Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy.

INTRODUCTION: Vascular neointimal hyperplasia, a pathological process observed in cardiovascular diseases such as atherosclerosis and pulmonary hypertension, involves the abundant presence of vascular smooth muscle cells (VSMCs). The proliferation, migration, and autophagy of VSMCs are associated with the development of neointimal lesions. Circular RNAs (circRNAs) play critical roles in regulating VSMC proliferation and migration, thereby participating in neointimal hyperplasia. However, the regulatory roles of circRNAs in VSMC autophagy remain unclear. OBJECTIVES: We aimed to identify circRNAs that are involved in VSMC autophagy-mediated neointimal hyperplasia, as well as elucidate the underlying mechanisms. METHODS: Dual-luciferase reporter gene assay was performed to validate two competing endogenous RNA axes, hsa_circ_0001402/miR-183-5p/FKBP prolyl isomerase like (FKBPL) and hsa_circ_0001402/miR-183-5p/beclin 1 (BECN1). Cell proliferation and migration analyses were employed to investigate the effects of hsa_circ_0001402, miR-183-5p, or FKBPL on VSMC proliferation and migration. Cell autophagy analysis was conducted to reveal the role of hsa_circ_0001402 or miR-183-5p on VSMC autophagy. The role of hsa_circ_0001402 or miR-183-5p on neointimal hyperplasia was evaluated using a mouse model of common carotid artery ligation. RESULTS: Hsa_circ_0001402 acted as a sponge for miR-183-5p, leading to the suppression of miR-183-5p expression. Through direct interaction with the coding sequence (CDS) of FKBPL, miR-183-5p promoted VSMC proliferation and migration by decreasing FKBPL levels. Besides, miR-183-5p reduced BECN1 levels by targeting the 3'-untranslated region (UTR) of BECN1, thus inhibiting VSMC autophagy. By acting as a miR-183-5p sponge, overexpression of hsa_circ_0001402 increased FKBPL levels to inhibit VSMC proliferation and migration, while simultaneously elevating BECN1 levels to activate VSMC autophagy, thereby alleviating neointimal hyperplasia. CONCLUSION: Hsa_circ_0001402, acting as a miR-183-5p sponge, increases FKBPL levels to inhibit VSMC proliferation and migration, while enhancing BECN1 levels to activate VSMC autophagy, thus alleviating neointimal hyperplasia. The hsa_circ_0001402/miR-183-5p/FKBPL axis and hsa_circ_0001402/miR-183-5p/BECN1 axis may offer potential therapeutic targets for neointimal hyperplasia.

Integrated analysis of tRNA-derived small RNAs in proliferative human aortic smooth muscle cells.

BACKGROUND: Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to vascular remodeling diseases. Recently, it has been discovered that tRNA-derived small RNAs (tsRNAs), a new type of noncoding RNAs, are related to the proliferation and migration of VSMCs. tsRNAs regulate target gene expression through miRNA-like functions. This study aims to explore the potential of tsRNAs in human aortic smooth muscle cell (HASMC) proliferation. METHODS: High-throughput sequencing was performed to analyze the tsRNA expression profile of proliferative and quiescent HASMCs. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the sequence results and subcellular distribution of AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076. Based on the microRNA-like functions of tsRNAs, we predicted target promoters and mRNAs and constructed tsRNA-promoter and tsRNA-mRNA interaction networks. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to reveal the function of target genes. EdU incorporation assay, Western blot, and dual-luciferase reporter gene assay were utilized to detect the effects of tsRNAs on HASMC proliferation. RESULTS: Compared with quiescent HASMCs, there were 1838 differentially expressed tsRNAs in proliferative HASMCs, including 887 with increased expression (fold change > 2, p < 0.05) and 951 with decreased expression (fold change < ½, p < 0.05). AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076 were increased in proliferative HASMCs and were mainly located in the nucleus. Bioinformatics analysis suggested that the four tsRNAs involved a variety of GO terms and pathways related to VSMC proliferation. AS-tDR-000067 promoted HASMC proliferation by suppressing p53 transcription in a promoter-targeted manner. AS-tDR-000076 accelerated HASMC proliferation by attenuating mitofusin 2 (MFN2) levels in a 3'-untranslated region (UTR)-targeted manner. CONCLUSIONS: During HASMC proliferation, the expression levels of many tsRNAs are altered. AS-tDR-000067 and AS-tDR-000076 act as new factors promoting VSMC proliferation.

