Targeted massively parallel sequencing of candidate regions on chromosome 22q predisposing to multiple schwannomas: An analysis of 51 individuals in a single-center experience.

Constitutional LZTR1 or SMARCB1 pathogenic variants (PVs) have been found in ∼86% of familial and ∼40% of sporadic schwannomatosis cases. Hence, we performed massively parallel sequencing of the entire LZTR1, SMARCB1, and NF2 genomic loci in 35 individuals with schwannomas negative for constitutional first-hit PVs in the LZTR1/SMARCB1/NF2 coding sequences; however, with 22q deletion and/or a different NF2 PV in each tumor, including six cases with only one tumor available. Furthermore, we verified whether any other LZTR1/SMARCB1/NF2 (likely) PVs could be found in 16 cases carrying a SMARCB1 constitutional variant in the 3'-untranslated region (3'-UTR) c.*17C>T, c.*70C>T, or c.*82C>T. As no additional variants were found, functional studies were performed to clarify the effect of these 3'-UTR variants on the transcript. The 3'-UTR variants c.*17C>T and c.*82C>T showed pathogenicity by negatively affecting the SMARCB1 transcript level. Two novel deep intronic SMARCB1 variants, c.500+883T>G and c.500+887G>A, resulting in out-of-frame missplicing of intron 4, were identified in two unrelated individuals. Further resequencing of the entire repeat-masked genomics sequences of chromosome 22q in individuals negative for PVs in the SMARCB1/LZTR1/NF2 coding- and noncoding regions revealed five potential schwannomatosis-predisposing candidate genes, that is, MYO18B, NEFH, SGSM1, SGSM3, and SBF1, pending further verification.

The transition from HIF-1 to HIF-2 during prolonged hypoxia results from reactivation of PHDs and HIF1A mRNA instability.

The hypoxia-inducible factors (HIF) are transcription factors that activate the adaptive hypoxic response when oxygen levels are low. The HIF transcriptional program increases oxygen delivery by inducing angiogenesis and by promoting metabolic reprograming that favors glycolysis. The two major HIFs, HIF-1 and HIF-2, mediate this response during prolonged hypoxia in an overlapping and sequential fashion that is referred to as the HIF switch. Both HIF proteins consist of an unstable alpha chain and a stable beta chain. The instability of the alpha chains is mediated by prolyl hydroxylase (PHD) activity during normoxic conditions, which leads to ubiquitination and proteasomal degradation of the alpha chains. During normoxic conditions, very little HIF-1 or HIF-2 alpha-beta dimers are present because of PHD activity. During hypoxia, however, PHD activity is suppressed, and HIF dimers are stable. Here we demonstrate that HIF-1 expression is maximal after 4 h of hypoxia in primary endothelial cells and then is dramatically reduced by 8 h. In contrast, HIF-2 is maximal at 8 h and remains elevated up to 24 h. There are differences in the HIF-1 and HIF-2 transcriptional profiles, and therefore understanding how the transition between them occurs is important and not clearly understood. Here we demonstrate that the HIF-1 to HIF-2 transition during prolonged hypoxia is mediated by two mechanisms: (1) the HIF-1 driven increase in the glycolytic pathways that reactivates PHD activity and (2) the much less stable mRNA levels of HIF-1α (HIF1A) compared to HIF-2α (EPAS1) mRNA. We also demonstrate that the alpha mRNA levels directly correlate to the relative alpha protein levels, and therefore to the more stable HIF-2 expression during prolonged hypoxia.

Genome-wide mRNA profiling identifies X-box-binding protein 1 (XBP1) as an IRE1 and PUMA repressor.

Accumulation of misfolded proteins in ER activates the unfolded protein response (UPR), a multifunctional signaling pathway that is important for cell survival. The UPR is regulated by three ER transmembrane sensors, one of which is inositol-requiring protein 1 (IRE1). IRE1 activates a transcription factor, X-box-binding protein 1 (XBP1), by removing a 26-base intron from XBP1 mRNA that generates spliced XBP1 mRNA (XBP1s). To search for XBP1 transcriptional targets, we utilized an XBP1s-inducible human cell line to limit XBP1 expression in a controlled manner. We also verified the identified XBP1-dependent genes with specific silencing of this transcription factor during pharmacological ER stress induction with both an N-linked glycosylation inhibitor (tunicamycin) and a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) (thapsigargin). We then compared those results to the XBP1s-induced cell line without pharmacological ER stress induction. Using next-generation sequencing followed by bioinformatic analysis of XBP1-binding motifs, we defined an XBP1 regulatory network and identified XBP1 as a repressor of PUMA (a proapoptotic gene) and IRE1 mRNA expression during the UPR. Our results indicate impairing IRE1 activity during ER stress conditions accelerates cell death in ER-stressed cells, whereas elevating XBP1 expression during ER stress using an inducible cell line correlated with a clear prosurvival effect and reduced PUMA protein expression. Although further studies will be required to test the underlying molecular mechanisms involved in the relationship between these genes with XBP1, these studies identify a novel repressive role of XBP1 during the UPR.

