Long noncoding RNAs (lncRNAs) play a critical role in the regulation of atherosclerosis. Here, we investigated the role of the lncRNA growth arrest-specific 5 (lncR-GAS5) in atherogenesis. We found that the enforced expression of lncR-GAS5 contributed to the development of atherosclerosis, which presented as increased plaque size and reduced collagen content. Moreover, impaired autophagy was observed, as shown by a decreased LC3II/LC3I protein ratio and an elevated P62 level in lncR-GAS5-overexpressing human aortic endothelial cells. By contrast, lncR-GAS5 knockdown promoted autophagy. Moreover, serine/arginine-rich splicing factor 10 (SRSF10) knockdown increased the LC3II/LC3I ratio and decreased the P62 level, thus enhancing the formation of autophagic vacuoles, autolysosomes, and autophagosomes. Mechanistically, lncR-GAS5 regulated the downstream splicing factor SRSF10 to impair autophagy in the endothelium, which was reversed by the knockdown of SRSF10. Further results revealed that overexpression of the lncR-GAS5-targeted gene miR-193-5p promoted autophagy and autophagic vacuole accumulation by repressing its direct target gene, SRSF10. Notably, miR-193-5p overexpression decreased plaque size and increased collagen content. Altogether, these findings demonstrate that lncR-GAS5 partially contributes to atherogenesis and plaque instability by impairing endothelial autophagy. In conclusion, lncR-GAS5 overexpression arrested endothelial autophagy through the miR-193-5p/SRSF10 signaling pathway. Thus, miR-193-5p/SRSF10 may serve as a novel treatment target for atherosclerosis.
Humans ; Atherosclerosis/genetics* ; Autophagy/genetics* ; Cell Cycle Proteins/metabolism* ; Endothelial Cells/metabolism* ; Endothelium/metabolism* ; MicroRNAs/metabolism* ; Repressor Proteins/metabolism* ; RNA Splicing Factors ; Serine-Arginine Splicing Factors/genetics* ; RNA, Long Noncoding/metabolism*

OBJECTIVES: Graves' ophthalmopathy (GO) is a multifactorial disease, and the mechanism of non coding RNA interactions and inflammatory cell infiltration patterns are not fully understood. This study aims to construct a competing endogenous RNA (ceRNA) network for this disease and clarify the infiltration patterns of inflammatory cells in orbital tissue to further explore the pathogenesis of GO. METHODS: The differentially expressed genes were identified using the GEO2R analysis tool. The Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology analysis were used to analyze differential genes. RNA interaction relationships were extracted from the RNA interactome database. Protein-protein interactions were identified using the STRING database and were visualized using Cytoscape. StarBase, miRcode, and DIANA-LncBase Experimental v.2 were used to construct ceRNA networks together with their interacted non-coding RNA. The CIBERSORT algorithm was used to detect the patterns of infiltrating immune cells in GO using R software. RESULTS: A total of 114 differentially expressed genes for GO and 121 pathways were detected using both the KEGG and gene ontology enrichment analysis. Four hub genes (SRSF6, DDX5, HNRNPC,and HNRNPM) were extracted from protein-protein interaction using cytoHubba in Cytoscape, 104 nodes and 142 edges were extracted, and a ceRNA network was identified (MALAT1-MIR21-DDX5). The results of immune cell analysis showed that in GO, the proportions of CD8⁺ T cells and CD4⁺ memory resting T cells were upregulated and downregulated, respectively. The proportion of CD4 memory resting T cells was positively correlated with the expression of MALAT1, MIR21, and DDX5. CONCLUSIONS This study has constructed a ceRNA regulatory network (MALAT1-MIR21-DDX5) in GO orbital tissue, clarifying the downregulation of the proportion of CD4⁺ stationary memory T cells and their positive regulatory relationship with ceRNA components, further revealing the pathogenesis of GO.
Humans ; CD8-Positive T-Lymphocytes ; RNA, Long Noncoding/genetics* ; Algorithms ; CD4-Positive T-Lymphocytes ; Down-Regulation ; Graves Ophthalmopathy/genetics* ; Gene Regulatory Networks ; MicroRNAs/genetics* ; Serine-Arginine Splicing Factors ; Phosphoproteins