Expression and Functional Analysis of lncRNAs Involved in Platelet-Derived Growth Factor-BB-Induced Proliferation of Human Aortic Smooth Muscle Cells.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a common feature of many vascular remodeling diseases. Because long non-coding RNAs (lncRNAs) play a critical role in cardiovascular diseases, we analyzed the key lncRNAs that regulate VSMC proliferation. Microarray analysis identified 2,643 differentially expressed lncRNAs (DELs) and 3,720 differentially expressed coding genes (DEGs) between fetal bovine serum (FBS) starvation-induced quiescent human aortic smooth muscle cells (HASMCs) and platelet-derived growth factor-BB (PDGF-BB)-stimulated proliferative HASMCs. Gene Ontology and pathway analyses of the identified DEGs and DELs demonstrated that many lncRNAs were enriched in pathways related to cell proliferation. One of the upregulated lncRNAs in proliferative HASMC was HIF1A anti-sense RNA 2 (HIF1A-AS2). HIF1A-AS2 suppression decreased HASMC proliferation via the miR-30e-5p/CCND2 mRNA axis. We have thus identified key DELs and DEGs involved in the regulation of PDGF-BB induced HASMC proliferation. Moreover, HIF1A-AS2 promotes HASMC proliferation, suggesting its potential involvement in VSMC proliferative vascular diseases.

Functional predication of differentially expressed circRNAs/lncRNAs in the prefrontal cortex of Nrf2-knockout mice.

In the central nervous system, nuclear factor erythroid-2-related factor 2 (Nrf2) protects neurons from oxidant injury, thereby ameliorating neurodegeneration. We explored the key circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) involved in Nrf2-induced neuroprotection. We used microarrays to examine the circRNAs (DEcircRNAs), lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) differentially expressed between Nrf2 (+/+) and Nrf2 (-/-) mice and identified DEcircRNA/DElncRNA-miRNA-DEmRNA interaction networks. In total, 197 DEcircRNAs, 685 DElncRNAs and 356 DEmRNAs were identified in prefrontal cortical tissues from Nrf2 (-/-) mice. The expression patterns of selected DEcircRNAs (except for mmu_circ_0003404) and DElncRNAs in qRT-PCR analyses were generally consistent with the microarray analysis results. Functional annotation of the DEmRNAs in the DEcircRNA/DElncRNA-miRNA-DEmRNA networks indicated that five non-coding RNAs (mmu_circ_0000233, ENSMUST00000204847, NONMMUT024778, NONMMUT132160 and NONMMUT132168) may contribute to Nrf2 activity, with the help of mmu_circ_0015035 and NONMMUT127961. The results also revealed that four non-coding RNAs (cicRNA.20127, mmu_circ_0012936, ENSMUST00000194077 and NONMMUT109267) may influence glutathione metabolism. Additionally, 44 DEcircRNAs and 7 DElncRNAs were found to possess coding potential. These findings provide clues to the molecular pathways through which Nrf2 protects neurons.

Differential Expression Profiles and Functional Prediction of Circular RNAs and Long Non-coding RNAs in the Hippocampus of Nrf2-Knockout Mice.