Triazoloacridone C-1305 impairs XBP1 splicing by acting as a potential IRE1α endoribonuclease inhibitor.

Inositol requiring enzyme 1 alpha (IRE1α) is one of three signaling sensors in the unfolding protein response (UPR) that alleviates endoplasmic reticulum (ER) stress in cells and functions to promote cell survival. During conditions of irrevocable stress, proapoptotic gene expression is induced to promote cell death. One of the three signaling stressors, IRE1α is an serine/threonine-protein kinase/endoribonuclease (RNase) that promotes nonconventional splicing of XBP1 mRNA that is translated to spliced XBP1 (XBP1s), an active prosurvival transcription factor. Interestingly, elevated IRE1α and XBP1s are both associated with poor cancer survival and drug resistance. In this study, we used next-generation sequencing analyses to demonstrate that triazoloacridone C-1305, a microtubule stabilizing agent that also has topoisomerase II inhibitory activity, dramatically decreases XBP1s mRNA levels and protein production during ER stress conditions, suggesting that C-1305 does this by decreasing IRE1α's endonuclease activity.

Primary endothelial cell-specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia.

During hypoxia, a cellular adaptive response activates hypoxia-inducible factors (HIFs; HIF-1 and HIF-2) that respond to low tissue-oxygen levels and induce the expression of a number of genes that promote angiogenesis, energy metabolism, and cell survival. HIF-1 and HIF-2 regulate endothelial cell (EC) adaptation by activating gene-signaling cascades that promote endothelial migration, growth, and differentiation. An HIF-1 to HIF-2 transition or switch governs this process from acute to prolonged hypoxia. In the present study, we evaluated the mechanisms governing the HIF switch in 10 different primary human ECs from different vascular beds during the early stages of hypoxia. The studies demonstrate that the switch from HIF-1 to HIF-2 constitutes a universal mechanism of cellular adaptation to hypoxic stress and that HIF1A and HIF2A mRNA stability differences contribute to HIF switch. Furthermore, using 4 genome-wide mRNA expression arrays of HUVECs during normoxia and after 2, 8, and 16 h of hypoxia, we show using bioinformatics analyses that, although a number of genes appeared to be regulated exclusively by HIF-1 or HIF-2, the largest number of genes appeared to be regulated by both.-Bartoszewski, R., Moszynska, A., Serocki, M., Cabaj, A., Polten, A., Ochocka, R., Dell'Italia, L., Bartoszewska, S., Króliczewski, J., Dabrowski, M., Collawn, J. F. Primary endothelial cell-specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia.

The effects of seed from Linum usitatissimum cultivar with increased phenylpropanoid compounds and hydrolysable tannin in a high cholesterol-fed rabbit.

