Digital Gene Expression Profiling Research Articles

Down-regulation of the insulin signaling pathway by SHC may correlate with congenital heart disease in Chinese populations.

Congenital heart disease (CHD) is one of the most common and severe congenital defects. The incidence of fetal cardiac malformation is increased in the context of maternal gestational diabetes mellitus (GDM). Therefore, we wanted to determine whether abnormalities in the insulin signaling pathway are associated with the occurrence of nonsyndromic CHD (ns-CHD). We used digital gene expression profiling (DGE) of right atrial myocardial tissue samples from eight ns-CHD patients and four controls. The genes potentially associated with CHD were validated by real-time fluorescence quantitative PCR analysis of right atrial myocardial tissues from 37 patients and 10 controls and the H9C2 cell line. The results showed that the insulin signaling pathway, which is mediated by the SHC gene family, was inhibited in the ns-CHD patients. The expression levels of five genes (PTPRF, SHC4, MAP2K2, MKNK2, and ELK1) in the pathway were significantly down-regulated in the patients' atrial tissues (P<0.05 for all). In vitro, the H9C2 cells cultured in high glucose (33 mmol/l) expressed less SHC4, MAP2K2, and Elk-1 than those cultured in low glucose (25 mmol/l). Furthermore, the high glucose concentration down-regulated the 25 genes associated with blood vessel development based on Gene Ontology (GO) term enrichment analyses of RNA-seq data. We considered that changes in the insulin signaling pathway mediated by SHC might be involved in the heart development process. This mechanism might account for the increase in the incidence of fetal cardiac malformations in the context of GDM.

Identification and differential expression analysis of anthocyanin biosynthetic genes in root-skin color variants of radish (Raphanus sativus L.).

Taproot skin color is a major trait for assessing the commercial and nutritional quality of radish, and red-skinned radish is confirmed to improve consumer's interest and health. However, little is known about the molecular mechanisms responsible for controlling the formation of red-skinned radish. This study aimed to identify the differentially expressed anthocyanin biosynthetic genes between red- and white-skinned radishes and understand the molecular regulatory mechanism underlying red-skinned radish formation. Based on the published complete genome sequence of radish, the digital gene expression profiles of Yangzhouyuanbai (YB, white-skinned) and Sading (SD, red-skinned) were analyzed using Illumina sequencing. A total of 3666 DEGs were identified in SD compared with YB. Interestingly, 46 genes encoded enzymes related to anthocyanin biosynthesis and 241 genes encoded transcription factors were identified. KEGG pathway analysis showed that the formation of red-skinned radish was mainly controlled by pelargonidin-derived anthocyanin biosynthetic pathway genes. This process included the upregulation of PAL, C4H, 4CL, CHS, CHI, F3H, DFR, LDOX, and UGT enzymes in SD. CHS genes were specifically expressed in SD, and it might be the key point for red pigment accumulation in red-skinned radish. Furthermore, MYB1/2/75, bHLH (TT8), and WD 40 showed higher expression in SD than in YB. Meanwhile, the corresponding low-abundance anthocyanin biosynthesis enzymes and upregulation of MYB4 might be the factors influencing the formation of white-skinned radish. These findings provide new insights into the molecular mechanisms and regulatory network of anthocyanin biosynthesis in red-skinned radish.

Exploration of targets regulated by miR-125b in porcine adipocytes.

MicroRNA (miRNA) has been proved to play a key role in lipid metabolism. In our previous study, miR-125b was validated to be differentially expressed in preadipocytes and adipocytes, which was also proved to involve in lipid metabolism. To explore the comprehensive targets of miR-125b in adipocytes, isobaric tag for relative and absolute quantitation (iTRAQ) analysis was performed to obtain differentially expressed proteins in adipocytes comparing negative control (NC) and miR-125b mimic, combining with digital gene expression (DGE) profiling of mRNA incorporated into RNA-induced silencing complex (RISC) pulled down by biotinylated miR-125b mimic and targets prediction of miR-125b by three algorithms, acyl-CoA dehydrogenase short chain (ACADS) and mitochondrial trans-2-enoyl-CoA reductase (MECR) were screened out as miR-125b direct targets. Luciferase reporter assay further validated that miR-125b mimic significantly inhibited the luciferase activity by targeting wild type (WT) 3'-UTR compared with NC. qPCR analysis of ACADS and MECR mRNA from adipose tissues of miR-125b knockout (KO) mice further confirmed the inhibition of miR-125b on ACADS and MECR expressions. Here we report miR-125b play a vital role in maintaining homeostasis of fatty acid metabolism by targeting key enzyme ACADS and MECR in the process of fatty acid elongation and degradation.

