Sogatella furcifera, a migratory pest of rice exhibits wing dimorphism, is a model insect for studying phenotypic plasticity of wing size. The Insullin-PI3K-Akt-FOXO signaling pathway plays a crucial role in the manipulation of wing size in the migratory insects. However, the regulatory mechanism via the pathway involved in wing dimorphism are still unexplored. RESULTS: Accompanied by special alternative splicing, genes involved in muscle contraction and energy metabolism were highly expressed in the wing hinges of macropters, demonstrating their adaptation for energy-demanding long-distance flights. Based on FOXO ChIP-Seq analysis, a total of 1259 putative target genes were observed in the wing hinges, including wing morph development, flight muscle and energy metabolism genes. An integrated gene interaction network was built by combining four heterogeneous datasets, and the IIS-PI3K-Akt-FOXO pathway was clustered in a divided functional module. In total, 45 genes in the module directly interacting with the IIS-PI3K-Akt-FOXO pathway showed differential expression levels between the two wing hinges, thus are regarded as potential Insulin pathway mediated wing dimorphism related genes (IWDRGs). Of the 45 IWDRGs, 5 were selected for verification by gene knockdown experiments, and played significant roles in the insect wing size regulation. CONCLUSIONS: We provided valuable insights on the genetic basis of wing dimorphism, and also demonstrated that network analysis is a powerful approach to identify new genes regulating wing dimorphic development via insulin signaling pathway in the migratory insect.

KLF9 suppresses gastric cancer cell invasion and metastasis through transcriptional inhibition of MMP28.

Gastric cancer (GC) is among the most lethal human malignancies, and the leading cause of GC mortality is metastasis. However, the precise mechanism of GC metastasis remains unclear. To screen key transcriptional factors (TFs) involved in GC metastasis, we performed bioinformatics analysis of The Cancer Genome Atlas database and found that Krüppel-like factor 9 (KLF9) is a GC metastasis-associated TF. KLF9 is significantly decreased in patients with GC with distant metastasis compared with those patients without distant metastasis. Ectopic expression of KLF9 evidently inhibited the migration and invasion capabilities of GC cells. Conversely, knockdown of KLF9 endowed GC cells with stronger invasive capacity. Moreover, tail intravenous injection confirmed that KLF9 strongly inhibits the lung metastasis process of GC in vivo. Mechanistically, chromatin immunoprecipitation coupled with high-throughput sequencing data from Encyclopedia of DNA Elements revealed that KLF9 specifically binds to the promoter region of matrix metalloproteinase (MMP)28. Further quantitative real-time PCR and dual-luciferase assay indicated that KLF9 directly inhibited MMP28 transcription. Importantly, decreased invasion and metastasis capability of GC cells caused by ectopic KLF9 expression could be rescued via reinforcing MMP28 expression in vivo. Collectively, our study indicates that KLF9 significantly suppresses GC cell invasion and metastasis through inhibiting MMP28 transcription.-Li, Y., Sun, Q., Jiang, M., Li, S., Zhang, J., Xu, Z., Guo, D., Gu, T., Wang, B., Xiao, L., Zhou, T., Zhuo, W. KLF9 suppresses gastric cancer cell invasion and metastasis through transcriptional inhibition of MMP28.

Long Noncoding RNA GMAN, Up-regulated in Gastric Cancer Tissues, Is Associated With Metastasis in Patients and Promotes Translation of Ephrin A1 by Competitively Binding GMAN-AS.

BACKGROUND & AIMS: We aimed to identify long noncoding RNAs (lncRNAs) that are up-regulated in gastric cancer tissues from patients and study their function in gastric tumor metastasis. METHODS: We collected gastric tumor and nontumor tissues from patients in China and analyzed levels of lncRNAs by microarray analysis, proteins by immunohistochemistry, and RNAs by quantitative reverse-transcription polymerase chain reaction; we compared these with survival times of patients and tumor progression. RNA levels were knocked down or knocked out in BGC-823, SGC-7901, and MKN45 cell lines using small interfering or short hairpin RNAs or clustered regularly interspaced short palindromic repeats (ie, CRISPR)/CRISPR associated protein 9 (ie, Cas9) vectors. Genes were overexpressed from transfected plasmids in HGC-27 cells. Cells were analyzed by Northern blot and immunoblot, polysome profiling assay, and cell invasion assay. Cells were injected into the tail veins or spleens of nude mice or SCID mice; lung and liver tissues were collected, and metastases were counted. lncRNAs were cloned by using rapid amplification of complementary DNA ends. Their interactions with other genes were determined by RNA pulldown and mapping assays. RESULTS: In microarray analyses, we identified 151 lncRNAs expressed at significantly higher levels in gastric tumor vs nontumor tissues. Levels of an lncRNA that we called gastric cancer metastasis associated long noncoding RNA (GMAN) were increased in gastric tumor tissues, compared with nontumor tissues; its up-regulation was associated with tumor metastasis and shorter survival times of patients. The GMAN gene overlaps with the ephrin A1 gene (EFNA1) and was highly expressed in BGC-823 and MKN45 cells. Knockdown of GMAN in these cells did not affect proliferation, colony formation, or adhesion but did reduce their invasive activity in Transwell assays. Ectopic expression of GMAN increased the invasive activity of HGC-27 cells. BGC-823 and MKN45 cells with knockdown of GMAN formed fewer metastases after injection into tail veins of nude mice. Knockdown or knockout of GMAN also reduced levels of ephrin A1 protein in cells. We found that GMAN promoted translation of ephrin A1 messenger RNA into protein by binding to the antisense GMAN RNA (GMAN-AS)-this antisense sequence is also complementary to that of ephrin A1 mRNA. Levels of ephrin A1 protein were also increased in gastric tumors from patients with metastases than in those without metastases. Knockout of ephrin A1 in BGC-823 cells reduced their invasive activity in Transwell assays and ability to form metastases after injection into SCID mice. Ectopic expression of ephrin A1 in BGC-823 cells with knockdown or knockout of GMAN restored their invasive activities and ability form metastases in nude or SCID mice. A CRISPR/Cas9-based strategy to disrupt the GMAN gene significantly reduced the numbers of metastases formed from SGC-7901 cells in mice. CONCLUSIONS: We identified an lncRNA, which we call GMAN, that is increased in gastric tumors from patients and associated with survival and formation of metastases. It regulates translation of ephrin A1 mRNA by binding competitively to GMAN-AS. Knockdown or knockout of GMAN or ephrin A1 in gastric cancer cell lines reduces their invasive activity and ability to form metastases after injection into mice. These genes might be targeted to prevent or reduce gastric cancer metastasis.