A transcriptional regulatory network for wild-type and ATP7B-knockout HepG2 cells exposed to copper was constructed by bioinformatics methods to explore the potential mechanism of key transcription factors in the pathogenesis of hepatolenticular degeneration. The differentially expressed genes (DEGs) for wild-type and ATP7B-knockout HepG2 cell lines without copper and exposed to copper were collected from the gene expression omnibus (GEO) database. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were performed for DEGs induced by copper. The key functional modules and genes were identified based on the protein-protein interaction (PPI) network. Moreover, the enrichment analysis of genes in functional modules was performed. Finally, a transcriptional regulatory network was constructed to screen the core transcription factors. A total of 1 034 genes, including 509 down-regulated genes and 525 up-regulated genes, were selected as DEGs. The up-regulated and down-regulated functional modules based on PPI network included 3 785 and 3 931 genes, respectively. Genes in key functional modules were enriched in cell-substrate junction, chromosomal region, spliceosomal complex and ribosome. They were involved in mRNA processing, histone modification, RNA splicing, regulation of DNA metabolic process, protein phosphorylation and other biological processes. Moreover, they were correlated to transcriptional coregulator activity, DNA-binding transcription factor binding, ubiquitin-like protein ligase binding and other molecular functions. KEGG analysis showed that genes in key functional modules were significantly enriched in hepatitis B, MAPK signaling pathway, cellular senescence and apoptosis, neurotrophin signaling pathway and pathways of neurodegeneration-multiple diseases. The transcriptional regulatory network contained 11 differentially expressed transcription factors and 96 DEGs. Among them, U2AF1, NFRKB, FUS, MAX, SRSF1, CEBPA and RXRA were the core transcription factors, which may facilitate the study of the biological function of relevant molecules in transcriptional regulation of hepatolenticular degeneration.
Humans ; Transcriptome ; Gene Expression Profiling/methods* ; Hepatolenticular Degeneration/genetics* ; Copper ; Gene Regulatory Networks ; Computational Biology/methods* ; Transcription Factors/genetics* ; DNA ; DNA-Binding Proteins/genetics* ; Serine-Arginine Splicing Factors/genetics*

Natural antisense transcripts (NAT) and alternative polyadenylation (APA) of messenger RNA (mRNA) are important contributors of transcriptome complexity, each playing a critical role in multiple biological processes. However, whether they have crosstalk and function collaboratively is unclear. We discovered that APA enriched in human sense-antisense (S-AS) gene pairs, and finally focused on RNASEH2C-KAT5 S-AS pair for further study. In cis but not in trans over-expression of the antisense KAT5 gene promoted the usage of distal polyA (pA) site in sense gene RNASEH2C, which generated longer 3' untranslated region (3'UTR) and produced less protein, accompanying with slowed cell growth. Mechanistically, elevated Pol II occupancy coupled with SRSF3 could explain the higher usage of distal pA site. Finally, NAT-mediated downregulation of sense gene's protein level in RNASEH2C-KAT5 pair was specific for human rather than mouse, which lacks the distal pA site of RNASEH2C. We provided the first evidence to support that certain gene affected phenotype may not by the protein of its own, but by affecting the expression of its overlapped gene through APA, implying an unexpected view for understanding the link between genotype and phenotype.
Cell Proliferation ; genetics ; Evolution, Molecular ; Gene Expression Regulation ; genetics ; HEK293 Cells ; Humans ; Polyadenylation ; genetics ; RNA, Antisense ; genetics ; RNA, Messenger ; genetics ; Ribonuclease H ; genetics ; Serine-Arginine Splicing Factors ; metabolism ; Transcription, Genetic ; Up-Regulation ; genetics

Objective: To verify whether the enzymatic activity of kynureninase (KYNU) could be changed by the Arg188Gln (G/A) mutation. Methods: The total RNA of human hepatic tissue was extracted and the KYNU gene cDNA was amplified by RT-PCR. Primers were designed according to the sequences around the site Arg188Gln of KYNU gene and the Arg188Gln (G/A) mutant KYNU cDNA was generated by site-directed mutagenesis. Both the wild-type and mutant-type KYNU genes were subcloned into pcDNA vectors and the recombinant plasmids were constructed. After being transfected into human embryonic kidney 293 (HEK293) cells, the expression of KYNU recombinant plasmids were assessed by Western blot. The enzymatic activities of KYNU were detected by high performance liquid chromatography (HPLC). Results: The KYNU enzyme activities were expressed in both wild and mutant HEK293 cells. Michaelis constants (Km) of the wild and mutant KYNU were (9.833±0.513) μmol/L and (29.900±0.265) μmol/L, respectively (P<0.05). The maximum velocities (Vmax) of the wild and mutant KYNU were (0.700±0.096) nmol·mg^-1·min^-1 and (0.084±0.003) nmol·mg^-1·min^-1, respectively (P<0.05). Conclusion: Arg188Gln (G/A) mutation can decrease the enzymatic activity of KYNU.
Arginine ; genetics ; Enzyme Activation ; genetics ; HEK293 Cells ; Humans ; Hydrolases ; genetics ; metabolism ; Mutation ; Plasmids

