MiR-501-5p alleviates cardiac dysfunction in septic patients through targeting NR4A3 to prevent its binding with Bcl-2.

Sepsis-induced myocardial dysfunction is a common complication in septic patients. To date, a limited number of biomarkers that could predict cardiomyocyte apoptosis have been explored. In this study, we successfully established a cecal ligation and puncture (CLP)-induced septic model, and it was found that miR-501-5p expression was down-regulated in peripheral blood samples of septic patients with cardiac dysfunction, lipopolysaccharide (LPS)-induced cardiomyocytes, and the myocardium and peripheral blood in the septic model. Moreover, it was revealed that miR-501-5p overexpression could increase left ventricular diastolic pressure (LVDP), fractional shortening (FS), ejection fraction (EF), and maximum rate of the rise of left ventricular pressure (+dp/dt) in vivo, while it decreased the levels of myocardial injury-related indicators. In addition, LPS induction accelerated apoptosis and elevated the inflammation in HL-1 and HCM cells, which could be reversed by miR-501-5p overexpression. Mechanistically, we considered nuclear receptor subfamily 4 group A member 3 (NR4A3) as the target of miR-501-5p, and it was found that miR-501-5p prevented the binding between NR4A3 and Bcl-2. It was found that miR-501-5p exerted an inhibitory effect on cardiomyocyte apoptosis and inflammation in a NR4A3-dependent manner. Overall, our results may provide evidence for consideration of miR-501-5p in the therapy of sepsis.

MicroRNA-770 affects proliferation and cell cycle transition by directly targeting CDK8 in glioma.

BACKGROUND: MicroRNAs play crucial roles in tumorigenesis and tumor progression. miR-770 has been reported to be downregulated in several cancers and affects cancer cell proliferation, apoptosis, metastasis and drug resistance. However, the role and underlying molecular mechanism of miR-770 in human glioma remain unknown and need to be further elucidated. METHODS: The expression of miR-770 in glioma tissues and cell lines was measured by quantitative real-time PCR (qRT-PCR) to explore the association of miR-770 expression with clinicopathological characteristics. The expression of CDK8 was detected by qRT-PCR and Western blotting in glioma tissues. A target prediction program and a dual-luciferase reporter assay were used to confirm that CDK8 is a target gene of miR-770. MTT and cell counting assays were used to assess the effect of miR-770 on glioma cell proliferation. The cell cycle distribution and apoptosis were examined by flow cytometry. CDK8 siRNA and overexpression were used to further confirm the function of the target gene. RESULTS: We demonstrated that miR-770 expression was downregulated in human glioma tissues and cell lines. The overexpression of miR-770 inhibited glioma cell proliferation and cell cycle G1-S transition and induced apoptosis. The inhibition of miR-770 facilitated cell proliferation and G1-S transition and suppressed apoptosis. miR-770 expression was inversely correlated with CDK8 expression in glioma tissues. CDK8 was confirmed to be a direct target of miR-770 by using a luciferase reporter assay. The overexpression of miR-770 decreased CDK8 expression at both the mRNA and protein levels, and the suppression of miR-770 increased CDK8 expression. Importantly, CDK8 silencing recapitulated the cellular and molecular effects observed upon miR-770 overexpression, and CDK8 overexpression eliminated the effects of miR-770 overexpression on glioma cells. Moreover, both exogenous expression of miR-770 and silencing of CDK8 resulted in suppression of the Wnt/ß-catenin signaling pathway. CONCLUSIONS: Our study demonstrates that miR-770 inhibits glioma cell proliferation and G1-S transition and induces apoptosis through suppression of the Wnt/ß-catenin signaling pathway by targeting CDK8. These findings suggest that miR-770 plays a significant role in glioma progression and serves as a potential therapeutic target for glioma.

Hepatic infarction induced by HELLP syndrome: a case report and review of the literature.

BACKGROUND: HELLP syndrome is a rare disease in China, and 20% of patients with severe preeclampsia have been accompanied with HELLP syndrome, which is characterized by the presence of hemolysis, elevated liver enzymes and low platelet count. CASE PRESENTATION: In this case, we reported that a patient with preeclampsia was diagnosed with HELLP syndrome. Furthermore, hepatic infarction also was found via the computed tomographic (CT) images, which showed peripheral wedge-shaped inhomogeneous low attenuation in the right hepatic lobes via plain CT scan, and the low-density shadow and mottled appearance in the same areas where vessels were seen coursing through them via contrast-enhanced CT scan. CONCLUSIONS: Besides typical clinical manifestations of the pregnant patient with preeclampsia, the typical laboratory evidences were elevated liver enzymes and thrombocytopenia. The abdominal CT scan showed imaging features of hepatic infarction, which was helpful to identify the rare complication of HELLP syndrome. Thus, we diagnosed a patient with HELLP syndrome with hepatic infarction, though the patient had no chance to do the liver biopsy.

Glycolipid Metabolism Disorder in the Liver of Obese Mice Is Improved by TUDCA via the Restoration of Defective Hepatic Autophagy.

