Hypoxic preconditioning of myoblasts implanted in a tissue engineering chamber significantly increases local angiogenesis via upregulation of myoblast vascular endothelial growth factor‐A expression and downregulation of miRNA‐1, miRNA‐206 and angiopoietin‐1

Vascularization is a major hurdle for growing three‐dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR‐1 and miR‐206 (p < 0.05) and angiopoietin‐1 (p < 0.05) with upregulation of vascular endothelial growth factor‐A (VEGF‐A; p < 0.05). The miR‐1 and angiopoietin‐1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR‐206 in L6 myoblasts caused a significant increase in VEGF‐A expression (p < 0.05), further establishing that changes in VEGF‐A expression are influenced by miR‐206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel? into isolated bilateral tissue engineering chambers incorporating a flow‐through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin‐1, miR‐1 and miR‐206. The relatively simple strategy of hypoxic preconditioning of implanted cells ‐ including non‐stem cell types – has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis.     

Multiple functions of miR‐8‐3p in the development and metamorphosis of the red flour beetle,Tribolium castaneum

The microRNA miR‐8‐3p is conserved among insects and closely involved in development and immunity, but its functions in vivo are unexplored in the red flour beetle, Tribolium castaneum. Here, we show that miR‐8‐3p was highly expressed in late larva and early adult stages, as determined by quantitative real‐time PCR. It was enriched in the fat body and cuticle in late larval tissues and abundant in the head and cuticle in early adult tissues, indicating this microRNA plays important roles during T. castaneum development. Specific inhibition of miR‐8‐3p in late larvae led to metamorphosis defects in the development of wings, eyes, legs and embryo. Moreover, a series of genes related to organism development were identified as miR‐8‐3p targets by computational prediction and microRNA–messenger RNA interaction validation, including Wingless, Eyg, Fpps and Sema‐1a. These genes were critical for the regulation of the larva‐to‐adult transition. Eyg, as a functional target of miR‐8‐3p, participates in eye development, which was further confirmed by luciferase assay and loss‐of‐function analyses. In brief, miR‐8‐3p is broadly involved in the development of wings, eyes and legs through its target genes and has extensive regulatory roles during T. castaneum development.     

miRNA‐1 and miRNA‐133a are involved in early commitment of pluripotent stem cells and demonstrate antagonistic roles in the regulation of cardiac differentiation

miRNA‐1 (miR‐1) and miRNA‐133a (miR‐133a) are muscle‐specific miRNAs that play an important role in heart development and physiopathology. Although both miRNAs have been broadly studied during cardiogenesis, the mechanisms by which miR‐1 and miR‐133a could influence linage commitment in pluripotent stem cells remain poorly characterized. In this study we analysed the regulation of miR‐1 and miR‐133a expression during pluripotent stem cell differentiation [P19.CL6 cells; embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] and investigated their role in DMSO and embryoid body (EB)‐mediated mesodermal and cardiac differentiation by gain‐ and loss‐of‐function studies, as well as in vivo, by the induction of teratomas. Gene expression analysis revealed that miR‐1 and miR‐133a are upregulated during cardiac differentiation of P19.CL6 cells, and also during ESC and iPSC EB differentiation. Forced overexpression of both miRNAs promoted mesodermal commitment and a concomitant decrease in the expression of neural differentiation markers. Moreover, overexpression of miR‐1 enhanced the cardiac differentiation of P19.CL6, while miR‐133a reduced it with respect to control cells. Teratoma formation experiments with P19.CL6 cells confirmed the influence of miR‐1 and miR‐133a during in vivo differentiation. Finally, inhibition of both miRNAs during P19.CL6 cardiac differentiation had opposite results to their overexpression. In conclusion, gene regulation involving miR‐1 and miR‐133a controls the mesodermal and cardiac fate of pluripotent stem cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Genetic polymorphisms of microRNA machinery genes predict overall survival of esophageal squamous carcinoma

Background

MicroRNA (miRNA)‐related single nucleotide polymorphisms (miR‐SNPs) in miRNA processing machinery genes are implicated in carcinogenesis, as they change the expression profiles of miRNA. Six miR‐SNPs in miRNA processing machinery genes, including Dicer (rs3742330), RAN (rs14035), XPO5 (rs11077), TNRC6B (rs9623117), GEMIN3 (rs197412), and GEMIN4 (rs2740348), were evaluated for their association with esophageal squamous cell carcinoma (ESCC).