The expression changes of Rars gene in ischemia-injured neurons were investigated by detecting its translational product arginyl-tRNA synthetase (ArgRS), and the inhibitory effects of ischemic preconditioning (IPC) on Rars gene were explored. Both IPC model and prolonged ischemia (PI) model were established by using the classic oxygen glucose deprivation (OGD) method. The primary cultured neurons were assigned into the following groups: the experimental group (IPC+PI group), undergoing PI after a short period of IPC; the conditional control group (PI control group), subjected to PI without IPC; blank control group, the normally cultured neurons. The Rars transcriptional activities and ArgRS expression levels were measured at different time points after re-oxygenation (3 h/6 h/12 h/24 h). Data were collected and statistically analyzed. Compared to the blank control group, the Rars activities and ArgRS levels were significantly increased in PI control group, peaking at the time point of 6 h after re-oxygenation. Rars activities and ArgRS levels were significantly lower in the experimental group than in the PI control group at different time points after re-oxygenation. PI insult can induce an escalating activity of Rars and lead to ArgRS over-expression in primary cultured neurons. IPC can inhibit the increased Rars activity and down-regulate ArgRS expression of ischemia-insulted neurons. This mechanism may confer ischemic tolerance on neurons.
Animals ; Arginine-tRNA Ligase ; biosynthesis ; genetics ; metabolism ; Brain Ischemia ; genetics ; metabolism ; pathology ; Gene Expression Regulation ; genetics ; Glucose ; metabolism ; Humans ; Ischemic Preconditioning ; methods ; Neurons ; metabolism ; pathology ; Oxygen ; metabolism ; Primary Cell Culture ; Rats

The expression of microRNA-19b (miR-19b) in acute necrotizing pancreatitis (ANP) and its functional role in acinar cell necrosis of SD rats were investigated. Twelve SD rats were divided into two groups randomly, including control group and ANP group. The rat ANP models were established by intraperitoneal injection of L-arginine (2400 mg/kg body weight), and equal volume of 0.9% NaCl was injected in the control group. MiRNA chip assay was performed to examine the expression of miRNAs in the pancreas in two different groups. Besides, to further explore the role of miR-19b in ANP in vitro, taurolithocholic acid 3-sulfate disodium salt (TLC-S) (200 μmol/L) was administrated to treat the rat pancreatic acinar cell line, AR42J, for establishing the ANP cells model. The quantitative real-time PCR (qRT-PCR) was adopted to measure the miR-19b expression. Moreover, the mimic miRNA, miRNA antisense oligonucleotide (AMO) and control vector were used to transfect AR42J cells, the expression of miR-19b was confirmed by qRT-PCR and the necrotizing rate of AR42J cells was detected with AO/EB method. The expression of miR-19b was significantly higher in ANP group than in control group as displayed by the miRNA chip assay. Furthermore, after inducing necrosis of AR42J cells in vitro, the expression of miR-19b was significantly increased by 2.51±0.14 times in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-19b was 5.94±0.95 times higher in the mimic miRNA group than in the control vector group, companied with an obviously increased acinar cell necrotizing rate (50.3%±1.5% vs. 39.6%±2.3%, P<0.05). Moreover, the expression of miR-19b in the miRNA AMO group was 0.38±0.15 times lower than in the control vector group, and the cell necrosis rate was much lower accordingly (23.1%±3.3% vs. 39.6%±2.3%, P<0.05). Besides, there was no significant difference between the control vector cells and the cells without treatment (P>0.05). The expression of miR-19b was significantly induced in ANP. In addition, up-regulation of miR-19b could promote the necrosis of pancreatic acinar cells and miR-19b deficiency could decrease the rate of pancreatic acinar cell necrosis.
Acinar Cells ; metabolism ; pathology ; Animals ; Arginine ; toxicity ; Cell Line ; MicroRNAs ; genetics ; metabolism ; Necrosis ; Pancreatitis, Acute Necrotizing ; etiology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Taurolithocholic Acid ; analogs & derivatives ; toxicity ; Up-Regulation

OBJECTIVETo study the distribution of single nucleotide polymorphisms (SNP) of arginine-vasopressin (AVP) gene rs66818855 and rs1078152 in Chinese Guangxi healthy population in comparison with that in different ethnic populations.