Genome-wide characterization of Rice Black Streaked Dwarf Virus-responsive genes in rice

Background: Rice black streaked dwarf virus (RBSDV) is an important pathogen disease in rice and gramineous planting regions all over the world. The major phenotypes of RBSDV infected rice were dwarf with dark green leaves, which generally resulted in seriously loss of yield. The RBSDV known so far is preoperatively transmitted to rice in a persistent manner by small brown planthopper (SBPH), instead of transmitting to offspring through ovary. Results: To identify RBSDV responsive genes and to explore and clarify the molecular mechanisms involved in plant-virus interaction, digital gene expression profile (DGE) analysis was performed by high-throughput sequencing using wild-type (WT) and RBSDV infected rice leaves (IRL) as materials. A total of 165,975 and 165,940 unique tags were obtained in IRL and WT libraries, respectively. In comparison with the control, 896 differentially expressed genes (DEGs) were obtained, of which 500 DEGs were up regulated and 396 DEGs were down regulated. Functional analysis showed that DEGs mainly classified into10 groups, including metabolism, stress pathogen and defense, signal transduction, Transporter, transcription and post-transcription, cell structure and division etc. To further validate reliability and authenticity of the data, 10 DEGs were randomly picked and Real-time RT-PCR was carried out, and the expression trends of 7 genes were in line with the RNA-seq results. By searching the RBSDV related miRNA database of rice, 10 targeted genes of 6 significantly changed miRNAs were also identified in these DEGs. Conclusions: The data derived from RNA-seq were valid and credible. Through this research, a series of candidate RBSDV-responsive genes were obtained, and special signal transduction and metabolism pathways were built and pulled out in rice. This study provided further insight into the molecular mechanisms during compatible and incompatible interactions between viruses and their host plants.

沙田柚自交不亲和花柱数字基因表达谱及相关基因分析

沙田柚自交不亲和花柱数字基因表达谱及相关基因分析

Over-Expression of Transferrin Receptor (TFRC/CD71) and Low Expression of Innate and Adaptive Immune Cell Subsets in HIV-Associated, GCB-DLBCL By Digital Gene Expression Profiling