Methods

The miR‐SNP of the miRNA processing genes were genotyped using the polymerase chain reaction‐ligase detection reaction (PCR‐LDR) assay, while the XPO5 expression levels in ESCC tissues were measured by immunochemistry methods.

Results

Patients carrying the rs11077 AA allele exhibited a significantly increased lifespan than AC+CC carriers, as determined by univariate and multivariate analyses (relative risk: 2.490; 95% confidence interval [CI]: 1.225‐5.058; P=.012). Furthermore, the rs11077 AA genotype displayed a trend for high XPO5 expression in ESCC tissues by immunochemistry analysis, and these high XPO5 expression levels were also associated with high survival rates among ESCC patients.

Conclusion

Our results suggested that the miRNA machinery gene expression‐associated miR‐SNPs would modify cancer outcomes; in this light, XPO5 may be an important new target for ESCC therapy.

     

MicroRNA miR‐252 targets mbt to control the developmental growth of Drosophila

Developmental growth is an intricate process involving the coordinated regulation of the expression of various genes, and microRNAs (miRNAs) play crucial roles in diverse processes throughout animal development. The ecdysone‐responsive miRNA, miR‐252, is normally upregulated during the pupal and adult stages of Drosophila development. Here, we found that overexpression of miR‐252 in the larval fat body decreased total tissue mass through a reduction in both cell size and cell number, causing a concomitant decrease in larval size. Furthermore, miR‐252 overexpression led to a delayed larval‐to‐pupal transition with defective anterior spiracle eversion, as well as a decrease in adult size and mass. Conversely, adult flies lacking miR‐252 showed an increase in mass compared with control flies. We found that miR‐252 directly targeted mbt, encoding a p21‐activated kinase, to repress its expression. Notably, co‐overexpression of mbt rescued the developmental and growth defects associated with miR‐252 overexpression, indicating that mbt is a biologically relevant target of miR‐252. Overall, our data support a role for the ecdysone/miR‐252/mbt regulatory axis in growth control during Drosophila development.     

MicroRNA miR‐8 regulates multiple growth factor hormones produced from Drosophila fat cells

Metabolic organs such as the liver and adipose tissue produce several peptide hormones that influence metabolic homeostasis. Fat bodies, the Drosophila counterpart of liver and adipose tissues, have been thought to analogously secrete several hormones that affect organismal physiology, but their identity and regulation remain poorly understood. Previous studies have indicated that microRNA miR‐8, functions in the fat body to non‐autonomously regulate organismal growth, suggesting that fat body‐derived humoral factors are regulated by miR‐8. Here, we found that several putative peptide hormones known to have mitogenic effects are regulated by miR‐8 in the fat body. Most members of the imaginal disc growth factors and two members of the adenosine deaminase‐related growth factors are up‐regulated in the absence of miR‐8. Drosophila insulin‐like peptide 6 (Dilp6) and imaginal morphogenesis protein‐late 2 (Imp‐L2), a binding partner of Dilp, are also up‐regulated in the fat body of miR‐8 null mutant larvae. The fat body‐specific reintroduction of miR‐8 into the miR‐8 null mutants revealed six peptides that showed fat‐body organ‐autonomous regulation by miR‐8. Amongst them, only Imp‐L2 was found to be regulated by U‐shaped, the miR‐8 target for body growth. However, a rescue experiment by knockdown of Imp‐L2 indicated that Imp‐L2 alone does not account for miR‐8's control over the insect's growth. Our findings suggest that multiple peptide hormones regulated by miR‐8 in the fat body may collectively contribute to Drosophila growth.     

Evaluation of the poly(ADP‐ribose) polymerase‐1 gene variants in Alzheimer's disease

