The role of miR-2∼13∼71 cluster in resistance to deltamethrin in Culex pipiens pallens

Excessive and continuous application of deltamethrin has resulted in the development of deltamethrin resistance among mosquitoes, which becomes a major obstacle for mosquito control. In a previous study, differentially expressed miRNAs between deltamethrin-susceptible (DS) strain and deltamethrin-resistant (DR) strain using illumina sequencing in Culex pipiens pallens were identified. In this study, we applied RNAi and the Centers for Disease Control and Prevention (CDC) bottle bioassay to investigate the relationship between miR-2∼13∼71 cluster (miR-2, miR-13 and miR-71) and deltamethrin resistance. We used quantitative real-time PCR (qRT-PCR) to measure expression levels of miR-2∼13∼71 clusters. MiR-2∼13∼71 cluster was down regulated in adult female mosquitoes from the DR strain and played important roles in deltamethrin resistance through regulating target genes, CYP9J35 and CYP325BG3. Knocking down CYP9J35 and CYP325BG3 resulted in decreased mortality of DR mosquitoes. This study provides the first evidence that miRNA clusters are associated with deltamethrin resistance in mosquitoes. Moreover, we investigated the regulatory networks formed between miR-2∼13∼71 cluster and its target genes, which provide a better understanding of the mechanism involved in deltamethrin resistance.     

Downregulation of microRNA-451 in non-alcoholic steatohepatitis inhibits fatty acid-induced proinflammatory cytokine production through the AMPK/AKT pathway

Mechanisms associated with the progression of non-alcoholic fatty liver disease (NAFLD) remain unclear. We attempted to identify the pattern of altered gene expression at different time points in a high fat diet (HFD)-induced NAFLD mouse model. The early up-regulated genes are mainly involved in the innate immune responses, while the late up-regulated genes represent the inflammation processes. Although recent studies have shown that microRNAs play important roles in hepatic metabolic functions, the pivotal role of microRNAs in the progression of NAFLD is not fully understood. We investigated the functions of miR-451, which was identified as a target gene in the inflammatory process in NAFLD. miR-451 expression was significantly decreased in the palmitate (PA)-exposed HepG2 cells and in liver tissues of HFD-induced non-alcoholic steatohepatitis (NASH) mice. Its decreased expressions were also observed in liver specimens of NASH patients. In vitro analysis of the effect of miR-451 on proinflammatory cytokine provided evidence for negative regulation of PA-induced interleukin (IL)-8 and tumor necrosis factor-alpha (TNF-α) production. Furthermore, miR-451 over-expression inhibited translocation of the PA-induced NF-κB p65 subunit into the nucleus. Our result showed that Cab39 is a direct target of miRNA-451 in steatotic cells. Further study showed that AMPK activated through Cab39 inhibits NF-κB transactivation induced in steatotic HepG2 cells. miR-451 over-expression in steatotic cells significantly suppressed PA-induced inflammatory cytokine. These results provide new insights into the negative regulation of miR-451 in fatty acid-induced inflammation via the AMPK/AKT pathway and demonstrate potential therapeutic applications for miR-451 in preventing the progression from simple steatosis to severely advanced liver disease.     

Cloning and preliminary functional studies of the JAM-A gene in grass carp (Ctenopharyngodon idellus)

Grass carp (Ctenopharyngodon idellus) is a very important aquaculture species in China and other South-East Asian countries; however, disease outbreaks in this species are frequent, resulting in huge economic losses. Grass carp hemorrhage caused by grass carp reovirus (GCRV) is one of the most serious diseases. Junction adhesion molecule A (JAM-A) is the mammalian receptor for reovirus, and has been well studied. However, the JAM-A gene in grass carp has not been studied so far. In this study, we cloned and elucidated the structure of the JAM-A gene in grass carp (GcJAM-A) and then studied its functions during grass carp hemorrhage. GcJAM-A is composed of 10 exons and 9 introns, and its full-length cDNA is 1833 bp long, with an 888 bp open reading frame (ORF) that encodes a 295 amino acid protein. The GcJAM-A protein is predicted to contain a typical transmembrane domain. Maternal expression pattern of GcJAM-A is observed during early embryogenesis, while zygote expression occurs at 8 h after hatching. GcJAM-A is expressed strongly in the gill, liver, intestine and kidney, while it is expressed poorly in the blood, brain, spleen and head kidney. Moreover, lower expression is observed in the gill, liver, intestine, brain, spleen and kidney of 30-month-old individuals, compared with 6-month-old. In a GcJAM-A-knockdown cell line (CIK) infected with GCRV, the expression of genes involved in the interferon and apoptosis pathways was significantly inhibited. These results suggest that GcJAM-A could be a receptor for GCRV. We have therefore managed to characterize the GcJAM-A gene and provide evidence for its role as a receptor for GCRV.     

