Joint Genome-Wide Profiling of miRNA and mRNA Expression in Alzheimer's Disease Cortex Reveals Altered miRNA Regulation

Although microRNAs are being extensively studied for their involvement in cancer and development, little is known about their roles in Alzheimer''s disease (AD). In this study, we used microarrays for the first joint profiling and analysis of miRNAs and mRNAs expression in brain cortex from AD and age-matched control subjects. These data provided the unique opportunity to study the relationship between miRNA and mRNA expression in normal and AD brains. Using a non-parametric analysis, we showed that the levels of many miRNAs can be either positively or negatively correlated with those of their target mRNAs. Comparative analysis with independent cancer datasets showed that such miRNA-mRNA expression correlations are not static, but rather context-dependent. Subsequently, we identified a large set of miRNA-mRNA associations that are changed in AD versus control, highlighting AD-specific changes in the miRNA regulatory system. Our results demonstrate a robust relationship between the levels of miRNAs and those of their targets in the brain. This has implications in the study of the molecular pathology of AD, as well as miRNA biology in general.     

Hepatic Gene Expression Profiling Reveals Key Pathways Involved in Leptin-Mediated Weight Loss in ob/ob Mice

Background

Leptin, a cytokine-like protein, plays an important role in the regulation of body weight through inhibition of food intake and stimulation of energy expenditure. Leptin circulates in blood and acts on the brain, which sends downstream signals to regulate body weight. Leptin therapy has been successful in treating leptin deficient obese patients. However, high levels of leptin have been observed in more common forms of obesity indicating a state of leptin resistance which limits the application of leptin in the treatment of obesity. If the central effect of leptin could be by-passed and genes which respond to leptin treatment could be regulated directly, new therapeutic targets for the treatment of obesity may be possible. The purpose of this study was to identify genes and subsequent pathways correlated with leptin-mediated weight loss.

Methodology/Principal Findings

We utilized microarray technology to compare hepatic gene expression changes after two types of leptin administration: one involving a direct stimulatory effect when administered peripherally (subcutaneous: SQ) and another that is indirect, involving a hypothalamic relay that suppresses food intake when leptin is administered centrally (intracerebroventricular: ICV). We identified 214 genes that correlate with leptin mediated weight loss. Several biological processes such as mitochondrial metabolic pathways, lipid metabolic and catabolic processes, lipid biosynthetic processes, carboxylic acid metabolic processes, iron ion binding and glutathione S-transferases were downregulated after leptin administration. In contrast, genes involved in the immune system inflammatory response and lysosomal activity were found to be upregulated. Among the cellular compartments mitochondrion (32 genes), endoplasmic reticulum (22 genes) and vacuole (8 genes) were significantly over represented.

Conclusions/Significance

In this study we have identified key molecular pathways and downstream genes which respond to leptin treatment and are involved in leptin-mediated weight loss. Many of these genes have previously been shown to be associated with obesity; however, we have also identified a number of other novel target genes. Further investigation will be required to assess the possible use of these genes and their associated protein products as therapeutic targets for the treatment of obesity.     

Quantitative Profiling of Drosophila melanogaster Dscam1 Isoforms Reveals No Changes in Splicing after Bacterial Exposure

The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens.     

Valproate-Induced Epigenetic Upregulation of Hypothalamic Fto Expression Potentially Linked with Weight Gain

Cellular and Molecular Neurobiology - Valproate (VPA), a widely-used antiepileptic drug, is a selective inhibitor of histone deacetylase (HDAC) that play important roles in epigenetic regulation....     

