circROCK1 Promotes septic myocardial injury through regulating miR-96-5p/OXSR1 axis.

OBJECTIVE: A recent high-throughput sequencing showed that circular RNA Rho-associated kinase 1 (circROCK1) is abnormally highly expressed in sepsis, but whether it is involved in sepsis development remains unclear. The objective of this study was to investigate the biological function of circROCK1 in sepsis-induced myocardial injury and reveal its potential downstream molecular mechanism. METHODS: Real-time reverse transcriptase-polymerase chain reaction was applied to detect circROCK1 and miR-96-5p expressions in the serum of septic patients. Spearman correlation analysis examined the correlation between circROCK1 and the clinicopathological characteristics of septic patients. The Cecal puncture and ligation (CLP) method was used to establish an in vivo sepsis model. circROCK1 and miR-96-5p expressions in mice were modified by injection of lentivirus or oligonucleotide. The left ventricular systolic pressure, left ventricular end-diastolic pressure, and the maximum increase/decrease rate of left ventricular pressure were checked. ELISA was applied to detect inflammatory factors levels as well as myocardial injury markers levels. Hematoxylin and eosin staining was performed to observe pathological changes in myocardial tissues, and Western blot examined phosphorylated nuclear factor (NF)-κB and oxidative stress-responsive 1 (OXSR1) expression. Dual luciferase reporter experiment was conducted to confirm the targeting relationship between circROCK1, OXSR1, and miR-96-5p. RESULTS: circROCK1 and OXSR1 were highly expressed in sepsis and miR-96-5p was under-expressed. circROCK1 was positively correlated with serum creatinine, C-reactive protein, procalcitonin, and sequential organ failure assessment scores in septic patients. Silencing circROCK1 could improve the diastolic and systolic function of CLP mice, as well as myocardial damage, reduce myocardial tissue edema and necrosis, and inhibit inflammatory factor level and phosphorylated NF-κB expression. Down-regulating miR-96-5p promoted myocardial injury in CLP mice. Silencing circROCK1 and miR-96-5p inhibited and promoted OXSR1 expression, respectively. Both circROCK1 and OXSR1 had a targeting relationship with miR-96-5p. CONCLUSION: CircROCK1 promotes myocardial injury in septic mice by regulating the miR-96-5p/OXSR1 axis, and it can be used as a potential target for treating septic myocardial dysfunction.

High expression of miR-374a-5p inhibits the proliferation and promotes differentiation of Rencell VM cells by targeting Hes1.

The proliferation and differentiation of NSCs are regulated by miRNAs. This study investigated the role of miR-374a-5p in the proliferation and differentiation of ReNcell VM cells. ReNcell VM cells were transfected with miR-374a-5p mimic, miR-374a-5p inhibitor and Hes1, respectively. Cell proliferation was detected by clone formation assay. Target gene for miR-374a-5p was predicted by TargetScan and confirmed by dual-luciferase reporter. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to detect the expressions of relative genes. After culturing the cells in differentiation medium, the ReNcell VM cells differentiated into ßIII-tubulin (Tuj1)-positive neurons and GFAP-positive astrocytes. The miR-374a-5p expression was increased as the cells continued to differentiate. Hes1, which was predicted to be the target gene for miR-374a-5p, was low-expressed during cell differentiation. The miR-374a-5p mimic decreased cell clones, inhibited the expressions of ki-67 and Nestin, but increased those of Tuj1 and GFAP. However, miR-374a-5p inhibitor produced the opposite effects to miR-374a-5p mimic. Hes1 increased the expressions of ki-67 and Nestin, but decreased those of Tuj1 and GFAP, moreover, Hes1 reversed the role of miR-374a-5p mimic. MiR-374a-5p inhibited the proliferation of Rencell VM cells and promoted the differentiation of NSCs by reducing the Hes1 expression.

miR-4286/TGF-ß1/Smad3-Negative Feedback Loop Ameliorated Vascular Endothelial Cell Damage by Attenuating Apoptosis and Inflammatory Response.

Atherosclerosis (AS), known as the chronic inflammatory disease, results from the dysfunction of vascular endothelial cells (VECs). Transforming growth factor-ß1 (TGF-ß1) has been reported to be induced by oxidized low-density lipoprotein (ox-LDL) and contribute to AS-related vascular endothelial cell damage. This work planned to study the mechanism of TGF-ß1 in vascular endothelial cell damage. We found that TGF-ß1 was activated by ox-LDL in human umbilical vascular endothelial cells (HUVECs). Silence of TGF-ß1 reversed the inductive effect of ox-LDL on apoptosis and inflammatory response of HUVECs. Mechanistically, microRNA-4286 (miR-4286) targeted and inhibited TGF-ß1 to inhibit Smad3, and Smad3 bound to the promoter of miR-4286 to repress its transcription. Rescue assays indicated that miR-4286 ameliorated the ox-LDL-induced apoptosis and inflammatory response through inhibiting TGF-ß1. In conclusion, our study first demonstrated that miR-4286/TGF-ß1/Smad3-negative feedback loop ameliorated vascular endothelial cell damage by attenuating apoptosis and inflammatory response, providing new thoughts for promoting the treatment of AS.

Transferrin plays a central role in coagulation balance by interacting with clotting factors.

