Effect of miR-19a on lipid catabolism in hepatocyte LO2

AIM: To observe the effect of microRNA-19a (miR-19a) on the lipid catabolism of hepatocyte LO2, and to explore the potential mechanism. METHODS: miR-19a was over-expressed or silenced by transfection of miR-19a mimics or miR-19a inhibitor into LO2 cells, then the mRNA level of miR-19a was detected by real-time PCR. The potential target of miR-19a was found by the method of bioinformatics through internet website. The effect of miR-19a on the 3' UTR of peroxisome proliferator-activated receptor α (PPARα) was measured by dual luciferase reporter assay, and the protein level of PPARα and its 2 major downstream rate-limiting enzymes involved in lipid catabolism, acyl-coenzyme a dehydrogenase (ACADM) and carnitine palmitoyltransferase 1A (CPT1A), were detected by Western blotting. Meanwhile, the effect of miR-19a on the generation of ketone body was measured by beta-hydroxybutyric acid (β-OHB) detection assay. RESULTS: The mRNA level of miR-19a was dramatically elevated by the transfection of miR-19a mimics, and sharply decreased by the transfection of miR-19a inhibitor (P<0.05). PPARα was found as a potential target of miR-19a, and dual luciferase reporter assay and Western blotting confirmed the regulatory effect of miR-19a on the expression of PPARα, with the protein level changes of ACADM and CPT1A. miR-19a mimics down-regulated, while miR-19a inhibitor up-regulated the concentration of β-OHB in LO2 cells (P<0.05). CONCLUSION: miR-19a regulates the lipid catabolism of hepatocytes by targeting the PPARα and its 2 downstream rate-limiting enzymes.     

MicroRNA-9-5p mediates sympathetic overactivity by targeting KCNN3 in paraventricular nucleus of rats with T2D

AIM To investigate whether microRNA-9-5p （miR-9-5p） mediates sympathetic overactivity by targeting KCNN3 （potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3） gene,which encoded small-conductance calcium-activated potassium channel 3 （SK3） protein, in paraventricular nucleus （PVN） of rats with type 2 diabetes mellitus （T2D）. METHODS A rat model of T2D was established by high-fat diet combined with intraperitoneal injection of 30 mg/kg streptozotocin. The levels of miR-9-5p and KCNN3 mRNA in PVN were detected by real-time PCR. The relationship between KCNN3 and miR-9-5p was predicted by TargetScan. Recombinant adeno-associated virus （rAAV）-miR-9-5p or KCNN3 were bilaterally microinjected into the PVN to observe the changes in plasma glucose levels and sympathetic drive indicators. The number of FosB and SK3 positive cells was measured by immunofluorescence staining. The protein expression of SK3 was determined by Western blot. The relationship between KCNN3 and miR-9-5p were confirmed by cell transfection and dual-luciferase reporter assay. RESULTS Compared with the rats in diabetes control （DC） group, the blood glucose, sympathetic drive indexes and the level of miR-9-5p in PVN were significantly increased, while the SK3 expression in PVN was obviously reduced in the diabetes mellitus （DM） rats. After microinjecion of rAAV-miR-9-5p in PVN, the sympathetic drive indexes, blood glucose, and the number of FosB-positive cells were increased significantly, but the SK3 protein expression was significantly reduced （P<0.05）. However, up-regulation of KCNN3 in PVN had the opposite effect. These responses were obviously enhanced in DM rats compared with DC rats. The results of cell transfection and dual-luciferase reporter assay demonstrated that miR-9-5p bound to the 3’-UTR of KCNN3 and inhibit its expression. CONCLUSION miR-9-5p was up-regulated in PVN of the rats with T2D, and it may mediate sympathoexcitation by targeting KCNN3.     

Sinomenine reduces MPP+-induced damage in SK-N-SH cells via ANRIL/miR-626 signaling pathway