Introduction: HIV infected individuals are 17x more likely to receive a diagnosis of Diffuse Large B cell Lymphoma (DLBCL) compared to their uninfected counterparts. Moreover, DLBCL is more aggressive in HIV infected individuals. However, the molecular pathology driving the aggressive nature of HIV related DLBCL is poorly understood. Previously, we demonstrated that HIV-associated [HIV(+)] germinal center B-cell (GCB) DLBCL tumors are more proliferative, with enhanced genomic stability and increased expression of DNA repair genes compared to their HIV-not associated [HIV(-)] counterparts (Maguire et al, Int J Cancer,2019). Given the immunocompromised nature of these patients, herein we assessed whether specific immunological signaling pathways are also altered in HIV(+) GCB DLBCL tumors, that may work in conjunction with, or independently of, increased DNA repair, to drive the enhanced aggressive nature of HIV related DLBCL. Methods: We used our previously studied cohort of patient samples, which included a total of 39 molecularly defined GCB DLBCL cases. Of these, 18 were HIV(+) cases from the AIDS Cancer Specimen Resource Network (https://acsr.ucsf.edu/) and 21 were HIV(-) institutional cases. Samples underwent hematopathologist review for diagnosis confirmation and tumor content assessment. Samples with &lt;60% tumor content were macrodissected, with a total of 4x 5µm FFPE sections per sample used for RNA/DNA extraction. Digital gene expression profiling was used to assess differential expression of 579 immune related genes using the NanoString PanCancer Immune Profiling panel. The data was normalized using nSolver, and differential expression analysis was performed using the R statistical software package NanoStringDiff. The resulting expression data were then analyzed using Gene Set Enrichment Analysis (GSEA), and GSEA Molecular Signatures Database (MSigDB) overlap analyses. Results: A total of 110 genes were differentially expressed between the HIV(+) and HIV(-) cohorts at p&lt;0.05. As expected due to immunosuppression in HIV(+) individuals, 100 genes (91%) were significantly reduced and 10 genes (9%) were significantly increased in the HIV(+) cohort compared to the HIV(-) cohort. GSEA and MSigDB analyses revealed that the genes with reduced expression in the HIV(+) cohort were associated with losses in both adaptive and innate immune signaling including cytokine interactions, the IL1 pathway and B-cell receptor signaling amongst others. MSigDB overlap analyses of the 10 genes upregulated in the HIV(+) cohort identified 3 Gene Ontology molecular functions, all of which were receptor signaling related; including Receptor Binding (CD8A, CD160, CCL8, C1QBP, MIF), Receptor Activity (CD8A, CD160, CCRL1, TFRC, KLRC2), and Cargo Receptor Activity (CCRL1, TFRC). Gene overlaps between the subsets include genes associated with NK and cytolytic T cells (CD8A, CD160), as well as the transferrin receptor TRFC/CD71, which with a fold increase of 3.9, was the most significantly increased gene in the HIV(+) cohort (p=0.002, FDR=0.0025). Conclusions: As expected, the results demonstrate a loss in both adaptive and innate immune signaling in the HIV(+) cohort compared to the HIV negative cohort as well as alterations in receptor signaling. In line with our previous observation that HIV(+) GCB DLBCL tumors have higher expression of proliferation genes and the Ki67 antigen than their HIV(-) counterparts, the results of this study also reveal overexpression of TRFC/CD71 mRNA in HIV(+) GCB DLBCL tumors compared to the HIV(-) cases. TFRC/CD71 is well known as a mediator of iron uptake in erythroid cells. Iron uptake through TFRC/CD71 is also necessary for, and positively regulates T and B cell proliferation; and is commonly expressed on aggressive B cell lymphomas. We suggest that TFRC/CD71 may play a role in the enhanced proliferative capabilities of HIV(+) GCB DLBCL. These effects may be mediated through the HIV protein Nef, which has known effects on TFRC/CD71 protein recycling in lymphoid populations (Madrid et al, J Biol Chem 2005), and has been shown to be transferred from HIV infected macrophages to uninfected B-cells via contact-dependent cellular conduits (Xu et al, Nat Immunol 2009). Nef transfer between cells has also been described to occur via microvesicle transfer and trogocytosis (reviewed in Ellwanger et al, Infect Genet Evol 2017). Further analysis of these pathways is ongoing. Disclosures Rimsza: NanoSting: Patents &amp; Royalties: Named inventor on a patent licensed to NanoSting [Institution].

Digital Immune Gene Expression Profiling Discriminates Allergic Rhinitis Responders from Non-Responders to Probiotic Supplementation.

Probiotic supplementation for eight weeks with a multi-strain probiotic by individuals with allergic rhinitis (AR) reduced overall symptom severity, the frequency of medication use and improved quality of life. The purported mechanism of action is modulation of the immune system. This analysis examined changes in systemic and mucosal immune gene expression in a subgroup of individuals, classified as either responders or non-responders based on improvement of AR symptoms in response to the probiotic supplement. Based on established criteria of a beneficial change in the mini-rhinoconjunctivitis quality of life questionnaire (mRQLQ), individuals with AR were classified as either responders or non-responders. Systemic and mucosal immune gene expression was assessed using nCounter PanCancer Immune Profiling (Nanostring Technologies, Seattle, WA, USA) kit on blood samples and a nasal lysate. There were 414 immune genes in the blood and 312 immune genes in the mucosal samples expressed above the limit of detection. Unsupervised hierarchical clustering of immune genes separated responders from non-responders in blood and mucosal samples at baseline and after supplementation, with key T-cell immune genes differentially expressed between the groups. Striking differences in biological processes and pathways were evident in nasal mucosa but not blood in responders compared to non-responders. These findings support the use of network approaches to understand probiotic-induced changes to the immune system.