Effects and mechanism of miR-23b on glucose-mediated epithelial-to-mesenchymal transition in diabetic nephropathy

MicroRNAs (miRNAs) play important roles in epithelial-to-mesenchymal transition (EMT). Moreover, hyperglycaemia induces damage to renal tubular epithelial cells, which may lead to EMT in diabetic nephropathy. However, the effects of miRNAs on EMT in diabetic nephropathy are poorly understood. In the present study, we found that the level of microRNA-23b (miR-23b) was significantly decreased in high glucose (HG)-induced human kidney proximal tubular epithelial cells (HK2) and in kidney tissues of db/db mice. Overexpression of miR-23b attenuated HG-induced EMT, whereas knockdown of miR-23b induced normal glucose (NG)-mediated EMT in HK2 cells. Mechanistically, miR-23b suppressed EMT in diabetic nephropathy by targeting high mobility group A2 (HMGA2), thereby repressing PI3K-AKT signalling pathway activation. Additionally, HMGA2 knockdown or inhibition of the PI3K-AKT signalling pathway with LY294002 mimicked the effects of miR-23b overexpression on HG-mediated EMT, whereas HMGA2 overexpression or activation of the PI3K-AKT signalling pathway with BpV prevented the effects of miR-23b on HG-mediated EMT. We also confirmed that overexpression of miR-23b alleviated EMT, decreased the expression levels of EMT-related genes, ameliorated renal morphology, glycogen accumulation, fibrotic responses and improved renal functions in db/db mice. Taken together, we showed for the first time that miR-23b acts as a suppressor of EMT in diabetic nephropathy through repressing PI3K-AKT signalling pathway activation by targeting HMGA2, which maybe a potential therapeutic target for diabetes-induced renal dysfunction.     

A transcriptional target of androgen receptor,miR-421 regulates proliferation and metabolism of prostate cancer cells

Prostate cancer is one of the most common malignancies, and microRNAs have been recognized to be involved in tumorigenesis of various kinds of cancer including prostate cancer (PCa). Androgen receptor (AR) plays a core role in prostate cancer progression and is responsible for regulation of numerous downstream targets including microRNAs. This study identified an AR-repressed microRNA, miR-421, in prostate cancer. Expression of miR-421 was significantly suppressed by androgen treatment, and correlated to AR expression in different prostate cancer cell lines. Furthermore, androgen-activated AR could directly bind to androgen responsive element (ARE) of miR-421, as predicted by bioinformatics resources and demonstrated by ChIP and luciferase reporter assays. In addition, over-expression of miR-421 markedly supressed cell viability, delayed cell cycle, reduced glycolysis and inhibited migration in prostate cancer cells. According to the result of miR-421 target genes searching, we focused on 4 genes NRAS, PRAME, CUL4B and PFKFB2 based on their involvement in cell proliferation, cell cycle progression and metabolism. The expression of these 4 downstream targets were significantly repressed by miR-421, and the binding sites were verified by luciferase assay. Additionally, we explored the expression of miR-421 and its target genes in human prostate cancer tissues, both in shared microarray data and in our own cohort. Significant differential expression and inverse correlation were found in PCa patients.     