Coagulation balance is maintained through fine-tuned interactions among clotting factors, whose physiological concentrations vary substantially. In particular, the concentrations of coagulation proteases (pM to nM) are much lower than their natural inactivator antithrombin (AT, ~ 3 µM), suggesting the existence of other coordinators. In the current study, we found that transferrin (normal plasma concentration ~40 µM) interacts with fibrinogen, thrombin, factor XIIa (FXIIa), and AT with different affinity to maintain coagulation balance. Normally, transferrin is sequestered by binding with fibrinogen (normal plasma concentration ~10 µM) at a molar ratio of 4:1. In atherosclerosis, abnormally up-regulated transferrin interacts with and potentiates thrombin/FXIIa and blocks AT's inactivation effect on coagulation proteases by binding to AT, thus inducing hypercoagulability. In the mouse model, transferrin overexpression aggravated atherosclerosis, whereas transferrin inhibition via shRNA knockdown or treatment with anti-transferrin antibody or designed peptides interfering with transferrin-thrombin/FXIIa interactions alleviated atherosclerosis. Collectively, these findings identify that transferrin is an important clotting regulator and an adjuster in the maintenance of coagulation balance and modifies the coagulation cascade.

[Phenotype Analysis of 78 Cases of Abnormal Hemoglobin E Homozygotes].

OBJECTIVE: To analyze the hematological characteristics of HbE homozygotes. METHODS: Complete blood cells count and hemoglobin electrophoresis were used for phenotypic analysis of 78 cases with HbE homozygotes from Yunnan province, China. The PCR-fluorescence hybridization was used to detect the common gene mutation of thalassemia. The hematological indexes, including MCV, MCH, Hb, HbA2, HbF and HbE were statistically analyzed between groups with different sex, ages and compound α thalassemia status. RESULTS: In HbE homozygotes (HbEE), 89.5% (17/19) children presented mild to moderate microcytic hypochromic anemia, and 10.5% of them presented moderate anemia. 39.6% (19/48) of women with HbEE developed mild anemia ,while 11 cases of male with HbE homozygotes were asymptomatic. The levels of MCV and MCH in HbE homozygotes increased by co-inheritance of α thalassemia mutation. CONCLUSION: The clinical phenotype of HbE homozygote shows highly heterogeneous, which is relates with age, sex and co-inheriting α-globin genotypes. In Hb EE women and children are more likely to develop mild to moderate anemia. The microcytic hypochromic anemia degree is relieved when HbEE combined with α- thalassemia.

[Analysis of clinical phenotype and genotype of unstable Hemoglobin Rush].

OBJECTIVE: To analyze the hematological and genetic characteristics of unstable hemoglobin Rush (Hb Rush) and compound heterozygote of Hb Rush and thalassemia. METHODS: Peripheral blood samples and genomic DNA from three patients (including two ethnic Dai and one Han Chinese) with anemia of undetermined origin were collected. Hematological phenotypes of these patients were determined through red blood cell analysis and hemoglobin electrophoresis. Genotypes of alpha- and beta-globin genes, -158 XmnⅠ polymorphic site of Gγ promoter region, and haplotypes of 7 polymorphic restriction sites in the beta-globin gene cluster were determined using PCR-based methods and DNA sequencing. RESULTS: All patients have presented hypochromic microcytic anemia and hemoglobin fraction with significant increased measurement (30.5%-59.2%) in the region of fetal hemoglobin during alkaline medium electrophoresis. DNA analysis suggested that all patients have carried mutations leading to the unstable hemoglobin Rush (HBB codon 101, GAG>CAG, Glu>Gln). Two of them were compound heterozygotes of Hb Rush and thalassemia mutations of -α 3.7,CD17 and Hb E, respectively. Hb Rush mutation was associated with various haplotypes of the ß-globin gene cluster. No significant association was found between increased abnormal hemoglobin fraction in the region of Hb F and the polymorphism of Gγ promoter or large deletion of the beta-globin gene cluster. CONCLUSION: This study has confirmed the distribution of Hb Rush among various Chinese populations and is the third report of its kind. Hb Rush can result in increased measurement of hemoglobin fraction in the region of fetal hemoglobin (Hb F) during routine hemoglobin electrophoresis under alkaline condition. Hb Rush heterozygote alone can lead to hypochromic microcytic anemia and thalassemia-like phenotype. Prenatal diagnosis of Hb Rush is necessary for carriers.

Mitochondrial DNA Haplogroup A Decreases the Risk of Drug Addiction but Conversely Increases the Risk of HIV-1 Infection in Chinese Addicts.

Drug addiction is one of the most serious social problems in the world today and addicts are always at a high risk of acquiring HIV infection. Mitochondrial impairment has been reported in both drug addicts and in HIV patients undergoing treatment. In this study, we aimed to investigate whether mitochondrial DNA (mtDNA) haplogroup could affect the risk of drug addiction and HIV-1 infection in Chinese. We analyzed mtDNA sequence variations of 577 Chinese intravenous drug addicts (289 with HIV-1 infection and 288 without) and compared with 2 control populations (n = 362 and n = 850). We quantified the viral load in HIV-1-infected patients with and without haplogroup A status and investigated the potential effect of haplogroup A defining variants m.4824A > G and m.8794C > T on the cellular reactive oxygen species (ROS) levels by using an allotopic expression assay. mtDNA haplogroup A had a protective effect against drug addiction but appeared to confer an increased risk of HIV infection in addicts. HIV-1-infected addicts with haplogroup A had a trend for a higher viral load, although the mean viral load was similar between carriers of haplogroup A and those with other haplogroup. Hela cells overexpressing allele m.8794 T showed significantly decreased ROS levels as compared to cells with the allele m.8794C (P = 0.03). Our results suggested that mtDNA haplogroup A might protect against drug addiction but increase the risk of HIV-1 infection. The contradictory role of haplogroup A might be caused by an alteration in mitochondrial function due to a particular mtDNA ancestral variant.