BACKGROUND: Dietary fat is considered one of the most important factors associated with blood lipid metabolism and plays a significant role in the cause and prevention of atherosclerosis that has been widely accepted as an inflammatory disease of the vascular system. The aim of the present study was to evaluate the effect of genetically modified flaxseed (W86) rich in phenylpropanoid compounds and hydrolysable tannin in high cholesterol-induced atherosclerosis rabbit models compared to parental cultivar Linola. METHODS: Twenty-Eight White New Zealand white rabbits aged 6 months were randomly divided into four groups, control group, high cholesterol group (10 g/kg), Linola flaxseed group (100 g/kg) and W86 flaxseed group (100 g/kg). The rabbits were fed a normal diet or a high cholesterol diet for 10 weeks. Levels of blood lipids, hematological values, total antioxidative status and superoxide dismutase activity in serum were determined. Moreover, body weight and feed intake were measured after sixth and tenth weeks. After each stage of the experiment atherogenic indexes (non-HDL-C/HDL-C, LDL-C/HDL-C, and atherogenic index of plasma) was calculated. RESULTS: The intake of a dyslipidaemic diet negatively influenced lipid profile in rabbits at the 10 weeks of feeding. W86 flaxseed significantly decreased total cholesterol, LDL-C, VLDL-C and TG serum levels in cholesterolemic rabbits compared with parental Linola after 10 weeks. Atherogenic indexes decreased over time with a significant difference between the diets and they were the best for W86 flaxseed. Similarly, the experimental addition of W86 significantly decreased atherogenic predictors such as heterophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and the mean platelet volume-to-lymphocyte ratio. In rabbits, W86 flaxseed increased the activity of superoxide dismutase and total antioxidative status compared to Linola. CONCLUSIONS: Results of the presented study suggest that the addition of W86 flaxseed alleviate serum lipid changes in high cholesterolemic diet-administered rabbits. W86 flaxseed significantly reduced atherogenic indexes, as compared with the Linola and indicate that W86 flaxseed more effectively red CVD risk factors during hypercholesterolemia. Moreover, the presented result suggested that W86 flaxseed can be a part of a heart-healthy and antiatherogenic diet for the human.

Chloroplast PetD protein: evidence for SRP/Alb3-dependent insertion into the thylakoid membrane.

BACKGROUND: In thylakoid membrane, each monomer of the dimeric complex of cytochrome b 6 f is comprised of eight subunits that are both nucleus- and plastid-encoded. Proper cytochrome b 6 f complex integration into the thylakoid membrane requires numerous regulatory factors for coordinated transport, insertion and assembly of the subunits. Although, the chloroplast-encoded cytochrome b 6 f subunit IV (PetD) consists of three transmembrane helices, the signal and the mechanism of protein integration into the thylakoid membrane have not been identified. RESULTS: Here, we demonstrate that the native PetD subunit cannot incorporate into the thylakoid membranes spontaneously, but that proper integration occurs through the post-translational signal recognition particle (SRP) pathway. Furthermore, we show that PetD insertion into thylakoid membrane involves the coordinated action of cpFTSY, cpSRP54 and ALB3 insertase. CONCLUSIONS: PetD subunit integration into the thylakoid membrane is a post-translational and an SRP-dependent process that requires the formation of the cpSRP-cpFtsY-ALB3-PetD complex. This data provides a new insight into the molecular mechanisms by which membrane proteins integration into the thylakoid membrane is accomplished and is not limited to PetD.

miR-200b downregulates CFTR during hypoxia in human lung epithelial cells.

BACKGROUND: Hypoxic conditions induce the expression of hypoxia-inducible factors (HIFs) that allow cells to adapt to the changing conditions and alter the expression of a number of genes including the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a low abundance mRNA in airway epithelial cells even during normoxic conditions, but during hypoxia its mRNA expression decreases even further. METHODS: In the current studies, we examined the kinetics of hypoxia-induced changes in CFTR mRNA and protein levels in two human airway epithelial cell lines, Calu-3 and 16HBE14o-, and in normal primary bronchial epithelial cells. Our goal was to examine the posttranscriptional modifications that affected CFTR expression during hypoxia. We utilized in silico predictive protocols to establish potential miRNAs that could potentially regulate CFTR message stability and identified miR-200b as a candidate molecule. RESULTS: Analysis of each of the epithelial cell types during prolonged hypoxia revealed that CFTR expression decreased after 12 h during a time when miR-200b was continuously upregulated. Furthermore, manipulation of the miRNA levels during normoxia and hypoxia using miR-200b mimics and antagomirs decreased and increased CFTR mRNA levels, respectively, and thus established that miR-200b downregulates CFTR message levels during hypoxic conditions. CONCLUSION: The data suggest that miR-200b may be a suitable target for modulating CFTR levels in vivo.

A new genotype of flax (Linum usitatissimum L.) with decreased susceptibility to fat oxidation: consequences to hematological and biochemical profiles of blood indices.