Spatial and temporal distribution of Patched-related protein in the Drosophila embryo

Patched-related (Ptr) encodes a protein with 12 potential transmembrane domains and a sterol-sensing domain that is closely related in predicted topology and domain organization to Patched, the canonical receptor of the Hedgehog pathway. Here we describe the production of an antibody specific for Drosophila Ptr and analyse its spatial and temporal distribution in the embryo. We find that at early developmental stages Ptr is predominantly localized at cell periphery but later on it becomes strongly and almost exclusively expressed in hemocytes. Interestingly Ptr null mutant embryos died without hatching. Our findings suggest that Ptr plays an essential function in Drosophila development, perhaps as a new receptor of embryonic hemocytes.     

mTOR/miR-145-regulated exosomal GOLM1 promotes hepatocellular carcinoma through augmented GSK-3β/MMPs

Golgi membrane protein 1 (GOLM1/GP73) is a serum marker of hepatocellular carcinoma (HCC). We have previously shown that mTOR promoted tumorigenesis of HCC through stimulating GOLM1 expression. In this study, we demonstrated that the mammalian target of rapamycin (mTOR) was a negative regulator of microRNA-145 (miR-145) expression. miR-145 inhibited GOLM1 expression by targeting a coding sequence of GOLM1 gene. GOLM1 and miR-145 were inversely correlated in human HCC tissues. GOLM1-enriched exosomes activated the glycogen synthase kinase-3β/matrix metalloproteinases (GSK-3β/MMPs) signaling axis of recipient cells and accelerated cell proliferation and migration. In contrast, miR-145 suppressed tumorigenesis and metastasis. We suggest that mTOR/miR-145/GOLM1 signaling pathway should be targeted for HCC treatment.     

Red blood cell membrane water permeability increases with length of ex vivo storage

Water transport across the red blood cell (RBC) membrane is an essential cell function that needs to be preserved during ex vivo storage. Progressive biochemical depletion during storage can result in significant conformational and compositional changes to the membrane. Characterizing the changes to RBC water permeability can help in evaluating the quality of stored blood products and aid in the development of improved methods for the cryopreservation of red blood cells. This study aimed to characterize the water permeability (L_p), osmotically inactive fraction (b), and Arrhenius activation energy (E_a) at defined storage time-points throughout storage and to correlate the observed results with other in vitro RBC quality parameters. RBCs were collected from age- and sex-matched blood donors. A stopped flow spectrophotometer was used to determine L_p and b by monitoring changes in hemoglobin autofluorescence when RBCs were exposed to anisotonic solutions. Experimental values of L_p were characterized at three different temperatures (4, 20 and 37 °C) to determine the E_a. Results showed that L_p, b, and E_a of stored RBCs significantly increase by day 21 of storage. Degradation of the RBC membrane with length of storage was seen as an increase in hemolysis and supernatant potassium, and a decrease in deformability, mean corpuscular hemoglobin concentration and supernatant sodium. RBC osmotic characteristics were shown to change with storage and correlate with changes in RBC membrane quality metrics. Monitoring water parameters is a predictor of membrane damage and loss of membrane integrity in ex vivo stored RBCs.     

LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes,fibH, fibL and fhx,in the silk gland of the silkworm,Bombyx mori

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.     

Fat accumulation in differentiated brown adipocytes is linked with expression of Hox genes

Homeobox (Hox) genes are involved in body plan of embryo along the anterior–posterior axis. Presence of several Hox genes in white adipose tissue (WAT) and brown adipose tissue (BAT) is indicative of involvement of Hox genes in adipogenesis. We propose that differentiation inducing agents viz. isobutyl-methyl-xanthine (IBMX), indomethacin, dexamethasone (DEX), triiodothyronine (T3) and insulin may regulate differentiation in brown adipose tissue through Hox genes. In vitro culture of brown fat stromalvascular fraction (SVF) in presence or absence of differentiation inducing agents was used for establishing relationship between fat accumulation in differentiated adipocytes and expression of Hox genes. Relative expression of Pref1, UCP1 and Hox genes was determined in different stages of adipogenesis. Presence or absence of IBMX, indomethacin and DEX during differentiation of proliferated pre-adipocytes resulted in marked differences in expression of Hox genes and lipid accumulation. In presence of these inducing agents, lipid accumulation as well as expression of HoxA1, HoxA5, HoxC4 & HoxC8 markedly enhanced. Irrespective of presence or absence of T3, insulin down regulates HoxA10. T3 results in over expression of HoxA5, HoxC4 and HoxC8 genes, whereas insulin up regulates expression of only HoxC8. Findings suggest that accumulation of fat in differentiated adipocytes is linked with expression of Hox genes.     