Amyloid peptide is thought to play a critical role in neuronal death in Alzheimer's disease (AD), most likely through oxidative stress. Free radical‐related injury leads to DNA breaks, which subsequently activates the repair enzyme poly(ADP‐ribose) polymerase‐1 (PARP‐1). In this study, the relationship between genetic variants situated at the PARP‐1 gene and AD development was investigated. We performed a case and control study from a Taiwanese population enrolled 120 AD patients and 111 healthy controls by using a polymerase chain reaction restriction fragment length polymorphism approach for two PARP‐1 exonic polymorphisms, 414C/T (rs1805404) and 2456T/C (rs1136410), corresponding to protein residues at positions 81Asp/Asp and762Val/Ala. There were no significant differences in allele or genotype frequencies for either PARP‐1 gene variant between the case and control groups; however, upon analysis of the haplotype distribution, four haplotypes (Hts) were identified. We found that the distributions of Ht3‐TT and Ht4‐CC were significantly associated with an increased risk of AD (P<0.0001), whereas the Ht1‐TC haplotype showed a protective effect for cases compared with the control group (P<0.05). These results reveal that the PARP‐1 gene is highly associated with AD susceptibility and might contribute to a critical mechanism that mediates cell survival or death as a response to cytotoxic stress. J. Clin. Lab. Anal. 24:182–186, 2010. © 2010 Wiley‐Liss, Inc.     

Transgenic characterization of two testis‐specific promoters in the silkworm,Bombyx mori

Sex‐specific regulatory elements are key components for developing insect genetic sexing systems. The current insect genetic sexing system mainly uses a female‐specific modification system whereas little success was reported on male‐specific genetic modification. In the silkworm Bombyx mori, a lepidopteran model insect with economic importance, a transgene‐based, female‐specific lethality system has been established based on sex‐specific alternative splicing factors and a female‐specific promoter BmVgp (vitellogenin promoter) has been identified. However, no male‐specific regulatory elements have yet been identified. Here we report the transgenic identification of two promoters that drive reporter gene expression in a testis‐specific manner in B. mori. Putative promoter sequences from the B. mori Radial spoke head 1 gene (BmR1) and beta‐tubulin 4 gene (Bmβ4) were introduced using piggybac‐based germline transformation. In transgenic silkworms, expression of the reporter gene enhanced green fluorescent protein (EGFP) directed by either BmR1 promoter (BmR1p) or Bmβ4p showed precisely testis‐specific manners from the larval to adult stage. Furthermore, EGFP expression of these two transgenic lines showed different localization in the testis, indicating that BmR1p or Bmβ4p might be used as distinct regulatory elements in directing testis‐specific gene expression. Identification of these testis‐specific promoters not only contributes to a better understanding of testis‐specific gene function in insects, but also has potential applications in sterile insect techniques for pest management.     

Identification of miRNAs as potential modulators of tissue factor expression in patients with systemic lupus erythematosus and antiphospholipid syndrome

Summary. Background: Tissue factor (TF) is the main initiator of the coagulation cascade and elements that may upregulate its expression might provoke thrombotic events. Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are autoimmune diseases characterized by a high TF expression in monocytes. Objectives: To examine the role of microRNAs (miRNAs) in TF expression and to evaluate their levels in SLE and APS patients. Methods: An in silico search was performed to find potential putative binding sites of miRNAs in TF mRNA. In vitro validation was performed transfecting cells expressing TF (THP‐1 and MDA‐MB‐231) with oligonucleotide miRNA precursors and inhibitors. Additionally, reporter assays were performed to test for the binding of miR‐20a to TF mRNA. Levels of miRNAs and TF were measured by quantitative (qRT‐PCR) in patients with APS and SLE. Results: Overexpression of miRNA precursors, but not inhibitors, of two of the members of cluster miR‐17～92, for example miR‐19b and miR‐20a, in cells expressing TF decreased TF mRNA, protein levels, and procoagulant activity between 30% and 60%. Reporter assays showed that miR‐20a binds to TF mRNA. Finally, we measured levels of miR‐19b and miR‐20a in monocytes from patients with APS and SLE and observed significantly lower miRNAs levels in comparison with healthy subjects inversely correlated with the levels of TF. Conclusions: Down‐regulation of miR‐19b and miR‐20a observed in patients with SLE and APS could contribute to increased TF expression and thus provoke the hypercoagulable state characteristic of these patients.     