BACKGROUND: Flaxseed is an alternative to marine products that provide the traditional dietary sources of ω-fatty acids. A new genotype of flax, W92, is rich in natural antioxidants as well as having a reduced content of α-linolenic acid and therefore shows decreased susceptibility to fat oxidation. The objective of this study was to evaluate the effect of a diet supplemented with W92 flaxseed on hematological and biochemical blood indices. RESULTS: A positive impact of diet with the addition of flaxseed was observed on erythrocyte indices, including red blood cell (RBC), hematocrit (HCT), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) values. There were no significant differences for white blood cell (WBC), total protein and glucose values. Aspartate aminotransferase and alanine aminotransferase estimations in serum were also carried out and no obvious toxicity to the liver was shown. Moreover, a lipid profile was performed in serum samples and a decrease in total cholesterol and low-density lipoprotein cholesterol (LDL-C), accompanied by an increase in high-density lipoprotein cholesterol (HDL-C), was observed in rabbits fed flaxseed diets. CONCLUSION: Based on the results obtained, it appeared that the inclusion of a new genetically modified type of flaxseed in the diet altered cholesterol metabolism and could reduce the possibility of cardiovascular diseases. Diet enrichment with W92 flaxseed may be a solution to the health issues that are a result of improper diet in humans and animals. © 2016 Society of Chemical Industry.

Codon bias and the folding dynamics of the cystic fibrosis transmembrane conductance regulator.

Synonymous or silent mutations are often overlooked in genetic analyses for disease-causing mutations unless they are directly associated with potential splicing defects. More recent studies, however, indicate that some synonymous single polynucleotide polymorphisms (sSNPs) are associated with changes in protein expression, and in some cases, protein folding and function. The impact of codon usage and mRNA structural changes on protein translation rates and how they can affect protein structure and function is just beginning to be appreciated. Examples are given here that demonstrate how synonymous mutations alter the translational kinetics and protein folding and/or function. The mechanism for how this occurs is based on a model in which codon usage modulates the translational rate by introducing pauses caused by nonoptimal or rare codons or by introducing changes in the mRNA structure, and this in turn influences co-translational folding. Two examples of this include the multidrug resistance protein (p-glycoprotein) and the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR is also used here as a model to illustrate how synonymous mutations can be examined using in silico predictive methods to identify which sSNPs have the potential to change protein structure. The methodology described here can be used to help identify "non-silent" synonymous mutations in other genes.

Ribosome nascent chain complexes of the chloroplast-encoded cytochrome b6 thylakoid membrane protein interact with cpSRP54 but not with cpSecY.

We analysed the interplay between the cpSecY, cpSRP54 and the chloroplast-encoded cytochrome b6 via isolation of chloroplast ribosome nascent chain complexes and the use of cross-linking factors, antibodies and mass spectroscopy analyses. We showed that the cytochrome b6 nascent polypeptide complex is tightly associated with ribosomes and that the translation of cytochrome b6 was discontinuous. The causes of ribosome pausing and the functional significance of this phenomenon may be related to proper protein folding, insertion into thylakoid membranes and the association of cofactors during this process. It was also found that cpSecY was not in the vicinity of cytochrome b6 intermediates during the elongation process and does not act with mature cytochrome b6 after translation. Using the approach of cross-linking during elongation of the cytochrome b6 protein, we showed that cpSRP54 interacts strongly with the elongating nascent chain.

Trodusquemine (MSI-1436) Restores Metabolic Flexibility and Mitochondrial Dynamics in Insulin-Resistant Equine Hepatic Progenitor Cells (HPCs).

Equine metabolic syndrome (EMS) is a significant global health concern in veterinary medicine. There is increasing interest in utilizing molecular agents to modulate hepatocyte function for potential clinical applications. Recent studies have shown promising results in inhibiting protein tyrosine phosphatase (PTP1B) to maintain cell function in various models. In this study, we investigated the effects of the inhibitor Trodusquemine (MSI-1436) on equine hepatic progenitor cells (HPCs) under lipotoxic conditions. We examined proliferative activity, glucose uptake, and mitochondrial morphogenesis. Our study found that MSI-1436 promotes HPC entry into the cell cycle and protects them from palmitate-induced apoptosis by regulating mitochondrial dynamics and biogenesis. MSI-1436 also increases glucose uptake and protects HPCs from palmitate-induced stress by reorganizing the cells' morphological architecture. Furthermore, our findings suggest that MSI-1436 enhances 2-NBDG uptake by increasing the expression of SIRT1, which is associated with liver insulin sensitivity. It also promotes mitochondrial dynamics by modulating mitochondria quantity and morphotype as well as increasing the expression of PINK1, MFN1, and MFN2. Our study provides evidence that MSI-1436 has a positive impact on equine hepatic progenitor cells, indicating its potential therapeutic value in treating EMS and insulin dysregulation.

The Effect of Saponite Clay on Ruminal Fermentation Parameters during In Vitro Studies.