Inflammatory responses to a pathogenic West Nile virus strain

BackgroundWest Nile virus (WNV) circulates across Australia and was referred to historically as Kunjin virus (WNVKUN). WNVKUN has been considered more benign than other WNV strains circulating globally. In 2011, a more virulent form of the virus emerged during an outbreak of equine arboviral disease in Australia.MethodsTo better understand the emergence of this virulent phenotype and the mechanism by which pathogenicity is manifested in its host, cells were infected with either the virulent strain (NSW2012), or less pathogenic historical isolates, and their innate immune responses compared by digital immune gene expression profiling. Two different cell systems were used: a neuroblastoma cell line (SK-N-SH cells) and neuronal cells derived from induced pluripotent stem cells (iPSCs).ResultsSignificant innate immune gene induction was observed in both systems. The NSW2012 isolate induced higher gene expression of two genes (IL-8 and CCL2) when compared with cells infected with less pathogenic isolates. Pathway analysis of induced inflammation-associated genes also indicated generally higher activation in infected NSW2012 cells. However, this differential response was not paralleled in the neuronal cultures.ConclusionNSW2012 may have unique genetic characteristics which contributed to the outbreak. The data herein is consistent with the possibility that the virulence of NSW2012 is underpinned by increased induction of inflammatory genes.

Identification of drought response genes by digital gene expression (DGE) analysis in Caragana korshinskii Kom.

Caragana korshinskii Kom. is a legume shrub that is widely distributed across desert habitats with gravely, sandy, and saline soils in Asia and Africa. C. korshinskii has highly developed roots and a strong tolerance to abiotic stress. At present, there are few genetic studies of C. korshinskii because of the limited availability of genomic resources. To understand the comprehensive mechanisms that are associated with drought tolerance, we used RNA-seq to survey the differentially expressed genes (DEGs) in comparisons of drought-treated and control plants. After analysing the sequencing results, we found 440 differentially expressed genes existing in drought-treated and control plants. Among the DEGs, 39 unigenes showed up-regulated expression after drought treatment, while 401 unigenes were down-regulated. We used the KEGG database to annotate these drought-induced genes; 126 unigenes were identified by KEGG pathway annotation, and approximately 28% of the unigenes with known function fell into categories related to fatty acid metabolism, starch, sucrose metabolism, and nitrogen metabolism, suggesting that these pathways or processes may be involved in the drought response. Finally, we confirmed that one gene has a potential function in drought tolerance.Our study is the first to provide transcriptomic resources for Caragana korshinskii and to determine its digital gene expression profile under conditions of drought stress using the assembled transcriptomic data for reference. These data provide a valuable resource for genetic and genomic studies of desert plants under abiotic stress conditions.

Digital gene expression profiling and validation study highlight Cyclin F as an important regulator for sperm motility of chickens

In poultry industry, around 5 to 12% roosters were eliminated from the breeding program because of low sperm motility. Relatively few studies have been directed toward understanding and explaining the genetics mechanisms involved in sperm motility regulation in chickens. In the present study, digital gene expression (DGE) profiling and bioinformation analysis were used to explore the globally differentially expressed genes (DEG) in the testis of low sperm motility and high sperm motility roosters. Further validation study of key candidate genes was also performed. The DGE identified 652 DEGs, including 473 up-regulated and 179 down-regulated genes in the low sperm motility testis. Those DEGs were enriched on 21 terms of biological process category, 10 terms of cellular component category, including motile cilium, and 13 terms of molecular function category including microtubule motor activity and ATP binding. The kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis indicated that these DEGs were involved in the FoxO signaling pathway and insulin resistance pathway. Quantitative real time PCR (qRT-PCR) studies of 8 DEGs were used to validate the DGE results. A key candidate gene Cyclin F (CCNF) was extremely low expressed in the low sperm motility testis (log2 ratio (low sperm motility/high sperm motility) = -5.23). The CCNF gene silencing in the chicken DF-1 cell line induced the reduced cell activity and proliferation. In summary, the present study provides insight into the potential genetic regulation of sperm motility and highlighted the underlying pathways (Insulin resistance and FoxO signaling pathways) and important candidate genes such as CCNF.

Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis.

The apoptosis that occurs in the immature testis under physiological conditions is necessary for male germ cell development, whereas improper activation of apoptosis can impair spermatogenesis and cause defects in reproduction. We previously demonstrated that in mice, the makorin-2 (Mkrn2) gene is expressed exclusively in the testis and its deletion leads to male infertility. To understand the potential molecular mechanism, in this study, we found that levels of apoptosis in the testis were abnormally high in the absence of Mkrn2. To identify specific gene(s) involved, we performed digital gene expression profiling (DGE) and pathway analysis via gene set enrichment analysis (GSEA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and we found that MKRN2 inhibits p53 apoptosis effector related to PMP22 (PERP) expression and that levels of the protein in sperm samples have an inverse correlation with infertility levels. GSEA additionally indicated that PERP is a negative regulator of spermatogenesis and that its ectopic expression induces male infertility. Further, Gene Expression Omnibus (GEO) dataset analysis showed that p53, upstream of PERP, was upregulated in oligoasthenoteratozoospermia (OAT). These observations suggest that Mkrn2 is crucial for protecting germ cells from excessive apoptosis and implicate Mkrn2-based suppression of the p53/PERP signaling pathway in spermatogenesis and male fertility.

