Circular RNAs (circRNAs) have been shown to play critical roles in cancer biology, but their functions in nonalcoholic steatohepatitis (NASH) remain unexplored. Full length of circRNA_002581 was amplified and sequenced, followed by RNA immunoprecipitation, RNA-Fluorescence in Situ Hybridization and dual luciferase reporter gene analysis to confirm the existence of the circRNA_002581-miR-122-CPEB1 regulatory axis in vitro. CircRNA_002581 knockdown was used to study its roles in high concentration of free fatty acids-induced NASH-like cell model and a methionine and choline deficiency (MCD) diet-induced NASH mice model. Autophagy flux and related potential PTEN-AMPK-mTOR pathway were tested by western blot. CircRNA_002581 overexpression significantly relieved the inhibitory role of miR-122 on its target CPEB1 by sponging miR-122. CircRNA_002581 knockdown markedly attenuated lipid droplet accumulation, reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), pro-inflammatory cytokines, apoptosis, H 2 O 2 , and increased ATP level in both mice and cellular models of NASH. Mechanistically, circRNA_002581 interference significantly rescue the defective autophagy evidenced by increased autophagosome number, upregulated LC3-II/I level, and decreased p62 level. Further chloroquine-mediated total autophagy inhibition antagonizes the protective effect of circRNA_002581 knockdown. Finally, CPEB1-PTEN-AMPK-mTOR pathway is shown to link the autophagy and circRNA_002581 knockdown-mediated NASH alleviation. CircRNA_002581-miR-122-CPEB1 axis actively participates in the pathogenesis of NASH through PTEN-AMPK-mTOR pathway-related autophagy suppression. Targeting circRNA_002581 is a potential therapeutic strategy for NASH through partial autophagy restoration.

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SUMMARY BackgroundHelicobacter pylori infection is a worldwide threat to human health with recurrence rates that vary widely. The precise correlation between H. pylori recurrence and socioeconomic development has not been determined.

Cymbidium ensifolium is a Chinese Cymbidium with an elegant shape, beautiful appearance, and a fragrant aroma. C. ensifolium has a long history of cultivation in China and it has excellent commercial value as a potted plant and cut flower. The development of C. ensifolium genomic resources has been delayed because of its large genome size. Taking advantage of technical and cost improvement of RNA-Seq, we extracted total mRNA from flower buds and mature flowers and obtained a total of 9.52 Gb of filtered nucleotides comprising 98,819,349 filtered reads. The filtered reads were assembled into 101,423 isotigs, representing 51,696 genes. Of the 101,423 isotigs, 41,873 were putative homologs of annotated sequences in the public databases, of which 158 were associated with floral development and 119 were associated with flowering. The isotigs were categorized according to their putative functions. In total, 10,212 of the isotigs were assigned into 25 eukaryotic orthologous groups (KOGs), 41,690 into 58 gene ontology (GO) terms, and 9,830 into 126 Arabidopsis Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 9,539 isotigs into 123 rice pathways. Comparison of the isotigs with those of the two related orchid species P. equestris and C. sinense showed that 17,906 isotigs are unique to C. ensifolium. In addition, a total of 7,936 SSRs and 16,676 putative SNPs were identified. To our knowledge, this transcriptome database is the first major genomic resource for C. ensifolium and the most comprehensive transcriptomic resource for genus Cymbidium. These sequences provide valuable information for understanding the molecular mechanisms of floral development and flowering. Sequences predicted to be unique to C. ensifolium would provide more insights into C. ensifolium gene diversity. The numerous SNPs and SSRs identified in the present study will contribute to marker development for C. ensifolium.

Homocysteine- (Hcy-) induced endothelial cell apoptosis has been suggested as a cause of Hcy-dependent vascular injury, while the proposed molecular pathways underlying this process are unclear. In this study, we investigated the adverse effects of Hcy on human umbilical vein endothelial cells (HUVEC) and the underlying mechanisms. Our results demonstrated that moderate-dose Hcy treatment induced HUVEC apoptosis in a time-dependent manner. Furthermore, prolonged Hcy treatment increased the expression of NOX4 and the production of intracellular ROS but decreased the ratio of Bcl-2/Bax and mitochondrial membrane potential (MMP), resulting in the leakage of cytochrome c and activation of caspase-3. Prolonged Hcy treatment also upregulated glucose-regulated protein 78 (GRP78), activated protein kinase RNA-like ER kinase (PERK), and induced the expression of C/EBP homologous protein (CHOP) and the phosphorylation of NF-κb. The inhibition of NOX4 decreased the production of ROS and alleviated the Hcy-induced HUVEC apoptosis and ER stress. Blocking the PERK pathway partly alleviated Hcy-induced HUVEC apoptosis and the activation of NF-κb. Taken together, our results suggest that Hcy-induced mitochondrial dysfunction crucially modulated apoptosis and contributed to the activation of ER stress in HUVEC. The excessive activation of the PERK pathway partly contributed to Hcy-induced HUVEC apoptosis and the phosphorylation of NF-κb.