Reducing the emission of global warming gases currently remains one of the strategic tasks. Therefore, the objective of our work was to determine the effect of saponite clay on fermentation in the rumen of cows. The pH, total gas production, CH4, and volatile fatty acid (VFA) production in ruminal fluid was determined in vitro. Saponite clay from the Tashkiv deposit (Ukraine) has a high content of silicon, iron, aluminum, and magnesium. The addition of 0.15 and 0.25 g of saponite clay to the incubated mixture did not change the pH but reduced the total production (19% and 31%, respectively) and CH4 (24% and 46%, respectively) in the ruminal fluid compared to the control group and had no significant effect on the total VFA levels, but propionic acid increased by 15% and 21% and butyric acid decreased by 39% and 32%, respectively. We observed a decrease in the fermentation rates, with a simultaneous increase in the P:B ratio and an increase in the fermentation efficiency (FE) in the groups fermented with saponite clay, probably a consequence of the high efficiency in the breakdown of starch in the rumen. Therefore, further in vivo studies to determine the effective dose and effect of saponite clay on cow productivity and the reduction of gas emissions are promising and important.

Hypoxia-inducible factor (HIF)-3a2 serves as an endothelial cell fate executor during chronic hypoxia.

The adaptive response to hypoxia involves the transcriptional induction of three transcription factors called hypoxia inducible factor alpha 1, 2 and 3 (HIF-1α, HIF-2α, and HIF-3α) which dimerize with constitutively expressed beta chains that together form the HIF-1, -2 and -3 transcription factors. During normoxic conditions, the alpha chain is expressed at low levels since its stability is regulated by prolyl-hydroxylation that promotes subsequent ubiquitination and degradation. During hypoxic conditions, however, the prolyl hydroxylases are less active, and the alpha chain accumulates through elevated protein stability and the elevated induction of expression. Two of the three HIFs isoforms present in mammals, HIF-1 and HIF-2, are well characterized and have overlapping functions that promote cell survival, whereas HIF-3's role remains less clear. The HIF-3 response is complicated because the HIF3A gene can utilize different promotors and alternate splicing sites that result in a number of different HIF-3α isoforms. Here, using human umbilical vein endothelial cells (HUVECs), we demonstrate that one of the isoforms of HIF-3α, isoform 2 (HIF-3α2) accumulates at a late stage of hypoxia and induces the expression of DNA damage inducible transcript 3 (DDIT4), a gene known to promote apoptosis. We also demonstrate that caspase 3/7 activity is elevated, supporting that the role of the HIF-3α2 isoform is to promote apoptosis. Furthermore, we provide evidence that HIF-3α2 is also expressed in seven other primary endothelial cell types, suggesting that this may be a common feature of HIF-3α2 in endothelial cells.

Genome-wide mRNA profiling identifies RCAN1 and GADD45A as regulators of the transitional switch from survival to apoptosis during ER stress.

Endoplasmic reticulum (ER) stress conditions promote a cellular adaptive mechanism called the unfolded protein response (UPR) that utilizes three stress sensors, inositol-requiring protein 1, protein kinase RNA-like ER kinase, and activating transcription factor 6. These sensors activate a number of pathways to reduce the stress and facilitate cell survival. While much is known about the mechanisms involved that modulate apoptosis during chronic stress, less is known about the transition between the prosurvival and proapoptotic factors that determine cell fate. Here, we employed a genetic screen that utilized three different pharmacological stressors to induce ER stress in a human-immortalized airway epithelial cell line, immortalized human bronchial epithelial cells. We followed the stress responses over an 18-h time course and utilized real-time monitoring of cell survival, next-generation sequencing, and quantitative real-time PCR to identify and validate genes that were upregulated with all three commonly employed ER stressors, inhibitor of calpain 1, tunicamycin, and thapsigargin. growth arrest and DNA damage-inducible alpha (GADD45A), a proapoptotic factor, and regulator of calcineurin 1 (RCAN1) mRNAs were identified and verified by showing that small interfering RNA (siRNA) knockdown of GADD45A decreased CCAAT-enhancer-binding protein homologous protein (a.k.a DDIT3), BCL2-binding component 3 (a.k.a. BBC3), and phorbol-12-myristate-13-acetate-induced protein 1 expression, 3 proapoptotic factors, and increased cell viability during ER stress conditions, whereas siRNA knockdown of RCAN1 dramatically decreased cell viability. These results suggest that the relative levels of these two genes regulate cell fate decisions during ER stress independent of the type of ER stressor.