N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression

Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.Colorectal cancer (CRC)¹ is a very prevalent and heterogeneous pathology with highly variable disease progression and clinical outcome among patients. It is the third most common cancer in men and the second most common in women (1) with a highly stage-specific patient survival (2). Treatments are often curative for patients with local disease stages (stage I-II), whereas a 5-year survival of only 13% is observed in patients with distant metastasis (stage IV) (2). As CRC is often asymptomatic in the first years, unfortunately, only 40% of the patients are diagnosed at stage I-II, thus pointing to the urgent need of sensitive diagnostic tools for early detection and consequently effective, curative treatment (3). In this context, understanding the complex mechanisms of CRC is an overriding condition for the development of new, more efficient means of detection, treatment, and prognosis of the disease.Altered glycosylation is a hallmark of cancer (4) and is known to occur with cancer progression (4, 5) as glycans are involved in many cancer-associated events such as adhesion, invasion, and cell signaling (6). As a result of altered glycan structures, cellular processes can be affected due to a change of interactions with glycan-binding proteins (7–9). Several CRC tissue-associated changes in N-glycans, O-glycans, and glycosphingolipid glycans have been reported and recently reviewed (7). For instance, N-glycans extracted from colorectal tumor tissues are characterized by an increase of sulfated glycans, (truncated) high-mannose-type glycans, and glycans containing sialylated Lewis type epitopes, while showing a decrease of bisection as compared with glycans from nontumor colorectal tissue of the same individuals (10). In accordance, elevated expression of sialyl Lewis A (NeuAcα2,3Galβ1,3[Fucα1,4]GlcNAc-R; NeuAc = N-acetylneuraminic acid, Gal = galactose, Fuc = fucose, GlcNAc = N-acetylglucosamine, R = rest) and pauci-mannosidic N-glycans (truncated high-mannose-type, Man1–4GlcNAc1–4GlcNAc; Man = mannose) was recently found to be correlated with poor prognosis in (advanced) colon carcinomas and N-glycomic profiling was successfully applied to distinguish colorectal adenomas from carcinomas (11).Due to limitations in accessibility of tumor materials and possibilities of in vivo studies on a large scale, cancer cell lines represent a relevant alternative and are widely used as model systems for studying the molecular mechanisms associated with cancer outcome and progression. Since the early 1960s, colorectal cancer cell lines have been established with HT29, LoVo, LS-180, LS-174T, and Co115 representing the first continuous cell lines derived from colon tumors and xenografts (12–14). Major benefits of cancer cell lines are their continuous availability, their fast growth, and relatively easy handling, making them suitable also for high-throughput screenings (15) and a large range of experimental possibilities (16). Of note, advantages and limitations of cell lines have been recently reviewed (15).In order to select suitable in vitro models, the characterization of molecular features and their comparison to tumor tissues are needed. A detailed Cancer Cell Line Encyclopedia was recently established containing a genomic dataset for 947 human cancer cell lines, from which 58 are colorectal cancer lineages (17). The Cancer Cell Line Encyclopedia includes data collections on genomic characterization, point mutation frequencies, DNA copy number, and mRNA expression levels. Comparison of these features between cell lines and primary tumors showed a high correlation in most cancer types, especially for colorectal cancer, suggesting that cell lines do represent tumor tissues quite reasonably at least on the genetic level. However, the number of publications characterizing cancer cell lines at a molecular level is far behind the number of articles using cancer cell lines as model systems (18), and only few studies have been conducted on whether in vitro cultured cell lines can serve as suitable models for human tumors (19–22). Furthermore, cell lines are well characterized genetically, but they are largely understudied with regard to their glycosylation profiles.Here, we developed and optimized a new analytical method for the more sensitive and higher throughput N-glycome profiling of cells. This method is based on the release of N-glycans in a 96-well plate format from a PVDF-membrane (23) starting from a low number of cells (250,000 cells), the chemical derivatization of released N-glycans enabling the stabilization and discrimination of α2,3- and α2,6-linked N-acetylneuraminic acids (24), followed by registration of the N-glycans by MALDI-TOF(/TOF)-MS. The method was applied to characterize the N-glycome of 25 different colorectal cell lines in a fast and robust manner, including biological and technical replicates for all the cell lines. We obtained the comprehensive N-glycan profiles of 21 cell lines derived from primary tumors, two from lymph node metastases, one from a lung metastasis, and one from ascites fluid to assess their potential as glycobiological tumor model systems. Cancer cell line glycosylation features were then correlated with cancer cell markers and phenotypes as well as glycosyltransferase expressions. This study provides new insights into colon-cancer-associated glycan changes and sets a basis for studies into the functions of N-glycans in CRC with cell lines as model systems.     

Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

Background

Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.

Methodology

Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays.

Results

When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression.

Conclusion

Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.     

用cDNA-AFLP及其改进的方法分析茶树花发育过程中的基因表达

利用cDNA-AFLP及其改进的cDNA-AFLP方法,分析茶树花发育过程中的基因表达。其发育过程中的基因表达可以分为3类:未成熟阶段发育特异基因;成熟阶段发育特异基因;茶树花发育过程中均表达的基因。利用改进的cDNA-AFLP方法,我们获得编码花药发育特异基因:pollen coat protein(Pcp)。用cDNA-AFLP方法,我们获得7个已知功能基因分别编码Cytchrome(P450),beta-primeverosidase,Dnaj-like protein,anthranilate phosphoribosyl transferase(AnPRT),Ribulose-1,5-bisphosphate carboxylase/oxygenasesmall subunit(RubpS),alpha-tubulin和Carbonic anhydrase。用RACE方法获得pollen coat protein(Pcp),DnaJ-like protein和Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit等3个基因的全长,并已提交GenBank。     