METHDOSPolymerase chain reaction-single base extension (PCR-SBE) and DNA sequencing were used to detect the allele and genotype frequencies of AVP gene among 303 Chinese healthy individuals in Guangxi, China, and the results were compared with the reported frequencies in 4 other populations (HapMap-CEU, HapMap-YRI, HapMap-JPT, and HapMap-HCB) from Human Genome Project group (HapMap) data.

RESULTSWe found significant AVP gene polymorphisms in this Guangxi healthy population. The frequencies of allele and genotype of AVP gene rs66818855 and rs1078152 polymorphisms in this Guangxi population differed significantly from those in HapMap-CEU population (P<0.01), and allele frequencies of AVP gene rs66818855 polymorphism differed significantly from those in HapMap-YRI populations (P<0.05).

CONCLUSIONThe distribution pattern of AVP gene polymorphisms in this Guangxi population is significantly different from that in other ethnic populations, which might account for the difference in the morbidity of AVP-related disease among different ethnic groups and may have important indications in the study of population genetics and anthropology.

Alleles ; Arginine Vasopressin ; genetics ; Asian Continental Ancestry Group ; China ; Gene Frequency ; Genetics, Population ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

OBJECTIVEIn Corynebacterium crenatum, the adjacent D311 and D312 of N-acetyl-L-glutamate kinase (NAGK), as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine, were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.

METHODSNAGK enzyme expression was evaluated using a plasmid-based method. Homologous recombination was employed to eliminate the proB.

RESULTSThe IC50 and enzyme activity of NAGK M4, in which the D311R and D312R amino acid substitutions were combined with the previously reported E19R and H26E substitutions, were 3.7-fold and 14.6% higher, respectively, than those of the wild-type NAGK. NAGK M4 was successfully introduced into the C. crenatum MT genome without any genetic markers; the L-arginine yield of C. crenatum MT-M4 was 26.2% higher than that of C. crenatum MT. To further improve upon the L-arginine yield, we constructed the mutant C. crenatum MT-M4 proB. The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield. After L-proline was added to the medium at 10 mmol/L, the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation, approximately 70.1% higher than the yield attained using C. crenatum MT.

CONCLUSIONFeedback inhibition of L-arginine on NAGK in C. crenatum is clearly alleviated by the M4 mutation of NAGK, and deletion of the proB in C. crenatum from MT to M4 results in a significant increase in arginine production.

Animals ; Arginine ; biosynthesis ; Corynebacterium ; genetics ; metabolism ; Escherichia coli ; Feedback, Physiological ; Gene Deletion ; Mutagenesis, Site-Directed ; Phosphotransferases (Carboxyl Group Acceptor) ; genetics ; Proline ; metabolism

BACKGROUNDProtein arginine methyltransferases 1 (PRMT1) is over-expressed in a variety of cancers, including lung cancer, and is correlated with a poor prognosis of tumor development. This study aimed to investigate the role of PRMT1 in nonsmall cell lung cancer (NSCLC) migration in vitro.

METHODSIn this study, PRMT1 expression in the NSCLC cell line A549 was silenced using lentiviral vector-mediated short hairpin RNAs. Cell migration was measured using both scratch wound healing and transwell cell migration assays. The mRNA expression levels of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 1, 2 (TIMP1, 2) were measured using quantitative real-time reverse transcription-polymerase chain reaction. The expression levels of protein markers for epithelial-mesenchymal transition (EMT) (E-cadherin, N-cadherin), focal adhesion kinase (FAK), Src, AKT, and their corresponding phosphorylated states were detected by Western blot.

RESULTSCell migration was significantly inhibited in the PRMT1 silenced group compared to the control group. The mRNA expression of MMP-2 decreased while TIMP1 and TIMP2 increased significantly. E-cadherin mRNA expression also increased while N-cadherin decreased. Only phosphorylated Src levels decreased in the silenced group while FAK or AKT remained unchanged.

CONCLUSIONSPRMT1-small hairpin RNA inhibits the migration abilities of NSCLC A549 cells by inhibiting EMT, extracellular matrix degradation, and Src phosphorylation in vitro.

Blotting, Western ; Carcinoma, Non-Small-Cell Lung ; enzymology ; genetics ; Cell Line ; Cell Movement ; genetics ; physiology ; Epithelial-Mesenchymal Transition ; genetics ; physiology ; Extracellular Matrix Proteins ; metabolism ; Humans ; Protein-Arginine N-Methyltransferases ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; physiology