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Objective: To study the expression patterns of long noncoding ribonucleic acid (RNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and the cell proliferation inhibition, apoptosis, and motility changes induced by silencing MALAT1 in urothelial carcinoma of the bladder., Materials and Methods: The expression levels of MALAT1 were determined using real-time quantitative polymerase chain reaction in cancerous tissues and paired normal tissues in a total of 36 patients with urothelial carcinoma of the bladder. Expression differences were analyzed according to the grade and stage. Bladder urothelial carcinoma T24 and 5637 cells were transfected with MALAT1 small interfering RNA or negative control small interfering RNA. The cell proliferation changes of the transfected bladder urothelial carcinoma cells were determined using the MTT assay. Apoptosis caused by silencing MALAT1 was evaluated using the flow cytometry assay and enzyme-linked immunosorbent assay. The motility changes induced by silencing MALAT1 were measured using the wound healing assay., Results: MALAT1 was upregulated in bladder urothelial carcinoma compared with matched normal urothelium (P=.008). The MALAT1 expression levels were greater in high-grade carcinomas than in low-grade carcinoma (P=.001). The MALAT1 expression levels were greater in invasive carcinoma than in noninvasive carcinoma (P=.018). Cell proliferation inhibition, increased apoptosis, and decreased motility were observed in MALAT1 small interfering RNA-transfected bladder urothelial carcinoma T24 and 5637 cells., Conclusion: MALAT1 plays an oncogenic role in urothelial carcinoma of the bladder. Silencing MALAT1 is a potential novel therapeutic approach for this cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Human KCNQ4 mutations known as DFNA2 cause non-syndromic, autosomal-dominant, progressive high-frequency hearing loss in which the cellular and molecular basis is unclear. We provide immunofluorescence data showing that Kcnq4 expression in the adult cochlea has both longitudinal (base to apex) and radial (inner to outer hair cells) gradients. The most intense labeling is in outer hair cells at the apex and in inner hair cells as well as spiral ganglion neurons at the base. Spatiotemporal expression studies show increasing intensity of KCNQ4 protein labeling from postnatal day 21 (P21) to P120 mice that is most apparent in inner hair cells of the middle turn. We have identified four alternative splice variants of Kcnq4 in mice. The alternative use of exons 9-11 produces three transcript variants (v1-v3), whereas the fourth variant (v4) skips all three exons; all variants have the same amino acid sequence at the C termini. Both reverse transcription-PCR and quantitative PCR analyses demonstrate that these variants have differential expression patterns along the length of the mouse organ of Corti and spiral ganglion neurons. Our expression data suggest that the primary defect leading to high-frequency loss in DFNA2 patients may be attributable to high levels of the dysfunctional Kcnq4_v3 variant in the spiral ganglion and inner hair cells in the basal hook region. Progressive hearing loss associated with aging may result from an increasing mutational load expansion toward the apex in inner hair cells and spiral ganglion neurons.

Functional interactions between ligand-gated, voltage-, and Ca(2+)-activated ion channels are essential to the properties of excitable cells and thus to the working of the nervous system. The outer hair cells in the mammalian cochlea receive efferent inputs from the brain stem through cholinergic nerve fibers that form synapses at their base. The acetylcholine released from these efferent fibers activates fast inhibitory postsynaptic currents mediated, to some extent, by small-conductance Ca(2+)-activated K+ channels (SK) that had not been cloned. Here we report the cloning, characterization, and expression of a complete SK2 cDNA from the mouse cochlea. The cDNAs of the mouse cochlea alpha9 and alpha10 acetylcholine receptors were also obtained, sequenced, and coexpressed with the SK2 channels. Human cultured cell lines transfected with SK2 yielded Ca(2+)-sensitive K+ current that was blocked by dequalinium chloride and apamin, known blockers of SK channels. Xenopus oocytes injected with SK2 in vitro transcribed RNA, under conditions where only outward K+ currents could be recorded, expressed an outward current that was sensitive to EGTA, dequalinium chloride, and apamin. In HEK-293 cells cotransfected with cochlear SK2 plus alpha9/alpha10 receptors, acetylcholine induced an inward current followed by a robust outward current. The results indicate that SK2 and the alpha9/alpha10 acetylcholine receptors are sufficient to partly recapitulate the native hair cell efferent synaptic response.

The protein phosphatase 2C (PP2C) gene family, which is known to participate in cellular processes, is one of the most conserved plant-specific gene families that regulate signal transduction in eukaryotic organisms. However, the PP2C gene family of Glycyrrhiza uralensis Fisch. has not yet been systematically characterized. In this study, 76 PP2C genes of G. uralensis (GuPP2Cs) were identified. Phylogenetic analysis divided these GuPP2Cs into 12 subfamilies. The gene structure and protein motifs of the GuPP2Cs were then analysed. The analysis of the transcriptomic data of the GuPP2Cs revealed that nine genes were highly sensitive to abscisic acid (ABA) stress and might respond to ABA treatment. These nine genes were validated via quantitative real-time polymerase chain reaction. This study provides a foundation for future studies on the functional PP2C genes of G. uralensis and the improvement of the cultivation and quality of G. uralensis. [ABSTRACT FROM AUTHOR]