Objective. Tauroursodeoxycholic acid (TUDCA) has been considered an important regulator of energy metabolism in obesity. However, the mechanism underlying how TUDCA is involved in insulin resistance is not fully understood. We tested the effects of TUDCA on autophagic dysfunction in obese mice. Material and Methods. 500 mg/kg of TUDCA was injected into obese mice, and metabolic parameters, autophagy markers, and insulin signaling molecular were assessed by Western blotting and real-time PCR. Results. The TUDCA injections in the obese mice resulted in a reduced body weight gain, lower blood glucose, and improved insulin sensitivity compared with obese mice that were injected with vehicle. Meanwhile, TUDCA treatment not only reversed autophagic dysfunction and endoplasmic reticulum stress, but also improved the impaired insulin signaling in the liver of obese mice. Additionally, the same results obtained with TUDCA were evident in obese mice treated with the adenoviral Atg7. Conclusions. We found that TUDCA reversed abnormal autophagy, reduced ER stress, and restored insulin sensitivity in the liver of obese mice and that glycolipid metabolism disorder was also improved via the restoration of defective hepatic autophagy.

Hypoxia-regulated neurotrophin-3 expression by multicopy hypoxia response elements reduces apoptosis in PC12 cells.

We have previously reported that 5 copies of the hypoxia response element (HRE) can conditionally regulate brain-derived neurotrophic factor gene expression under hypoxic/ischemic conditions in mice. In the present study, we investigated the controlled expression of neurotrophin-3 (NT-3) by HRE under hypoxic conditions and determined the protective effects of conditionally expressed NT-3 on hypoxia-induced apoptosis in PC12 cells. Five copies of the HRE (5HRE) and the simian virus 40 minimal promoter (SV40mp) were employed to construct a cassette, and transfer of therapeutic gene, NT-3, into PC12 cells was achieved using a retroviral vector. Our results showed that the retroviral vector, pLNC-5HRE-NT3, was successfully constructed and transfected into PC12 cells. Compared with normal conditions, in which NT-3 was expressed at low levels, the expression of NT-3 significantly increased under hypoxic conditions in 5HRE-NT3 transgenic PC12 cells (P<0.05). By contrast, in NT-3 transgenic PC12 cells without HRE, we found no significant difference in NT-3 expression between the normoxic and hypoxic groups. The conditional adjustment of NT-3 expression by 5HRE significantly reduced apoptosis induced by hypoxia in 5HRE-NT3 transgenic PC12 cells (P<0.05) but not in 5HRE-enhanced green fluorescent protein (EGFP) transgenic PC12 cells and PC12 cells without gene transfer. In addition, the hypoxia-induced upregulation of both p38 and caspase-3 activities was suppressed in 5HRE-NT3 transgenic PC12 cells under hypoxic conditions (P<0.05). Taken together, these results demonstrate that 5HRE-SV40mp regulates NT-3 gene expression in response to hypoxia in PC12 cells. The data presented in this study may prove useful in future gene therapy studies for the treatment of ischemic diseases.

Adenovirus-mediated brain-derived neurotrophic factor expression regulated by hypoxia response element protects brain from injury of transient middle cerebral artery occlusion in mice.

Some gene expression may be regulated by hypoxia-responsive element (HRE) that is bound by hypoxia-inducible factor-1 (HIF-1) which is up-regulated during cerebral ischemia. To explore ischemia/hypoxia-controlled expression and the neuroprotective effects of brain-derived neurotrophic factor (BDNF) after ischemic brain injury, an adenoviral vector using five copies of hypoxia response element (HRE) in the vascular endothelial growth factor gene to regulate the expression of BDNF gene (Ad5HRE:BDNF) was constructed, and its efficacy was verified for driving BDNF expression in cultured Hela cells under hypoxic condition by ELISA. We found that the concentration of BDNF in the Ad5HRE:BDNF-transfected culture media was 28-fold greater in a hypoxic condition than under normoxia. To examine the effect of Ad5HRE:BDNF on ischemic brain injury in vivo, Ad5HRE:BDNF was injected into right caudate putamen of adult mice 7 days prior to 60 min transient middle cerebral artery occlusion (MCAO). It was found that exogenous BDNF expression was increased in the Ad5HRE-BDNF-treated group and infarct volume of the Ad5HRE:BDNF-treated group at 3 days after MCAO was significantly smaller than that of vehicle- or AdNull-treated groups. Moreover, Ad5HRE:BDNF injection resulted in significantly improved sensorimotor scores 7 days after MCAO and induced a reduction in the number of Fluoro-Jade B-positive neurons and TUNEL-positive cells, compared with vehicle- or AdNull-injection. Our findings suggest that BDNF expression could be regulated in hypoxia/ischemia condition with five copies of HRE and ameliorates ischemic brain injury in a mouse focal cerebral ischemia model.

[The expression of enhanced green fluorescent protein gene on cultured human fetal retinal progenitor cells].

PURPOSE: To investigate the transfer efficiency and transient expression of plasmid vector coding enhanced green fluorescent protein (EGFP)gene pEGFP-N1 which was transferred into primary cultured human fetal retinal progenitor cells. To build up a tracking method for study of retinal progenitor cells transplantation. METHODS: pEGFP-N1 plasmid was amplified in E.coli, primary cultured human fetal retinal progenitor cells transferred with pEGFP-N1 by means of CaCl2. The transfer efficiency was evaluated by flow cytometry. Transient expression in vitro was observed by fluorescent microscope and laser scanning confocal microscope. RESULTS: The transfer efficiency of pEGFP-N1 into primary cultured human fetal retinal progenitor cells could maximumly reach to 32.45% during 48 hours. There was obvious expression of pEGFP-N1 in vitro at 48th hour after transferring. CONCLUSION: pEGFP-N1 is an ideal transient expression vector for primary cultured human fetal retinal progenitor cells in vitro. It is a useful tracer for transplantation of retinal progenitor cells.