The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep

Male Kazak sheep and Xinjiang fine wool sheep, six for each different age group (days 2, 30, 60, 90 and 120), were used in the present study to investigate the tissue distribution and developmental changes of ghrelin mRNA expression in abomasum; however, there was no 120-day-old Kazak sheep. After measurement of body weight, the tissues such as hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle were sampled. And the total RNA of different tissues was extracted to determine the abundance of ghrelin mRNA by RT-PCR and real-time PCR. The results showed that (1) for both breeds, body weight among different ages was significantly different (P<0.05). And from day 30 to 90, the body weight of Kazak was significantly higher than that of Xinjiang (P<0.01); (2) Ghrelin mRNA existed in all the above tissues and was significantly higher in the abomasum than in other tissues (P<0.05); (3) the temporal patterns of abomasum ghrelin mRNA expression in Kazak and Xinjiang were similar. From day 2 to 60 in Kazak and 2 to 90 in Xinjiang, there was a steady increase in the ghrelin mRNA level. By day 60 in Kazak and day 90 in Xinjiang, the level reached a plateau and remained steady. These results also demonstrated that from birth to day 90, ghrelin mRNA level was significantly higher in Kazak than in Xinjiang (P<0.01).     

Integration of SNP and mRNA Arrays with MicroRNA Profiling Reveals That MiR-370 Is Upregulated and Targets NF1 in Acute Myeloid Leukemia

Background

Deregulated miRNA expression plays a crucial role in carcinogenesis. Recent studies show different mechanisms leading to miRNA deregulation in cancer; however, alterations affecting miRNAs by DNA copy number variations (CNV) remain poorly studied.

Results

Our integrative analysis including data from high resolution SNPs arrays, mRNA expression arrays, and miRNAs expression profiles in 16 myeloid cell lines highlights that CNV are alternative mechanisms to deregulate the expression of miRNAs in acute myeloid leukemia (AML), and represent a novel approach to identify novel candidate genes involved in AML. We found association between the expression levels of 19 miRNAs and CNVs affecting their loci. Functional analysis showed that NF1 is a direct target of miR-370, and that overexpression of miR-370 has similar effects that NF1 inactivation, increasing proliferation and colony formation in AML cells. Moreover, real time RT-PCR showed that NF1 downregulation is a recurrent event in AML (30.8%), and western blot analysis confirmed this result. MiR-370 overexpression and deletions affecting the NF1 locus were identified as alternative mechanisms to downregulate NF1.

Conclusions

NF1 downregulation is a common event in AML, and both deletions in the NF1 locus and overexpression of miR-370 are alternative mechanisms to downregulate NF1 in this disease. Our results suggest a leukemogenic role of miR-370 through NF1 downregulation in AML cells. Since NF1 deficiency leads to RAS activation, patients with AML and overexpression of miR-370 may potentially benefit from additional treatment with either RAS or mTOR inhibitors.     

维甲酸诱导的脊柱裂小鼠的脊髓全基因组表达谱分析(英文)

关于维甲酸胚胎病理学的研究很多,维甲酸受体在器官发生、发育及神经管闭合过程中发挥重要作用。但维甲酸影响这些过程的机制还不清楚。在本研究中,我们发现,小鼠怀孕8天时,给予母体连续3次维甲酸灌胃,将导致胎儿脊柱裂,发生率为96.77%。本研究应用微阵列技术,在维甲酸诱导的脊柱裂小鼠胎儿的脊髓组织中发现了134个差异表达在1.5倍以上的基因。基因富集分析显示,母亲暴露于维甲酸导致的胎儿脊柱裂,与促凋亡和抗凋亡、细胞增殖、迁徙、细胞骨架成分以及细胞或局部粘附等基因功能簇相关,提示这些细胞成分和生物学的功能缺陷促使脊柱发育异常。我们的研究提供了脊柱裂的全基因组基因表达模式,有助于理解神经管缺陷的病因和病理学。     

A Low Sympathoadrenal Activity is Associated with Body Weight Gain and Development of Central Adiposity in Pima Indian Men

TATARANNI P ANTONIO JAMES B YOUNG, CLIFTON BOGARDUS, ERIC RAVUSSIN. A low sympathoadrenal activity is associated with body weight gain and development of central adiposity in Pima Indian men. To investigate the possible role of impaired sympathetic nervous system and/or adrenal medullary function in the etiology of human obesity, we studied 64 Pima Indian men (28 ± 6 years, 101 ± 25 kg, 34 ± 9% body fat, mean ± SD) in whom sympathoadrenal function was estimated at baseline by measurements of 24-hour urinary norepinephrine (NE) and epinephrine (Epi) excretion rates under weight-maintenance conditions. Body weight, body composition (hydrodensitometry), and body fat distribution (waist-to-thigh circumference ratio, W/T) were measured at baseline and follow-up. Follow-up data were available on 44 subjects who gained on average 8.4 ± 9.5 kg over 3.3 ± 2.1 years. In these subjects, baseline NE excretion rate, adjusted for its determinants (i.e., fat free mass, fat mass, and W/T), correlated negatively with bodyweight gain (r=?0.38; p=0.009). Baseline Epi excretion rate correlated negatively with changes in W/T (r=?0.44; p=0.003). In conclusion, our data show for the first time that a low sympathetic nervous system activity is associated with body weight gain in humans. Also, a low activity of the adrenal medulla is associated with the development of central adiposity.     

