Circulating Extracellular-Vesicle-Incorporated MicroRNAs as Potential Biomarkers for Ischemic Stroke in Patients With Cancer.

BACKGROUND AND PURPOSE: This study aimed to evaluate whether extracellular-vesicle-incorporated microRNAs (miRNAs) are potential biomarkers for cancer-related stroke. METHODS: This cohort study compared patients with active cancer who had embolic stroke of unknown sources (cancer-stroke group) with patients with only cancer, patients with only stroke, and healthy individuals (control groups). The expression profiles of miRNAs encapsulated in plasma exosomes and microvesicles were evaluated using microarray and validated using quantitative real-time polymerase chain reaction. The XENO-QTM miRNA assay technology was used to determine the absolute copy numbers of individual miRNAs in an external validation cohort. RESULTS: This study recruited 220 patients, of which 45 had cancer-stroke, 76 were healthy controls, 39 were cancer controls, and 60 were stroke controls. Three miRNAs (miR-205-5p, miR-645, and miR-646) were specifically incorporated into microvesicles in patients with cancer-related stroke, cancer controls, and stroke controls. The area under the receiver operating characteristic curves of these three miRNAs were 0.7692-0.8510 for the differentiation of patients with cancer-stroke from cancer-controls and 0.8077-0.8846 for the differentiation of patients with cancer-stroke from stroke controls. The levels of several miRNAs were elevated in the plasma exosomes of patients with cancer, but were lower than those in plasma microvesicles. An in vivo study showed that systemic injection of miR-205-5p promoted the development of arterial thrombosis and elevation of D-dimer levels. CONCLUSION: Stroke due to cancer-related coagulopathy was associated with deregulated expression of miRNAs, particularly microvesicle-incorporated miR-205-5p, miR-645, and miR-646. Further prospective studies of extracellular-vesicle-incorporated miRNAs are required to confirm the diagnostic role of miRNAs in patients with stroke and to screen the roles of miRNAs in patients with cancer.

The E-box-like sterol regulatory element mediates the insulin-stimulated expression of hepatic clusterin.

Clusterin (also known as apolipoprotein J) is a highly conserved glycoprotein involved in various biological processes, including attenuation of complement activity, sperm maturation, apoptosis, and reverse lipid transport. Although clusterin is reportedly associated with metabolic diseases, the metabolic regulation of clusterin expression is largely unknown. We investigated the effect of insulin on hepatic clusterin expression and its underlying mechanisms. Insulin increased the mRNA and protein levels of clusterin in primary hepatocytes and hepatoma cell lines. Serial deletion and mutant analysis of the clusterin promoter demonstrated that insulin-stimulated transactivation is mediated via a non-canonical E-box (NCE-box) motif in the proximal upstream region. Interestingly, sterol regulatory element binding protein-1c (SREBP-1c) co-transfection showed the same transactivation pattern as insulin stimulation in serial deletion and mutant promoter analysis. In contrast, co-transfection with a dominant negative form of SREBP-1c inhibited insulin-stimulated clusterin expression. Furthermore, insulin increased the recruitment of SREBP-1c to the NCE-box of the clusterin promoter region. Taken together, our results suggest that an NCE-box within the clusterin promoter is necessary for insulin-stimulated hepatic expression of clusterin via SREBP-1c.

Hepatic TRAP80 selectively regulates lipogenic activity of liver X receptor.