AIM To investigate the effect of sinomenine (SN) on the damage of human neuroblastoma SK-N-SH cells induced by 1-methyl-4-4 phenylpyridine (MPP⁺) and its mechanism for exploring the pathogenesis of Parkinson disease. METHODS SN was used to treat MPP⁺-induced SK-N-SH cells. The levels of malondialdehyde (MDA) and glutathione (GSH) in cell culture supernatants were measured by ELISA. The apoptosis was analyzed by flow cytometry. The protein expression levels of Bcl-2 and Bax were determined by Western blot. The expression levels of long noncoding RNA ANRIL and microRNA-626 (miR-626) were detected by RT-qPCR. Dual-luciferase reporter assay was used to evaluate the relationship between ANRIL and miR-626. After ANRIL small interfering RNA was transfected into SK-N-SH cells, the effects of ANRIL expression knock-down on MPP⁺-induced SK-N-SH cell apoptosis, the protein expression levels of Bcl-2 and Bax, and the levels of MDA and GSH in cell culture supernatants were examined. RESULTS After treatment with MPP⁺, the apoptotic rate, Bax protein level and ANRIL expression in SK-N-SH cells were increased (P<0.05), and the Bcl-2 protein level and miR-626 expression were decreased (P<0.05). The level of MDA in cell culture supernatants was increased (P<0.05), and the level of GSH was decreased (P<0.05). After SN treatment or ANRIL expression knock-down, decreased apoptotic rate, Bax protein level and ANRIL expression (P<0.05), and increased Bcl-2 protein level and miR-626 expression in MPP⁺-induced SK-N-SH cells were observed (P<0.05). The level of MDA in the cell culture supernatants was decreased (P<0.05), and the level of GSH was increased (P<0.05). CONCLUSION SN attenuates MPP⁺-induced damage in SK-N-SH cells by regulating ANRIL/miR-626 signaling pathway.     

Bortezomib attenuates LPS-induced inflammatory response of human monocytes through regulating miR-223/NLRP3 signaling pathway

AIM To investigate the effects of bortezomib (BTZ) on microRNA-223 (miR-223)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) signaling pathway and lipopolysaccharide (LPS)-induced inflammatory response of human monocytes. METHODS Monocytes were isolated and purified from peripheral blood of rheumatodid arthritis (RA) patients. The levels of interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in supernatants of the monocytes were determined by ELISA, and the optimal induction time of LPS and the optimal treatment concentration of BTZ were selected according to the levels of IL-6, IL-1β and TNF-α. The monocytes were divided into control group, LPS induced group and BTZ group. The level of miR-223 in the monocytes was measured by RT-qPCR, and the protein levels of NLRP3, caspase-1, suppressor of cytokine signaling 1 (SOCS1) and SH2 domain-containing inositol phosphatase-1 (SHIP-1) in the monocytes were determined by Western blot. RESULTS The monocytes successfully isolated and purified from the peripheral blood of RA patients were spherical, evenly distributed and regular in shape.And after LPS induction for 24 h, the cells were mostly spindle-shaped and aggregated. According to the levels of IL-6, IL-1β and TNF-α, the optimal induction time of LPS was 24 h, and the optimal concentration of BTZ was 50 nmol/L. Compared with control group, the levels of miR-223, SOCS1 and SHIP-1 in LPS induction group were significantly decreased (P<0.05), and the levels of NLRP3 and caspase-1 were significantly increased (P<0.05). Compared with LPS induction group, the levels of miR-223, SOCS1 and SHIP-1 in BTZ group were significantly increased (P< 0.05), and the levels of NLRP3 and caspase-1 were significantly lowered (P<0.05). CONCLUSION Bortezomib may block the activation of miR-223/NLRP3 signaling pathway, thus reducing the secretion of inflammatory factors in LPS-induced human monocytes.     

MicroRNA-142-3p modulates atherosclerosis-associated endothelial cell apoptosis via targeting Rictor

AIM To investigate the role of microRNA-142-3p （miR-142-3p） in endothelial cell apoptosis during atherosclerosis （AS） and the underlying mechanism. METHODS Human aortic endothelial cells （HAECs） were treated with oxidized low-density lipoprotein （ox-LDL）. The expression level of miR-142-3p was detected by RT-qPCR. Apoptosis was determined via flow cytometry （FCM） and caspase-3 activity assay. Prediction of the binding site between miR-142-3p and 3’-UTR of Rictor mRNA was performed by bioinformatics analysis and confirmed by dual-luciferase reporter assay. RESULTS The expression of miR-142-3p was substantially up-regulated during the ox-LDL-elicited apoptosis in HAECs （P<0.05,P<0.01）. Forced expression of miR-142-3p exacerbated apoptosis in HAECs whereas inhibition of miR-142-3p partly alleviated apoptotic cell death mediated by ox-LDL. Further analysis identified Rictor as a direct target gene of miR-142-3p, and Rictor knock-down abolished the anti-apoptotic effect of miR-142-3p inhibitor. Moreover, the Akt/endothelial nitric oxide synthase （eNOS） signaling pathway was found to mediate the beneficial effect of miR-142-3p inhibitor on endothelial cells apoptosis. CONCLUSION Down-regulation of miR-142-3p inhibits endothelial cell apoptosis and atherosclerotic development by up-regulating the expression of Rictor and activating the Akt/eNOS signaling pathway.     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）, and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）, the apoptotic rate was significantly increased （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）, and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