Joint and Independent Associations of Gestational Weight Gain and Pre-Pregnancy Body Mass Index with Outcomes of Pregnancy in Chinese Women: A Retrospective Cohort Study

Objective

To explore the joint and independent effects of gestational weight gain (GWG) and pre-pregnancy body mass index (BMI) on pregnancy outcomes in a population of Chinese Han women and to evaluate pregnant women’s adherence to the 2009 Institute of Medicine (IOM) gestational weight gain guidelines.

Methods

This was a multicenter, retrospective cohort study of 48,867 primiparous women from mainland China who had a full-term singleton birth between January 1, 2011 and December 30, 2011. The independent associations of pre-pregnancy BMI, GWG and categories of combined pre-pregnancy BMI and GWG with outcomes of interest were examined using an adjusted multivariate regression model. In addition, women with pre-pregnancy hypertension were excluded from the analysis of the relationship between GWG and delivery of small-for-gestational-age (SGA) infants, and women with gestational diabetes (GDM) were excluded from the analysis of the relationship between GWG and delivery of large-for-gestational-age (LGA) infants.

Results

Only 36.8% of the women had a weight gain that was within the recommended range; 25% and 38.2% had weight gains that were below and above the recommended range, respectively. The contribution of GWG to the risk of adverse maternal and fetal outcomes was modest. Women with excessive GWG had an increased likelihood of gestational hypertension (adjusted OR 2.55; 95% CI = 1.92–2.80), postpartum hemorrhage (adjusted OR 1.30; 95% CI = 1.17–1.45), cesarean section (adjusted OR 1.31; 95% CI = 1.18–1.36) and delivery of an LGA infant (adjusted OR 2.1; 95% CI = 1.76–2.26) compared with women with normal weight gain. Conversely, the incidence of GDM (adjusted OR 1.64; 95% CI = 1.20–1.85) and SGA infants (adjusted OR 1.51; 95% CI = 1.32–1.72) was increased in the group of women with inadequate GWG. Moreover, in the obese women, excessive GWG was associated with an apparent increased risk of delivering an LGA infant. In the women who were underweight, poor weight gain was associated with an increased likelihood of delivering an SGA infant. After excluding the mothers with GDM or gestational hypertension, the ORs for delivery of LGA and SGA infants decreased for women with high GWG and increased for women with low GWG.

Conclusions

GWG above the recommended range is common in this population and is associated with multiple unfavorable outcomes independent of pre-pregnancy BMI. Obese women may benefit from avoiding weight gain above the range recommended by the 2009 IOM. Underweight women should avoid low GWG to prevent delivering an SGA infant. Pregnant women should therefore be monitored to comply with the IOM recommendations and should have a balanced weight gain that is within a range based on their pre-pregnancy BMI.     

建鲤IGFBP 3 基因多态性对生长的影响

胰岛素样生长因子结合蛋白3(insulin-like growth factor binding protein,IGFBP3)是调节动物生长和代谢的重要基因.本实验查找了建鲤(Cyprinus carpio var.jian)IGFBP3s基因上的SNP位点.使用PCRRFLP检测了其中5个位点(IGFBP3a-I3...     

猪肌肉组织Col3a1基因表达的发育性变化及其对肌内胶原特性的影响

以30—90妇体重莱芜猪和40—100kg体重鲁莱黑猪共84头去势公猪为试验对象（每组6头）,采用相对定量RT-PCR方法,以β-actin作为内标,研究肌肉中编码Ⅲ型胶原的Col3al基因表达的发育性变化及其对肌肉中胶原蛋白含量和性质（溶解度）的影响。结果表明：研究的两个品种猪肌肉中Col3al基因表达的发育性变化基本一致,即随体重的增加,肌肉中Col3al mRNA表达呈逐渐增加趋势,但莱芜猪和鲁莱黑猪分别在70妇和80妇体重组表达量略有下降。总体上莱芜猪肌肉组织Col3al mRNA表达丰度显著高于鲁莱黑猪（P〈0．05）。相关分析表明,莱芜猪肌肉组织Col3al mRNA表达的发育性变化与总胶原和不溶性胶原含量呈极显著正相关（P〈0．01）,与胶原溶解度呈极显著负相关（P〈0．01）。鲁莱黑猪肌肉组织Col3al mRNA表达的发育性变化与不溶性胶原和胶原溶解度分别呈显著正相关和负相关妒〈0．05）。研究结果提示：猪肌肉组织中Col3al基因表达具有明显的体重发育和品种特征,其mRNA表达对于肌内胶原的含量和性质有重要影响。