Inflammation in response to excess low-density lipoproteins in the blood is an important driver of atherosclerosis development. Due to its ability to enhance ATP-binding cassette A1-dependent (ABCA1-dependent) reverse cholesterol transport (RCT), liver X receptor (LXR) is an attractive target for the treatment of atherosclerosis. However, LXR also upregulates the expression of sterol regulatory element-binding protein 1c (SREBP-1c), leading to increased hepatic triglyceride synthesis, an independent risk factor for atherosclerosis. Here, we developed a strategy to separate the favorable and unfavorable effects of LXR by exploiting the specificity of the coactivator thyroid hormone receptor-associated protein 80 (TRAP80). Using human hepatic cell lines, we determined that TRAP80 selectively promotes the transcription of SREBP-1c but not ABCA1. Adenovirus-mediated expression of shTRAP80 inhibited LXR-dependent SREBP-1c expression and RNA polymerase II recruitment to the LXR responsive element (LXRE) of SREBP-1c, but not to the LXRE of ABCA1. In murine models, liver-specific knockdown of TRAP80 ameliorated liver steatosis and hypertriglyceridemia induced by LXR activation and maintained RCT stimulation by the LXR ligand. Together, these data indicate that TRAP80 is a selective regulator of hepatic lipogenesis and is required for LXR-dependent SREBP-1c activation. Moreover, targeting the interaction between TRAP80 and LXR should facilitate the development of potential LXR agonists that effectively prevent atherosclerosis.

Regulation of hepatic insulin sensitivity by activating signal co-integrator-2.

ASC-2 (activating signal co-integrator-2, also known as AIB3 and NCoA6) is a transcriptional co-activator and regulates insulin secretion and ß-cell survival. The present study was performed to elucidate the role of ASC-2 in the regulation of insulin sensitivity. Although islet cells from 10-week-old ASC-2+/- mice secreted less insulin than wild-type islets, there was no significant difference in glucose tolerance between ASC-2+/- and wild-type mice. However, ASC-2+/- mice did show increased insulin sensitivity compared with wild-type mice in insulin tolerance tests. Consistently, the levels of phosphorylated Akt were higher in ASC-2+/- hepatocytes than in wild-type hepatocytes after insulin treatment. Moreover, decreases in phosphoenol pyruvate carboxykinase mRNA in refed mice were more prominent in ASC-2+/- livers than in wild-type livers. Interestingly, the expression levels of SOCS1 (suppressor of cytokine signalling 1) and SOCS3, well-known insulin signalling inhibitors, were decreased in ASC-2+/- hepatocytes and increased in ASC-2-overexpressing hepatocytes. Furthermore, ASC-2 was recruited to the promoter region of SOCS1 and potentiated the transcription by SREBP-1c (sterol-regulatory-element-binding protein-1c). This transcription-activating function of ASC-2 was diminished by mutations of SREBP-1c-binding sites in the SOCS1 promoter. Taken together, these results suggest that ASC-2 negatively affects hepatic insulin sensitivity, at least in part, through induction of the insulin signalling inhibitors SOCS1 and SOCS3.

SREBP-1c regulates glucose-stimulated hepatic clusterin expression.

Clusterin is a stress-response protein that is involved in diverse biological processes, including cell proliferation, apoptosis, tissue differentiation, inflammation, and lipid transport. Its expression is upregulated in a broad spectrum of diverse pathological states. Clusterin was recently reported to be associated with diabetes, metabolic syndrome, and their sequelae. However, the regulation of clusterin expression by metabolic signals was not addressed. In this study we evaluated the effects of glucose on hepatic clusterin expression. Interestingly, high glucose concentrations significantly increased clusterin expression in primary hepatocytes and hepatoma cell lines, but the conventional promoter region of the clusterin gene did not respond to glucose stimulation. In contrast, the first intronic region was transcriptionally activated by high glucose concentrations. We then defined a glucose response element (GlRE) of the clusterin gene, showing that it consists of two E-box motifs separated by five nucleotides and resembles carbohydrate response element (ChoRE). Unexpectedly, however, these E-box motifs were not activated by ChoRE binding protein (ChREBP), but were activated by sterol regulatory element binding protein-1c (SREBP-1c). Furthermore, we found that glucose induced recruitment of SREBP-1c to the E-box of the clusterin gene intronic region. Taken together, these results suggest that clusterin expression is increased by glucose stimulation, and SREBP-1c plays a crucial role in the metabolic regulation of clusterin.