BACKGROUND: Nrf2 (nuclear factor, erythroid 2 like 2) is believed to play a major role in neurodegenerative diseases. The present study attempts to investigate the hippocampal circRNA and lncRNA expression profiles associated with Nrf2-mediated neuroprotection. METHODS: The hippocampal mRNA, circRNA and lncRNA expression profiles of Nrf2 (-/-) mice were determined by a microarray analysis. Bioinformatics analyses, including identification of differentially expressed mRNAs (DEmRNAs), circRNAs (DEcircRNAs) and lncRNAs (DElncRNAs), DEcircRNA-miRNA-DEmRNA interaction network construction, DElncRNA-DEmRNA co-expression network construction, and biological function annotation, were conducted. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the dysregulated expression of circRNAs and lncRNAs derived from the microarray data of the hippocampus of Nrf2 (-/-) mice. RESULTS: Compared to wild-type Nrf2 (+/+) mice, 412 DEmRNAs (109 up- and 303 down-regulated mRNAs), 1279 DEcircRNAs (632 up- and 647 down-regulated circRNAs), and 303 DElncRNAs (50 up- and 253 down-regulated lncRNAs) were identified in the hippocampus of Nrf2 (-/-) mice. Additionally, in the qRT-PCR validation results, the expression patterns of selected DEcircRNAs and DElncRNAs were generally consistent with results in the microarray data. The DEcircRNA-miRNA-DEmRNA interaction networks revealed that mmu_circRNA_44531, mmu_circRNA_34132, mmu_circRNA_000903, mmu_circRNA_018676, mmu_circRNA_45901, mmu_circRNA_33836, mmu_circRNA_ 34137, mmu_circRNA_34106, mmu_circRNA_008691, and mmu_circRNA_003237 were predicted to compete with 47, 54, 45, 57, 63, 81, 121, 85, 181, and 43 DEmRNAs, respectively. ENSMUST00000125413, NR_028123, uc008nfy.1, AK076764, AK142725, AK080547, and AK035903 were co-expressed with 178, 89, 149, 179, 142, 55, and 112 DEmRNAs in the Nrf2 (-/-) hippocampus, respectively. CONCLUSION: Our study might contribute to exploring the key circRNAs and lncRNAs associated with Nrf2-mediated neuroprotection.

circ-Sirt1 controls NF-κB activation via sequence-specific interaction and enhancement of SIRT1 expression by binding to miR-132/212 in vascular smooth muscle cells.

NF-κB-mediated inflammatory phenotypic switching of vascular smooth muscle cells (VSMCs) plays a central role in atherosclerosis and neointimal formation. However, little is known about the roles of circRNAs in the regulation of NF-κB signaling. Here, we identify the involvement of circ-Sirt1 that was one of transcripts of SIRT1 host gene in VSMC inflammatory response and neointimal hyperplasia. First, in the cytoplasm, circ-Sirt1 directly interacts with and sequesters NF-κB p65 from nuclear translocation induced by TNF-α in a sequence-dependent manner. The inhibitory complex of circ-Sirt1-NF-κB p65 is not dependent on IκBα. Second, circ-Sirt1 binds to miR-132/212 that interferes with SIRT1 mRNA, and facilitates the expression of host gene SIRT1. Increased SIRT1 results in deacetylation and inactivation of the nuclear NF-κB p65. These findings illustrate that circ-Sirt1 is a novel non-coding RNA regulator of VSMC phenotype.

The Differentially Expressed Circular RNAs in the Substantia Nigra and Corpus Striatum of Nrf2-Knockout Mice.