Artificial miRNA-mediated silencing of ecdysone receptor (EcR) affects larval development and oogenesis in Helicoverpa armigera

The insect pests are real threat to farmers as they affect the crop yield to a great extent. The use of chemical pesticides for insect pest control has always been a matter of concern as they pollute the environment and are also harmful for human health. Bt (Bacillus thuringensis) technology helped the farmers to get rid of the insect pests, but experienced a major drawback due to the evolution of insects gaining resistance towards these toxins. Hence, alternative strategies are high on demand to control insect pests. RNA-based gene silencing is emerging as a potential tool to tackle with this problem. In this study, we have shown the use of artificial microRNA (amiRNA) to specifically target the ecdysone receptor (EcR) gene of Helicoverpa armigera (cotton bollworm), which attacks several important crops like cotton, tomato chickpea, pigeon pea, etc and causes huge yield losses. Insect let-7a precursor miRNA (pre-miRNA) backbone was used to replace the native miRNA with that of amiRNA. The precursor backbone carrying the 21 nucleotide amiRNA sequence targeting HaEcR was cloned in bacterial L4440 vector for in vitro insect feeding experiments. Larvae fed with Escherichia coli expressing amiRNA-HaEcR showed a reduction in the expression of target gene as well as genes involved in the ecdysone signaling pathway downstream to EcR and exhibited mortality and developmental defects. Stem-loop RT-PCR revealed the presence of amiRNA in the insect larvae after feeding bacteria expressing amiRNA-HaEcR, which was otherwise absent in controls. We also found a significant drop in the reproduction potential (oogenesis) of moths which emerged from treated larvae as compared to control. These results demonstrate the successful use of an insect pre-miRNA backbone to express amiRNA for gene silencing studies in insects. The method is cost effective and can be exploited as an efficient and alternative tool for insect pest management.     

Know your ABCs: Characterization and gene expression dynamics of ABC transporters in the polyphagous herbivore Helicoverpa armigera

Polyphagous insect herbivores are adapted to many different secondary metabolites of their host plants. However, little is known about the role of ATP-binding cassette (ABC) transporters, a multigene family involved in detoxification processes. To study the larval response of the generalist Helicoverpa armigera (Lepidoptera) and the putative role of ABC transporters, we performed developmental assays on artificial diet supplemented with secondary metabolites from host plants (atropine-scopolamine, nicotine and tomatine) and non-host plants (taxol) in combination with a replicated RNAseq experiment. A maximum likelihood phylogeny identified the subfamily affiliations of the ABC transporter sequences. Larval performance was equal on the atropine-scopolamine diet and the tomatine diet. For the latter we could identify a treatment-specific upregulation of five ABC transporters in the gut. No significant developmental difference was detected between larvae fed on nicotine or taxol. This was also mirrored in the upregulation of five ABC transporters when fed on either of the two diets. The highest number of differentially expressed genes was recorded in the gut samples in response to feeding on secondary metabolites. Our results are consistent with the expectation of a general detoxification response in a polyphagous herbivore. This is the first study to characterize the multigene family of ABC transporters and identify gene expression changes across different developmental stages and tissues, as well as the impact of secondary metabolites in the agricultural pest H. armigera.     

Wolbachia infection in Aedes aegypti mosquitoes alters blood meal excretion and delays oviposition without affecting trypsin activity

Blood feeding in Aedes aegypti is essential for reproduction, but also permits the mosquito to act as a vector for key human pathogens such as the Zika and dengue viruses. Wolbachia pipientis is an endosymbiotic bacterium that can manipulate the biology of Aedes aegypti mosquitoes, making them less competent hosts for many pathogens. Yet while Wolbachia affects other aspects of host physiology, it is unclear whether it influences physiological processes associated with blood meal digestion. To that end, we examined the effects of wMel Wolbachia infection in Ae. aegypti, on survival post-blood feeding, blood meal excretion, rate of oviposition, expression levels of key genes involved in oogenesis, and activity levels of trypsin blood digestion enzymes. We observed that wMel infection altered the rate and duration of blood meal excretion, delayed the onset of oviposition and was associated with a greater number of eggs being laid later. wMel-infected Ae. aegypti also had lower levels of key yolk protein precursor genes necessary for oogenesis. However, all of these effects occurred without a change in trypsin activity. These results suggest that Wolbachia infection may disrupt normal metabolic processes associated with blood feeding and reproduction in Ae. aegypti.