Purpose: Abnormal expression of miRNAs is closely related to a variety of human cancers. The purpose of this study is to identify new tumor suppressor miRNA and elucidate its physiological function and mechanism in renal cell carcinoma (RCC). Methods: The expression of miR-145 in 45 RCC and adjacent normal tissues was performed by quantitative RT-PCR. Cell proliferation, migration, invasion, apoptosis and cycle assays were carried out for functional analysis after miR-145 transfection. Two target genes of miR-145 were identified by luciferase reporter assay. The altered expression of 84 epithelial to mesenchymal transition (EMT)-related genes after miR-145 transfection was detected by RT Profiler EMT PCR array. Results: The expression of miR-145 was downregulated in RCC compared to their normal adjacent tissues. Restoring miR-145 expression in RCC cell lines dramatically suppressed cell proliferation, migration and invasion, and induced cell apoptosis and G2-phase arrest. We further validated those miR-145 targets two oncogenes, ANGPT2 and NEDD9 in RCC. In addition, miR-145 was found to regulate numerous genes involved in the EMT. Conclusions: These findings demonstrate that miR-145 functions as tumor suppressor in RCC, suggesting that miR-145 may be a potential therapeutic target for RCC. [ABSTRACT FROM AUTHOR]

KCNQ4, a voltage-gated potassium channel, plays an important role in maintaining cochlear ion homoeostasis and regulating hair cell membrane potential, both essential for normal auditory function. Mutations in the KCNQ4 gene lead to DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss across all frequencies. Despite recent advances in the identification of pathogenic KCNQ4 mutations, the molecular aetiology of DFNA2 remains unknown. We report here that decreased cell surface expression and impaired conductance of the KCNQ4 channel are two mechanisms underlying hearing loss in DFNA2. In HEK293T cells, a dramatic decrease in cell surface expression was detected by immunofluorescent microscopy and confirmed by Western blot for the pathogenic KCNQ4 mutants L274H, W276S, L281S, G285C, G285S, G296S and G321S, while their overall cellular levels remained normal. In addition, none of these mutations affected tetrameric assembly of KCNQ4 channels. Consistent with these results, all mutants showed strong dominant-negative effects on the wild-type ( WT) channel function. Most importantly, overexpression of HSP90β, a key component of the molecular chaperone network that controls the KCNQ4 biogenesis, significantly increased cell surface expression of the KCNQ4 mutants L281S, G296S and G321S. KCNQ4 surface expression was restored or considerably improved in HEK293T cells mimicking the heterozygous condition of these mutations in DFNA2 patients. Finally, our electrophysiological studies demonstrated that these mutations directly compromise the conductance of the KCNQ4 channel, since no significant change in KCNQ4 current was observed after KCNQ4 surface expression was restored or improved. [ABSTRACT FROM AUTHOR]

Loss-of-function mutations in the KCNQ4 channel cause DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss. Previous studies have demonstrated that the majority of the pathogenic KCNQ4 mutations lead to trafficking deficiency and loss of KCNQ4 currents. Over the last two decades, various strategies have been developed to rescue trafficking deficiency of pathogenic mutants; the most exciting advances have been made by manipulating activities of molecular chaperones involved in the biogenesis and quality control of the target protein. However, such strategies have not been established for KCNQ4 mutants and little is known about the molecular chaperones governing the KCNQ4 biogenesis. To identify KCNQ4-associated molecular chaperones, a proteomic approach was used in this study. As a result, two major molecular chaperones, HSP70 and HSP90, were identified and then confirmed by reciprocal co-immunoprecipitation assays, suggesting that the HSP90 chaperone pathway might be involved in the KCNQ4 biogenesis. Manipulating chaperone expression further revealed that two different isoforms of HSP90, the inducible HSP90α and the constitutive HSP90β, had opposite effects on the cellular level of the KCNQ4 channel; that HSP40, HSP70, and HOP, three key components of the HSP90 chaperone pathway, were crucial in facilitating KCNQ4 biogenesis. In contrast, CHIP, a major E3 ubiquitin ligase, had an opposite effect. Collectively, our data suggest that HSP90α and HSP90β play key roles in controlling KCNQ4 homeostasis via the HSP40-HSP70-HOP-HSP90 chaperone pathway and the ubiquitin-proteasome pathway. Most importantly, we found that over-expression of HSP90β significantly improved cell surface expression of the trafficking-deficient, pathogenic KCNQ4 mutants L274H and W276S. KCNQ4 surface expression was restored by HSP90β in cells mimicking heterozygous conditions of the DFNA2 patients, even though it was not sufficient to rescue the function of KCNQ4 channels. [ABSTRACT FROM AUTHOR]

An outcome of the intricate K+ regulation in the cochlear duct is the endocochlear potential (EP), ∼80 mV, the "battery" that runs hair-cell transduction; however, the detailed molecular mechanisms for the generation of the EP remain unclear. We provide strong evidence indicating that the intermediate cells (ICs) of the stria vascularis (StV) express outward K+ current that rectifies inwardly at positive potentials. The channel belongs to the ether-a-go-go-related gene (erg) family of K+ channels. We cloned an ERG1a channel in the mouse inner ear (MERG1a). The cellular distribution of MERG1a in the cochlea displayed the highest levels of immunoreactivity in the ICs and modest reactivity in the marginal cells as well as in several extrastrial cells (e.g., hair cells). Functional expression of the StV-specific MERG1a channel reveals a current that activates at relatively negative potentials (approximately -50 mV) and shows rapid inactivation reflected as inward rectification at depolarized potentials. The current was sensitive to the methanesulfonanilide drug E-4031 (IC50, ∼165 nM) and the recombinant peptide rBeKm-1 (IC50, ∼16 nM), and the single-channel conductance in symmetrical K+ was ∼14 pS. The site of expression of MERG1a and its functional phenotype (e.g., modulation of the current by external K+) make it one of the most likely candidates for establishing the high throughput of K+ ions across ICs to generate EP. In addition, the property of the channel that produces marked K+ extrusion in increased external K+ may be important in shaping the dynamics of K+ cycling in the inner ear. [ABSTRACT FROM AUTHOR]