A MC motif in silkworm Argonaute 1 is indispensible for translation repression

Small RNA‐mediated gene silencing is a fundamental gene regulatory mechanism, which is conserved in many organisms. Argonaute (Ago) family proteins in the RNA‐induced silencing complex (RISC) play crucial roles in RNA interference (RNAi) pathways. In the silkworm Bombyx mori, four Ago proteins have been identified, named as Ago1, Ago2, Ago3 and Siwi. Ago2 participates in double‐stranded RNA (dsRNA)‐induced RNAi, whereas Ago3 and Siwi are involved in the Piwi‐interacting RNA (piRNA) pathway. However, there is no experimental evidence concerning silkworm Ago1 (BmAgo1) in the RNAi mechanism. In the present study, we analysed the function of BmAgo1 in the microRNA (miRNA)‐mediated RNAi pathway using tethering and miRNA sensor reporter assays. These results clearly demonstrate that BmAgo1 plays an indispensable role in translation repression in silkworm. Moreover, coimmunoprecipitation data indicated that BmAgo1 interacts with BmDcp2, an orthologue of mRNA‐decapping enzyme 2 (Dcp2) protein in the Drosophila processing‐bodies (P‐bodies). Substitutions of two conserved phenylalanines (F522 and F557) by valines in the MC motif strongly impaired the function of BmAgo1 in translation repression and its localization in P‐bodies, suggesting that these two amino acid residues in the MC motif of BmAgo1 are prerequisites for mRNA translation repression in B. mori.     

MicroRNA‐132 directs human periodontal ligament‐derived neural crest stem cell neural differentiation

Neurogenesis is the basis of stem cell tissue engineering and regenerative medicine for central nervous system (CNS) disorders. We have established differentiation protocols to direct human periodontal ligament‐derived stem cells (PDLSCs) into neuronal lineage, and we recently isolated the neural crest subpopulation from PDLSCs, which are pluripotent in nature. Here, we report the neural differentiation potential of these periodontal ligament‐derived neural crest stem cells (NCSCs) as well as its microRNA (miRNA) regulatory mechanism and function in NCSC neural differentiation. NCSCs, treated with basic fibroblast growth factor and epidermal growth factor‐based differentiation medium for 24 days, expressed neuronal and glial markers (βIII‐tubulin, neurofilament, NeuN, neuron‐specific enolase, GFAP, and S100) and exhibited glutamate‐induced calcium responses. The global miRNA expression profiling identified 60 upregulated and 19 downregulated human miRNAs after neural differentiation, and the gene ontology analysis of the miRNA target genes confirmed the neuronal differentiation‐related biological functions. In addition, overexpression of miR‐132 in NCSCs promoted the expression of neuronal markers and downregulated ZEB2 protein expression. Our results suggested that the pluripotent NCSCs from human periodontal ligament can be directed into neural lineage, which demonstrate its potential in tissue engineering and regenerative medicine for CNS disorders.     

Transient Bcl-2 gene down-expression in circulating mononuclear cells of severe sepsis patients who died despite appropriate intensive care

Objective To assess the levels of expression of the antiapoptotic gene Bcl-2 and the proapoptotic gene Bax in circulating mononuclear cells (CMNC) harvested during the course of severe sepsis (SS) in formerly non-immunocompromised patients undergoing hospital-acquired infection, in parallel to cytokine levels.Design Prospective study.Setting Intensive care unit.Participants A total of 24 patients without immunodeficiency undergoing standard goal-directed therapy for nosocomial SS, 10 critically ill patients without sepsis, and 10 healthy controls.Interventions Blood was collected before infection and within 12 h, 1, 3 and 7 days after fever onset, to determine plasma concentrations of IL-6, IL-10, TNF-alpha, C-reactive protein, whole blood cell counts, lymphocyte subsets, annexin V labelling for apoptosis, and Bax and Bcl-2 relative RNA expression by real-time polymerase chain reaction.Results SS patients displayed increased cytokine concentrations, TNF-alpha being significantly increased at full-blown sepsis. Within 12 h after onset of infection, lymphocyte counts were lower in SS patients than in critically ill controls (p=0.001), and this phenomenon was marked in CD4+ and CD8+ subsets (p<0.001). This was associated with enhanced apoptosis in CMNC (15.7±8.7% vs 3.4±2.1%, p<0.001) and a significant down-expression of the Bcl-2 gene throughout the study (p<0.05). In contrast, the expression of Bax did not change significantly. Within 12 h of fever onset, non-survivors expressed a 10-fold down-expression of Bcl-2 when compared to survivors (p<0.001).Conclusions An early transient down-expression of the gene Bcl-2 occurred in CMNC harvested from SS patients who died despite intensive care. In contrast, the expression of the gene Bax did not change significantly.This work was supported by the Délégation à la Recherche Clinique des Hôpitaux Universitaires de Strasbourg (PHRC Régional 2001-Grant 2629).Pascal Bilbault and Thomas Lavaux contributed equally to this study.     