The mechanism of damage in the midgut of Bombyx mori after chlorantraniliprole exposure.

Silkworm (Bombyx mori) is an economic insect of the Lepidoptera. Chlorantraniliprole (CAP) exposure results in reduced growth and development of B. mori and failure in cocooning, seriously affecting the development of sericulture. To study the mechanisms underlying the damage to silkworm caused by sublethal doses of CAP, we examined the oxidative damage, the activities of digestive enzymes in midgut, and the expressions of midgut-related genes at the mRNA level. We found that CAP exposure inhibited the growth of silkworm, decreased the body mass and caused the accumulation of reactive oxygen species (ROS) [the levels of O2-, H2O2 and lipid peroxidation (MDA) were increased by 1.62-, 1.87- and 1.46-fold, respectively]. Moreover, we also found that the midgut cells were disintegrated, microvilli disappeared, the stroma became thinner, and the chromatin of nucleus became aggregated after CAP exposure by the analysis of transmission electron microscopy (TEM). In addition, the activities of digestive enzymes were dysregulated in midgut (the activities of α-amylase and trypsin were decreased 0.69- and 0.20-fold, respectively). Furthermore, digital gene expression (DGE) profiling analysis revealed that the expressions of oxidative phosphorylation pathway and antioxidant defense system related genes in midgut were decreased, indicating that it was the oxidative damage in midgut caused by CAP that mainly affected the growth of silkworm, rather than the toxicological effects of CAP. Collectively, this study provided valuable insights into the toxic effects of CAP on insects.

Comparative transcriptome analyses reveal candidate genes regulating wood quality in Japanese larch (Larix kaempferi)

We studied the molecular mechanism of the quality traits of wood formation in larch. We used the immature latewood cells of two Japanese larch (Larix kaempferi) clones with significant differences in density and in microfibrillar angle (MFA) as materials to analyze their gene expression profiles. A total of 1735 differentially expressed genes were detected in immature latewood cells of the two clones, among which, 971 were up-regulated and 764 were down-regulated. Digital gene expression profiling analysis revealed that genes encoding transcription factor members NAC66 and R2R3-MYB4, microtubule-associated protein, actin-related protein, cell wall protein members, arabinogalactan protein, Fasciclin-like arabinogalactan protein and glycine-rich protein, and several cell-wall-synthesis genes affected wood density and MFA by regulating latewood formation at transcriptional level. Our study results represent a basis for selection of quality traits and genetic improvement of larch wood.

Transcriptomic responses of Caco-2 cells to Lactobacillus rhamnosus GG and Lactobacillus plantarum J26 against oxidative stress

Oxidative stress is the basic reason for aging and age-related diseases. In this study, we investigated the protective effect of 2 strains of lactic acid bacteria (LAB), Lactobacillus rhamnosus GG and L. plantarum J26, against oxidative stress in Caco-2 cells, and gave an overview of the mechanisms of lactic acid bacteria antioxidant activity using digital gene expression profiling. The 2 LAB strains provided significant protection against hydrogen peroxide (H2O2)-induced reduction in superoxide dismutase activity and increase in glutathione peroxidase activity in Caco-2 cells. However, inactive bacteria had little effect on alleviating oxidation stress in Caco-2 cells. Eight genes related to oxidative stress-FOSB, TNF, PPP1R15A, NUAK2, ATF3, TNFAIP3, EGR2, and FBN2-were significantly upregulated in H2O2-induced Caco-2 cells compared with untreated Caco-2 cells. After incubation of the H2O2-induced Caco-2 cells with L. rhamnosus GG and L. plantarum J26, 5 genes (TNF, EGR2, NUAK2, FBN2, and TNFAIP3) and 2 genes (NUAK2 and FBN2) were downregulated, respectively. In addition, the Kyoto Encyclopedia of Genes and Genomes indicated that some signaling pathways associated with inflammation, immune response, and apoptosis, such as Janus kinase/signal transducers and activators of transcription (Jak-STAT), mitogen-activated protein kinase (MAPK), nuclear factor-κB, and tumor necrosis factor, were all negatively modulated by the 2 strains, especially L. rhamnosus GG. In this paper, we reveal the mechanism of LAB in relieving oxidative stress and provide a theoretical basis for the rapid screening and evaluation of new LAB resources.