BACKGROUND/AIMS: The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a protective role in both acute neuronal damage and chronic neurodegeneration-related oxidative stress. Circular RNAs (circRNAs) are involved with various diseases in the central nervous system (CNS). This study aimed to identify the key circRNAs involved in Nrf2-neuroprotection against oxidative stress. METHODS: The differentially expressed circRNAs (DEcircRNAs) in the substantia nigra and corpus striatum between Nrf2 (-/-) and Nrf2 (+/+) mice were identified by microarray analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) was then used to validate the expression of selected DEcircRNAs in the substantia nigra and corpus striatum between Nrf2 (-/-) and Nrf2 (+/+) mice. Based on our previous microarray analysis of the differentially expressed mRNAs (DEmRNAs) in the substantia nigra and corpus striatum between Nrf2 (-/-) and Nrf2 (+/+) mice, the DEcircRNA-miRNA-DEmRNA interaction network was constructed. Functional annotation of DEmRNAs that shared the same binding miRNAs with DEcircRNAs was performed using gene ontology (GO) and pathway analyses. RESULTS: A total of 65 and 150 significant DEcircRNAs were obtained in the substantia nigra and corpus striatum of Nrf2 (-/-) mice, respectively, and seventeen shared DEcircRNAs were found in both these two tissues. The qRT-PCR results were generally consistent with the microarray results. The DEcircRNA-miRNA-DEmRNA interaction network and pathway analysis indicated that mmu_circRNA_34132, mmu_circRNA_017077 and mmu-circRNA-015216 might be involved with Nrf2-mediated neuroprotection against oxidative stress. Mmu_circRNA_015216 and mmu_circRNA_017077 might play roles in the Nrf2-related transcriptional misregulation and Nrf2-mediated processes of rheumatoid arthritis, respectively. In addition to these two processes, mmu_circRNA_34132 may be a potential regulator of Nrf2-mediated protection for diabetes mellitus and Nrf2-mediated defence against ROS in hearts. CONCLUSION: In conclusion, our study identified the key DEcircRNAs in the substantia nigra and corpus striatum of Nrf2 (-/-) mice, which might provide new clues for further exploring the mechanism of Nrf2-mediated neuroprotection against oxidative stress and other Nrf2-mediated processes.

TRAF6-Mediated SM22α K21 Ubiquitination Promotes G6PD Activation and NADPH Production, Contributing to GSH Homeostasis and VSMC Survival In Vitro and In Vivo.

RATIONALE: Vascular smooth muscle cell (VSMC) survival under stressful conditions is integral to promoting vascular repair, but facilitates plaque stability during the development of atherosclerosis. The cytoskeleton-associated smooth muscle (SM) 22α protein is involved in the regulation of VSMC phenotypes, whereas the pentose phosphate pathway plays an essential role in cell proliferation through the production of dihydronicotinamide adenine dinucleotide phosphate. OBJECTIVE: To identify the relationship between dihydronicotinamide adenine dinucleotide phosphate production and SM22α activity in the development and progression of vascular diseases. METHODS AND RESULTS: We showed that the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) are promoted in platelet-derived growth factor (PDGF)-BB-induced proliferative VSMCs. PDGF-BB induced G6PD membrane translocation and activation in an SM22α K21 ubiquitination-dependent manner. Specifically, the ubiquitinated SM22α interacted with G6PD and mediated G6PD membrane translocation. Furthermore, we found that tumor necrosis factor receptor-associated factor (TRAF) 6 mediated SM22α K21 ubiquitination in a K63-linked manner on PDGF-BB stimulation. Knockdown of TRAF6 decreased the membrane translocation and activity of G6PD, in parallel with reduced SM22α K21 ubiquitination. Elevated levels of activated G6PD consequent to PDGF-BB induction led to increased dihydronicotinamide adenine dinucleotide phosphate generation through stimulation of the pentose phosphate pathway, which enhanced VSMC viability and reduced apoptosis in vivo and in vitro via glutathione homeostasis. CONCLUSIONS: We provide evidence that TRAF6-induced SM22α ubiquitination maintains VSMC survival through increased G6PD activity and dihydronicotinamide adenine dinucleotide phosphate production. The TRAF6-SM22α-G6PD pathway is a novel mechanism underlying the association between glucose metabolism and VSMC survival, which is beneficial for vascular repair after injury but facilitates atherosclerotic plaque stability.

miR-146a and Krüppel-like factor 4 form a feedback loop to participate in vascular smooth muscle cell proliferation.