MicroRNA‐145 regulates disabled‐2 and Wnt3a expression in cardiomyocytes under hyperglycaemia

Aims

MicroRNA‐145 (miR‐145) could protect cardiomyocyte apoptosis against oxidative stress and repair infarcted myocardium. Angiotensin II (Ang II), a pro‐inflammatory cytokine could modulate myocardial remodelling. However, the role of hyperglycaemia on miR‐145 expression in cardiomyocyte or diabetes is not known. The effect of Ang II on miR‐145 expression under hyperglycaemia in cardiomyocytes remains unknown. We sought to investigate the effect of hyperglycaemia and Ang II on miR‐145 expression in cardiomyocytes.

Methods

Rat cardiomyocytes were cultured under high glucose concentration (25 mmol/L), and streptozotocin‐induced diabetic rats were established. TaqMan^® MicroRNA real‐time quantitative assay was used to quantitate miR‐145.

Results

Sustained high glucose concentration (hyperglycaemia) significantly decreased miR‐145 expression in cardiomyocytes. Hyperglycaemia significantly increased Ang II mRNA expression and secretion from rat cardiomyocytes. Ang II suppressed miR‐145 expression in cardiomyocytes. Hyperglycaemia increased Dab2 and decreased Wnt3a/ß‐catenin expression in cardiomyocytes. Repression of miR‐145 expression by Ang II resulted in increased Dab2 and decreased Wnt3a and ß‐catenin expression under hyperglycaemia. In contrast, overexpression of miR‐145 significantly decreased Dab2 mRNA and protein expression, whereas the mRNA and protein levels for Wnt3a and ß‐catenin were significantly reduced in left ventricular myocardium from 5 days to 28 days in diabetic rats. The protein expression patterns of Dab2 and Wnt3a/ß‐catenin in left ventricular myocardium of diabetic rats could be reversed upon treatment with valsartan.

Conclusions

Ang II downregulates miR‐145 to regulate Dab2 and Wnt3a/ß‐catenin expression in cardiomyocytes under high glucose concentration. Ang II plays a critical role in the regulation of miR‐145 in cardiomyocytes under hyperglycaemic conditions.     

Human platelet antigen polymorphisms (HPA‐1, ‐2, ‐3, ‐4, ‐5 and ‐15) in major ethnic groups of Pakistan

Objectives: Gene frequencies of human platelet antigens (HPA) determine the magnitude of platelet immunological disorders like neonatal alloimmune thrombocytopenia, platelet refractoriness and ease of availability of particular HPA‐typed platelet donors in a given community. Background: However, the pattern of HPA in Pakistani population is not known. Aim: The aim of present study was to determine the gene frequencies of HPA (HPA‐1 to ‐5 and ‐15) in individuals belonging to major ethnic groups and castes of Pakistani population. Materials and Methods: HPA genotyping was done in 593 individuals belonging to all ethnic groups of Pakistan, by polymerase chain reaction‐sequence specific primers with detection on polyacrylamide electrophoresis. Results: The gene frequencies of the ‘a’ and ‘b’ alleles of HPA‐1 to ‐5 and ‐15 in Pakistanis were as follows: HPA‐1a/b, 0·885/0·115; HPA‐2a/b, 0·92/0·08; HPA‐3a/b, 0·69/0·31; HPA‐4a/b, 1/0; HPA‐5a/b, 0·9/0·1; HPA‐15a/b, 0·59/0·41. Except for significant difference regarding gene frequency of HPA‐3 between Pathans and Sindhis, there was no significant difference of HPA‐1 to ‐5 and ‐15 between major ethnic groups of Pakistan. The estimated mismatch probability regarding platelet antigens 1–5 and 15 in Pakistanis, after transfusion of random donor platelets, is from 14 to 37%. The expected incidence of neonatal alloimmune thrombocytopenia due to anti‐HPA‐1a in Pakistani pregnant females is < 1 of 1000 pregnancies and 8–12 of 1000 in case of anti‐HPA‐5b. Homozygosity of HPA‐1b, ‐2b and ‐5b genotypes ranged from 1 to 2% in the Pakistani population, whereas homozygosity of HPA‐3b and ‐15b was 11 and 18%. Conclusions: There is a need to establish donor registries typed for HPA in the transfusion centres of the country.