The transcriptomic responses of the ark shell, Anadara broughtonii, to sulfide and hypoxia exposure.

Sulfide and hypoxia threaten marine organisms in various ways. Anadara broughtonii, a commercial marine bivalve in China which has great potential exposure to sulfide and hypoxia, was selected to test the responses to these stresses. Digital gene expression profile (DGE) analysis was performed on the juveniles' gills after exposed to normal condition (CG group), hypoxia (LO group), and low/high concentration of sulfide (LS/HS group, administered in hypoxia), respectively, using RNA-seq technology. A total of over 30 million clean reads were filtered from each DGE library and over 90% of them were annotated successfully. In total, 774 significant differentially expressed genes (DEGs) were detected and assigned to Gene ontology (GO) classification and KEGG Pathway enrichment analysis. The results show that many of the upregulated DEGs are related to hemoglobin, immunology, and stress responding. In the stressed A. broughtonii, cytochrome P450 and phosphoenolpyruvate carboxykinase may stimulate the glycolysis process to reduce oxygen consumption; Aminoacyl-tRNA synthetases, heat shock protein and protein disulfide isomerase probably help to maintain the genome integrity; Baculoviral IAP repeat-containing protein 2/3, mitogen-activated protein kinase and tumor necrosis factor pathways were probably responsible for protein repair, proteolysis, apoptosis and immune responses to high concentration of sulfide. Combined challenges also induced alternative oxidase and sushi repeat-containing protein, which have indistinct but probably indispensable function in invertebrates. For the first time, comprehensive transcriptome information on A. broughtonii in response to sulfide and hypoxia were provided. Our research offers new insights into the molecular mechanism behind the resistance of shellfish to sulfide and hypoxia.

The protein elicitor Hrip1 enhances resistance to insects and early bolting and flowering in Arabidopsis thaliana.

The elicitor Hrip1 isolated from necrotrophic fungus Alternaria tenuissima, could induce systemic acquired resistance in tobacco to enhance resistance to tobacco mosaic virus. In the present study, we found that the transgenic lines of Hrip1-overexpression in wild type (WT) Arabidopsis thaliana were more resistant to Spodoptera exigua and were early bolting and flowering than the WT. A profiling of transcription assay using digital gene expression profiling was used for transgenic and WT Arabidopsis thaliana. Differentially expressed genes including 40 upregulated and three downregulated genes were identified. In transgenic lines of Hrip1-overexpression, three genes related to jasmonate (JA) biosynthesis were significantly upregulated, and the JA level was found to be higher than WT. Two GDSL family members (GLIP1 and GLIP4) and pathogen-related gene, which participated in pathogen defense action, were upregulated in the transgenic line of Hrip1-overexpression. Thus, Hrip1 is involved in affecting the flower bolting time and regulating endogenous JA biosynthesis and regulatory network to enhance resistance to insect.

Sodium new houttuyfonate suppresses metastasis in NSCLC cells through the Linc00668/miR-147a/slug axis

BackgroundAs most lung cancer patients present with invasive, metastatic disease, it is vital to investigate anti-metastatic treatments for non-small cell lung cancer (NSCLC). Houttuynia cordata is commonly used as a Chinese anticancer medicine in the clinic, and sodium new houttuyfonate (SNH), a main compound of this herb, has long been found to have antibiotic effects, although its anticancer effects have not been investigated. Here, we tried to address this lack of research from the perspective of the competing endogenous RNA (ceRNA) theory.MethodsThe effects of SNH on NSCLC cells were analysed with Cell Counting Kit-8 assays and colony formation assays. In addition, transwell assays and wound healing assays were used to determine the effects of SNH on migration and invasion in NSCLC cells. The levels of key genes and proteins were examined by quantitative real-time PCR, western blotting, immunofluorescence staining and IHC staining. Through transcriptome screening and digital gene expression profiling, Linc00668 was identified to be regulated by SNH. Dual-luciferase reporter assays and RNA immunoprecipitation assays verified the binding efficiency between miR-147a and Linc00668 or Slug.ResultsIn the present study, SNH regulated NSCLC cells in multiple ways, the most prominent of which was suppressing the expression of Linc00668, which was indicated to promote migration and invasion in NSCLC cells. Functional studies demonstrated that Linc00668 acted as a ceRNA by sponging miR-147a to further regulate Slug mRNA levels, thereby influencing the progression of the epithelial-mesenchymal transition. Consistently, the results of in vivo animal models showed that SNH depressed Linc00668 and suppressed the metastasis of NSCLC.ConclusionsSNH suppressed metastasis of NSCLC cells and the mechanism may involve with the Linc00668/miR-147a/Slug axis.