MicroRNAs are phenotypic regulators of vascular smooth muscle cells (VSMCs). In this paper, we demonstrate that miR-146a targets the Krüppel-like factor 4 (KLF4) 3'-untranslated region and has an important role in promoting VSMC proliferation in vitro and vascular neointimal hyperplasia in vivo. Silencing of miR-146a in VSMCs increases KLF4 expression, whereas overexpression of miR-146a decreases KLF4 levels. Furthermore, we demonstrate that KLF4 competes with Krüppel-like factor 5 (KLF5) to bind to and regulate the miR-146a promoter, and that KLF4 and KLF5 exert opposing effects on the miR-146a promoter. Overexpression of KLF4 in VSMCs decreases miR-146a transcription levels. By using both gain-of-function and loss-of-function approaches, we found that miR-146a promotes VSMC proliferation in vitro. Transfection of antisense miR-146a oligonucleotide into balloon-injured rat carotid arteries markedly decreased neointimal hyperplasia. These findings suggest that miR-146a and KLF4 form a feedback loop to regulate each other's expression and VSMC proliferation.

Krüppel-like factor 4 promotes differentiation by transforming growth factor-beta receptor-mediated Smad and p38 MAPK signaling in vascular smooth muscle cells.

KLF4 (Krüppel-like factor 4) has been implicated in vascular smooth muscle cell (VSMC) differentiation induced by transforming growth factor beta (TGF-beta). However, the role of KLF4 and mechanism of KLF4 actions in regulating TGF-beta signaling in VSMCs remain unclear. In this study, we showed that TGF-beta1 inhibited cell cycle progression and induced differentiation in cultured rat VSMCs. This activity of TGF-beta1 was accompanied by up-regulation of KLF4, with concomitant increase in TbetaRI (TGF-beta type I receptor) expression. KLF4 was found to transduce TGF-beta1 signals via phosphorylation-mediated activation of Smad2, Smad3, and p38 MAPK. The activation of both pathways, in turn, increased the phosphorylation of KLF4, which enabled the formation of KLF4-Smad2 complex in response to TGF-beta1. Chromatin immunoprecipitation studies and oligonucleotide pull-down assays showed the direct binding of KLF4 to the KLF4-binding sites 2 and 3 of the TbetaRI promoter and the recruitment of Smad2 to the Smad-responsive region. Formation of a stable KLF4-Smad2 complex in the promoter's Smad-responsive region mediated cooperative TbetaRI promoter transcription in response to TGF-beta1. These results suggest that KLF4-dependent regulation of Smad and p38 MAPK signaling via TbetaRI requires prior phosphorylation of KLF4 through Smad and p38 MAPK pathways. This study demonstrates a novel mechanism by which TGF-beta1 regulates VSMC differentiation.

Distribution and expression of phosphorylated histone H3 during porcine oocyte maturation.

Phosphorylation modification of core histones is correlated well with diverse chromatin-based cell activities. However, its distribution pattern and primary roles during mammalian oocyte meiosis are still in dispute. In this study, by performing immunofluorescence and Western blotting, spatial distribution and temporal expression of phosphorylated serine 10 or 28 on histone H3 during porcine oocyte meiotic maturation were examined and distinct subcellular distribution patterns between them were presented. Low expression of phosphorylated H3/ser10 was detected in germinal vesicle. Importantly, following gradual dephosphorylation from germinal vesicle (GV) to late germinal vesicle (L-GV) stage, a transient phosphorylation at the periphery of condensed chromatin was re-established at early germinal vesicle breakdown (E-GVBD) stage, and then the dramatically increased signals covered whole chromosomes from pre-metaphase I (Pre-MI) to metaphase II (MII). Similarly, hypophosphorylation of serine 28 on histone H3 was also monitored from GV to E-GVBD, indicating dephosphorylation of histone H3 maybe involved in the regulation of meiotic resumption. Moreover, the rim staining on the chromosomes and high levels of H3/ser28 phosphorylation were observed in Pre-MI, MI, and MII stage oocytes. Based on above results, such stage-dependent dynamics of phosphorylation of H3/ser 10 and 28 may play specific roles during mammalian oocyte maturation.

Efficient generation of transgenic mice by direct intraovarian injection of plasmid DNA.