Utilizing Genome-Wide mRNA Profiling to Identify the Cytotoxic Chemotherapeutic Mechanism of Triazoloacridone C-1305 as Direct Microtubule Stabilization.

Rational drug design and in vitro pharmacology profiling constitute the gold standard in drug development pipelines. Problems arise, however, because this process is often difficult due to limited information regarding the complete identification of a molecule's biological activities. The increasing affordability of genome-wide next-generation technologies now provides an excellent opportunity to understand a compound's diverse effects on gene regulation. Here, we used an unbiased approach in lung and colon cancer cell lines to identify the early transcriptomic signatures of C-1305 cytotoxicity that highlight the novel pathways responsible for its biological activity. Our results demonstrate that C-1305 promotes direct microtubule stabilization as a part of its mechanism of action that leads to apoptosis. Furthermore, we show that C-1305 promotes G2 cell cycle arrest by modulating gene expression. The results indicate that C-1305 is the first microtubule stabilizing agent that also is a topoisomerase II inhibitor. This study provides a novel approach and methodology for delineating the antitumor mechanisms of other putative anticancer drug candidates.

Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis.

Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and (1)H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.

[Structure and biogenesis of cytochrome b6f complex]. / Budowa i biogeneza kompleksu cytochromów b6f.

Both photosynthetic cytochrome b6f complex, and respiratory cytochrome bc1 belong to the family of cytochrome bc complexes. Both protein supercomplexes participate in the transport of electrons, proton translocation through the biological membrane, and they catalyze chinon oxidation as well. The function, composition, spatial organization and biosynthesis of cytochrome b6f complex has been being the subject of research for years. The obtained crystal structures revealed the presence of the third haem in the cytochrome b6, whereas mutagenic experiments indicated the participation of the additional protein factor (TCA) engaged in the regulation of b6f cytochrome complex synthesis through the interaction between TCA and 5'UTR of the PETA transcript. The following compendium is the collection of the current data and knowledge with reference to the structure and biogenesis of the above mentioned protein complex.

miRNA networks modulate human endothelial cell adaptation to cyclic hypoxia.

Solid tumor microenvironments are often subjected to various levels of hypoxia. Although regulation of gene expression has been examined extensively, most studies have focused on prolonged hypoxia. The tumor microenvironment, however, experiences waves of hypoxia and reoxygenation that stimulate the expression of pro-angiogenic factors that promote blood vessel formation. In this study, we examined human umbilical vascular endothelial cells (HUVECs) under waves of intermittent (cyclic) hypoxia to determine how this process compares to prolonged hypoxia, and more importantly, how this influences the microRNA profiles that potentially affect the posttranscriptional regulation of angiogenic genes. The rationale for these studies is that cancer cells subjected to cyclic hypoxia appear to have increased metastatic potential and endothelial cells exhibit a higher radiation resistance and greater migration potential. This indicates that gene regulatory networks in cyclic hypoxia may be different from prolonged hypoxia. Here we examined the consequences of cyclic hypoxia on miRNA gene expression and how these changes in miRNA expression influence angiogenesis. Using Next Generation Sequencing, our results demonstrate that cyclic hypoxia has very different effects on the miRNA networks compared to prolonged hypoxia, and that the in silico predicted effects on the certain mRNA target genes are more similar than might be expected. More importantly, these studies indicate that identifying potential miRNAs (including hsa-miR-19a-5p) as therapeutic targets for inhibiting angiogenesis and tumor progression will require this type of physiologically relevant analysis.

microRNA single polynucleotide polymorphism influences on microRNA biogenesis and mRNA target specificity.

microRNAs (miRNAs) are nowadays recognized as an essential component of gene regulatory networks. Furthermore, deregulation of miRNAs expression often contributes to human pathologies. Recently, a substantial number of single nucleotide polymorphism (SNPs) and rare mutations within pri-, pre- and mature miRNA sequences have been reported. These miRNA SNPs have often been associated with human disease. However, due to the complexity of miRNA biogenesis and the genome-wide functional effects of miRNAs, the determination of biological consequences of these miRNA SNPs remains challenging. Despite an increasing number of reports linking miRNA SNPs with human pathologies, few reports have analyzed the mechanism by which miRNA-SNPs contribute to disease pathogenesis. In this review, we discuss how single polynucleotide polymorphisms in miRNAs genes may influence miRNAs expression and function and thus potentially alter disease pathogenesis.