Screening and analysis on the differentially expression genes between diploid and autotetraploid watermelon by using of digital gene expression profile.

Synthetic polyploids are key breeding materials for watermelon. Compared with diploid watermelon, the tetraploid watermelon often exhibit wide phenotypic differences and differential gene expression. Digital gene expression (DGE) profile technique was performed in this study to present gene expression patterns in an autotetraploid and its progenitor diploid watermelon, and deferentially expressed genes (DEGs) related to the abiotic and biotic stress were also addressed. Altogether, 4,985 DEGs were obtained in the autotetraploid against its progenitor diploid, and 66.02% DEGs is up-regulated. GO analysis shows that these DEGs mainly distributed in 'metabolic process', 'cell' and 'catalytic activity'. KEGG analysis revealed that these DEGs mainly cover 'metabolic pathways', 'secondary metabolites' and 'ribosome'. Moreover, 134 tolerance related DEGs were identified which cover osmotic adjustment substance, protective enzymes/protein, signaling proteins and pathogenesis-related proteins. This study present the differential expression of stress related genes and global gene expression patterns at background level in autotetraploid watermelons. These new evidences could supplement the molecular theoretical basis for the better resistance after the genome doubling in the gourd family.

Gene expression profiling of ovary identified eggshell proteins regulated by 20-hydroxyecdysone in Bactrocera dorsalis

The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive pests worldwide. The frequent use of chemical insecticides has led B. dorsalis to develop resistance to many insecticides in recent decades. New high-throughput-sequenced transcriptomes, as well as genomes, have revealed a large number of reference genes for functional target identification. Here, we performed digital gene expression profiling of ovary and testis of B. dorsalis adults. Various genes were identified to be highly expressed in B. dorsalis ovary. The genes encoding components of eggshell, vitelline membrane proteins (Vmps) and chorion-related proteins, were identified to be tissue-specifically expressed in ovary. Five cytochrome P450 genes were also identified to be highly expressed in ovary. Three of them were ecdysone synthesis pathway genes indicating the ovary as a potential synthesis site of female. The up-regulated expression of Vmps by exogenous 20-hydroxyecdysone implied the hormonal regulation of eggshell formation during ovarian development. Many other genes with potential functions in ovarian development were also identified, including vitellogenin receptor, insulin receptor, NASP protein, and odorant binding protein. These findings should promote our understanding of the regulation of vitellogenesis and eggshell formation and enable exploration of potentially novel pest control targets.

Digital gene expression profiling in larvae of Tribolium castaneum at different periods post UV-B exposure

UV-B radiation is an important environmental factor. Exposure to excess UV-B radiation can cause serious effects on the development, survival, and reproduction of different organisms. Plants and animals have developed many different strategies to cope with UV-B-induced damage, but the physiological response of insects to UV-B remains unclear. In the present study, the red flour beetle Tribolium castaneum (Herbst) was used to assess the stress response of UV-B. The underlying molecular mechanisms were explored using RNA sequencing. We investigated the transcriptomic profile of T. castaneum larvae at 4 and 24 h after treatment with UV-B radiation via digital gene expression analysis. The 310 and 996 differentially expressed genes were detected at 4 and 24 h, respectively. Then the biological functions and associated metabolic processes of these genes were determined by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The reliability of the data was verified using qRT-PCR. The results indicated that several differentially expressed genes are involved in antioxidation, DNA repair, protein folding, carbon flux diversion, and the extracellular matrix to protect against UV-B-induced damage. This study will increase our understanding of the molecular mechanism underlying insect response to UV-B radiation.