We established a rapid procedure for obtaining transgenic mice by directly injecting an enhanced green fluorescent protein (EGFP)-expressing plasmid (pIRES-EGFP) into the ovaries of fertile mice. The frequency of transgenic mouse production was determined by pair-mating, and by polymerase chain reaction (PCR) and sequence analysis of DNA taken from the tails of the offspring. The mice that received the EGFP gene transmitted it to their offspring (F(1)). Genetic and PCR analyses of F(1) progeny confirmed that the inserted EGFP was stably inherited. Of six female F(1) mice, all were able to pass the foreign DNA on to the next generation (F(2)). In situ hybridization using paraffin-embedded sections of ovarian and testicular tissues from the F(1) and F(2) progeny showed that the introduced gene was expressed in the gonads of the animals. The chromosomal location of the injected DNA was determined by fluorescence in situ hybridization, and the frequency of multiple site versus single site insertions is 85.71% (18/21) analyzed by FISH. We anticipate great progress in murine genetic engineering using this technique.

Copper-mediated C-N bond formation via direct aminolysis of dithioacetals.

Mediated by copper acetate, an efficient approach to the C-N bond formation via direct aminolysis of dithioacetals 2 and 5 with ammonia, primary or secondary amines are developed under mild conditions. Enaminones 3 and 6 were thus obtained in high to excellent yields with high chemoselectivity. This type of aminolysis reaction presents a new synthetic application of the dithioacetal functionality. [reaction: see text]

[Cloning and characterization of the promoter region of Musca domestica yolk protein-1 gene].

A partial Musca domestica genomic library was constructed. It was consisted of 1.2 x 10(5) recombinants with insert length ranging from 10 kb to 23 kb(15 kb average). High molecular weight genomic DNA with more than 50 kb size was extracted from the larva hatched 36 h and digested with unfrequently cutting restriction enzyme Bcl I. DNA fragments of 10 approximately 23 kb were recovered by agarose gel electrophoresis and ligated with EMBL3 BamH I Arms CIPase treated. Then the products of ligation were packed in vitro using packing protein. The cloning efficiency of the genomic library was 5 x 10(4) pfu/mL. The genomic library was screened by hybridization using a probe of a 768 bp partial cDNA fragment of Musca domestica yolk protein 1 (mdYP1) gene obtained by PCR and the probe was labeled with Digoxigen. A positive plaque was chosed and purified by in situ hybridization. A genomic DNA fragment about 4.0 kb mdYP1 was isolated from purified positive plaque by southern blotting analysis. Sequence analysis revealed that mdYP1 genomic gene was composed of 5'-upstream region about 1.7 kb with typical CAAT/TATA box. The promoter of the mdYP1 gene was characterized by examining the ability of 5'-upstream fragments to regulate expression of green fluorescent protein (GFP) in Musca domestica larva. Four fragments of the promoter region, P1 (+296/+7), P2 (+684/+7), P3(+1165/+7) and P4 (+1616/+7) ,were obtained by PCR specific amplification using template of recombinant A-lambdaNA containing mdYP1 gene sequence. Then the four fragments were respectively subcloned into pCMV-GFP reporter vector deleted CMV promoter. All the fragments showed no promoter activity when the four recombinant vectors were transfected into Sf9 and BHK -2 cells respectively, but three of them, P2, P3 and P4, showed significant promoter activity when they were respectively introduced into Musca domestica larva by electroporation. The two fragments, P5 (+684/+302) and P6 (+165/+302), obtained by digesting P2 and P3 with Spe I and Hind III, were also subcloned into pGFP vector, and they showed no promoter activity in Sf9 cells, BHK -21 cells and Musca domestica larva. The results demonstrated that the core promoter spanned 302bp and contained a CCAAT box and a TATA box upstream translation initiation codon (ATG), but itself had no transcriptional activity, and that regulatory promoters or enhancers and other cis-elements presented from +302 to +1616 were necessary to maintain the specific expression.