Advances on studies of miR-21 in vascular endothelial function and angiogenesis

MicroRNAs (miRNAs)are a class of non-coding, endogenous, single-stranded small RNA molecules composed of 19~25 nucleotides. miRNAs are widely involved in the process of human life activities. Recent studies have shown that part of miRNAs regulate the vascular endothelial function and angiogenesis. High expression of miRNA-21 is found to play important roles in the cell proliferation, cell apoptosis, cell growth and death of vascular endothelial cells. This review will focus on the recent progress related to miRNAs in vascular endothelial function and angiogenesis, providing a new insight in cardiovascular disease prevention, clinical diagnosis, prognosis and target therapeutics.     

Liraglutide ameliorates liver injury of type 2 diabetic mice via micro-RNA-33-AMPK pathway

AIM: To observe the effects of liraglutide on the level of microRNA-33 (miR-33) and the expression of AMP-activated protein kinase (AMPK) and apoptosis-related proteins in mice with type 2 diabetes mellitus (T2DM), and to explore its possible mechanism. METHODS: High-fat diet and intraperitoneal injection of streptozocin were used to establish the type 2 diabetic model in C57BL/6 mice. The mice were randomly divided into 4 groups (n=15):in control group, the normal mice were subcutaneously injected with equivalent volume of saline; in model group, the T2DM mice were subcutaneously injected with equivalent volume of saline; in low-and high-dose liraglutide treatment groups, the T2DM mice were subcutaneously injected with 100 and 200 μg·kg^-1·d^-1, respectively. After 4 weeks of administration, the levels of FBG, TG, TC, HDL-C, LDL-C, ALT and AST were determined. HE staining was used to observe the pathological changes of the liver tissues. The protein level of cleaved caspase-3 in the liver tissue was detected by the technique of immunofluorescence. The protein levels of p-AMPK/AMPK and apoptosis-related proteins were detected by Western blot. The expression of miR-33 in the liver tissues was detected by real-time PCR. RESULTS: Compared with model group, the contents of FBG, TG, TC, LDL-C, ALT and AST were decreased significantly, while the content of HDL-C was increased significantly in low-dose liraglutide group and high-dose liraglutide group (P<0.05). The protein levels of phosphorylated AMPK and Bcl-2 were up-regulated significantly, and the expression of cleaved caspase-3 was down-regulated significantly (P<0.05). The level of miR-33 was decreased significantly (P<0.01). CONCLUSION: Liraglutide alleviates liver injury in type 2 diabetic mice, and the mechanism may be associated with reducing the level of miR-33 and increasing the phosphorylation of AMPK in the liver tissues, thereby inhibiting hepatocyte apoptosis.     

Effect of miRNA-33 on ABCA1/ABCG1 expression in RAW264.7 macrophage-derived foam cells after Hcy treatment

AIM:To study whether homocysteine (Hcy) inhibits the expression of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) by microRNA-33 (miRNA-33) signaling, and reduces the efficiency of reverse cholesterol transport (RCT).METHODS:RAW264.7 macrophages were induced by oxidized low-density lipoprotein (ox-LDL) to establish foam cell model. Oil red O staining was used to determine whether the model was established successfully. miRNA-33 mimics and miRNA-33 inhibitor were transfected into the cells by Lipofectamine 2000, and the cells were exposed to Hcy at concentration of 5 mmol/L for 24 h. The intracellular lipid droplets were observed by Oil red O staining. The expression of ABCA1 and ABCG1 at mRNA and protein levels was determined by real-time PCR and Western blot. The cellular cholesterol content was analyzed by HPLC, and effluent rate of cholesterol was detected by the method of liquid scintillation counting.RESULTS:Compared with blank control group, the lipid content in miRNA-33 mimics group was increased, and the expression of ABCA1 and ABCG1 at mRNA and protein levels was decreased (P<0.05). The intracellular cholesterol content was increased gradually (P<0.05), and the cellular cholesterol efflux rate was gradually decreased (P<0.05) in miRNA-33 mimics group. Compared with blank control group, the testing results in miRNA-33 inhibitor group were the opposition of those in miRNA-33 mimics group (P<0.05). No diffe-rence of the above indexes among blank control group, miRNA-33 mimics-NC group and miRNA-33 inhibitor-NC group was observed.CONCLUSION:Hcy inhibits the mRNA and protein expression of ABCA1 and ABCG1 through miRNA-33 signaling, and reduces the efficiency of RCT in RAW264.7 macrophage-derived foam cells.