Long noncoding RNA MANTIS relieved the protein-bound uremic toxin-induced injury on human umbilical vein endothelial cells in chronic kidney disease and end-stage renal disease.
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This study aimed to explore the role of long, noncoding RNA MANTIS in regulating the protein-bound, uremic toxin-induced injury on human umbilical vein endothelial cells (HUVECs) in chronic kidney disease (CKD) and end-stage renal disease (ESRD). The MANTIS expression in patients with normal kidney function, stage 3 CKD, stage 4 CKD and ESRD was detected. In addition, HUVECs were stimulated with various concentrations of HSA-bound P-cresol (20, 40 and 80 μg/ml) and then transfected with pcDNA-MANTIS, sh-MANTIS and their controls to further investigate the effects of MANTIS overexpression and knockdown on HSA-bound P-cresol-induced HUVECs injury. Furthermore, the regulatory relationships between MANTIS and Sox18, as well as between MANTIS and p38 MAPK or p65 NF-κB pathways were elucidated. MANTIS expression was down-regulated in patients with CKD and ESRD and might be associated with disease severity. In addition, HSA-bound P-cresol induced HUVECs injury and decreased MANTIS expression. Overexpression of MANTIS relieved HSA-bound P-cresol induced HUVECs injury by increasing HUVECs viability, migration and invasion, and inhibiting cell autophagy. Moreover, the effects of MANTIS on HSA-bound P-cresol induced HUVECs injury were through positive regulation of Sox18. Besides, MANTIS overexpression markedly inhibited the activation of p38 MAPK and p65 NF-κB pathways in HSA-bound P-cresol-stimulated HUVECs, which were reversed after overexpression of MANTIS and knockdown of Sox18 synchronously. Our findings reveal that lncRNA MANTIS may relieve the protein-bound uremic toxins-induced HUVECs injury in CKD and ESRD via positive regulation of Sox18 and inhibition of p38 MAPK and p65 NF-κB pathways.Keywords:
Mantis
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Gene targeting
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Ectopic expression
Schneider 2 cells
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Canine mast cell tumours often express KIT mutations that result in constitutive activation of the c-kit receptor and which are associated with more aggressive disease. The aim of the current studywas to determinewhether small inhibitory RNA (SiRNA)molecules could specifically target canine KIT mRNA for knock-down. Canine beta-2 microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and KIT sequences were cloned into the psiCHECKTM-2 vector. SiRNA molecules, designed to target gene-specific sequences, were co-transfected with plasmid DNA into Chinese hamster ovary (CHO) cells. Renilla and firefly luciferase activity was measured using the Dual-GLO® Luciferase Assay (Promega). Using this reporter system, canine housekeeping gene-specific SiRNA molecules demonstrated knockdown of their targets (72.0% knockdown for B2M and 94.5% knockdown for GAPDH). An SiRNA molecule targeting exon 2 of canine KIT successfully knocked-down reporter gene expression of a KIT26–407 construct (90.8% knockdown). An SiRNA molecule targeting a 48 base-pair in-tandemduplicationmutation in KIT exon 11 selectively knocked down expression of the KIT1569–1966mutant construct (93.1% knockdown) but had no effect on the KIT1569–1918wild-type construct. The results show that RNA interference can be used to inhibit canine KIT mRNA expression and has the potential to selectively target the mutant version of KIT that is expressed by some malignant mast cells. © 2011 Elsevier B.V. All rights reserved.
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Abstract RhoGDIβ, a Rho GDP dissociation inhibitor, induced hypertrophic growth and cell migration in a cultured cardiomyoblast cell line, H9c2. We demonstrated that RhoGDIβ plays a previously undefined role in regulating Rac1 expression through transcription to induce hypertrophic growth and cell migration and that these functions are blocked by the expression of a dominant-negative form of Rac1. We also demonstrated that knockdown of RhoGDIβ expression by RNA interference blocked RhoGDIβ-induced Rac1 expression and cell migration. We demonstrated that the co-expression of ZAK and RhoGDIβ in cells resulted in an inhibition in the activity of ZAK to induce ANF expression. Knockdown of ZAK expression in ZAK-RhoGDIβ-expressing cells by ZAK-specific RNA interference restored the activities of RhoGDIβ.
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Objective To construct the short hairpin RNA(shRNA) expression vector of survivin and down-regulate the expression of survivin through RNA interference in neuroblastoma cell line SH-SY5Y. Methods Two pairs of oligonucleotide sequences specific for human survivin mRNA were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pBSHH1 plasmid. After being identified by restriction enzyme digestion and sequencing, the recombinant plasmids pBSHH1-S1 and pBSHH1-S2 were transfected into SH-SY5Y cells, respectively. Survivin expression in the transfected cells was assayed by both RT-PCR and western blot. Results Enzyme digestion analysis and DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pBSHH1 plasmid, and survivin expression in the transfected cells was knocked down significantly by pBSHH1-S1 or pBSHH1-S2 at both the protein and mRNA level. Conclusion The shRNA expression vectors of survivin were successfully constructed, and could down-regulate survivin expression in SH-SY5Y cells, which lay a foundation for further research on gene therapy for tumors such as neuroblastoma.
Survivin
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The nematode cuticle is a tough extracellular matrix composed primarily of cross-linked collagens and non-collagenous cuticulins. It is required for nematode motility and protection from the external environment. Little is known about how the complex process of cuticle formation has been adapted to the specialized requirements of the nematode cuticle, which is structurally and compositionally unique from other organisms. The C. elegans gene cuti-1 (CUTicle and epithelial Integrity) encodes a nematode-specific protein. We have shown that CUTI-1 is expressed in the epithelia and in seam cells. Within these tissues the expression of cuti-1 mRNA cycles throughout development in line with the molting cycle, a process that involves synthesis of a new cuticle. In addition, knockdown of cuti-1 by RNA interference (RNAi) results in worms that display post-embryonic phenotypes related to cuticle dysfunction and defects in epithelial integrity. This is one of the first reports of a nematode-specific protein involved in extracellular matrix formation. It provides further insight into how novel ways have evolved to regulate the formation of the cuticle, which is the primary protective barrier and skeletal component of nematodes.
Cuticle (hair)
Arthropod cuticle
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The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei. Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.
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Prothoracic gland
Ecdysteroid
20-Hydroxyecdysone
Ecdysone receptor
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A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNAi knockdown method, we have analyzed the function of the homodimeric IFT kinesin-2, Kin5, in Tetrahymena ciliary transport. In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality.
Intraflagellar Transport
Ciliogenesis
Kinesin
Immunoprecipitation
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