Lineage-Specific Changes in Biomarkers in Great Apes and Humans

Although human biomedical and physiological information is readily available, such information for great apes is limited. We analyzed clinical chemical biomarkers in serum samples from 277 wild- and captive-born great apes and from 312 healthy human volunteers as well as from 20 rhesus macaques. For each individual, we determined a maximum of 33 markers of heart, liver, kidney, thyroid and pancreas function, hemoglobin and lipid metabolism and one marker of inflammation. We identified biomarkers that show differences between humans and the great apes in their average level or activity. Using the rhesus macaques as an outgroup, we identified human-specific differences in the levels of bilirubin, cholinesterase and lactate dehydrogenase, and bonobo-specific differences in the level of apolipoprotein A-I. For the remaining twenty-nine biomarkers there was no evidence for lineage-specific differences. In fact, we find that many biomarkers show differences between individuals of the same species in different environments. Of the four lineage-specific biomarkers, only bilirubin showed no differences between wild- and captive-born great apes. We show that the major factor explaining the human-specific difference in bilirubin levels may be genetic. There are human-specific changes in the sequence of the promoter and the protein-coding sequence of uridine diphosphoglucuronosyltransferase 1 (UGT1A1), the enzyme that transforms bilirubin and toxic plant compounds into water-soluble, excretable metabolites. Experimental evidence that UGT1A1 is down-regulated in the human liver suggests that changes in the promoter may be responsible for the human-specific increase in bilirubin. We speculate that since cooking reduces toxic plant compounds, consumption of cooked foods, which is specific to humans, may have resulted in relaxed constraint on UGT1A1 which has in turn led to higher serum levels of bilirubin in humans.     

Human-specific amino acid changes found in 103 protein-coding genes

We humans have many characteristics that are different from those of the great apes. These human-specific characters must have arisen through mutations accumulated in the genome of our direct ancestor after the divergence of the last common ancestor with chimpanzee. Gene trees of human and great apes are necessary for extracting these human-specific genetic changes. We conducted a systematic analysis of 103 protein-coding genes for human, chimpanzee, gorilla, and orangutan. Nucleotide sequences for 18 genes were newly determined for this study, and those for the remaining genes were retrieved from the DDBJ/EMBL/GenBank database. The total number of amino acid changes in the human lineage was 147 for 26,199 codons (0.56%). The total number of amino acid changes in the human genome was, thus, estimated to be about 80,000. We applied the acceleration index test and Fisher's synonymous/nonsynonymous exact test for each gene tree to detect any human-specific enhancement of amino acid changes compared with ape branches. Six and two genes were shown to have significantly higher nonsynonymous changes at the human lineage from the acceleration index and exact tests, respectively. We also compared the distribution of the differences of the nonsynonymous substitutions on the human lineage and those on the great ape lineage. Two genes were more conserved in the ape lineage, whereas one gene was more conserved in the human lineage. These results suggest that a small proportion of protein-coding genes started to evolve differently in the human lineage after it diverged from the ape lineage.     

microRNAs in Circulation Are Altered in Response to Influenza A Virus Infection in Humans

Changes in microRNA expression have been detected in vitro in influenza infected cells, yet little is known about them in patients. microRNA profiling was performed on whole blood of H1N1 patients to identify signature microRNAs to better understand the gene regulation involved and possibly improve diagnosis. Total RNA extracted from blood samples of influenza infected patients and healthy controls were subjected to microRNA microarray. Expression profiles of circulating microRNAs were altered and distinctly different in influenza patients. Expression of highly dysregulated microRNAs were validated using quantitative PCR. Fourteen highly dysregulated miRNAs, identified from the blood of influenza infected patients, provided a clear distinction between infected and healthy individuals. Of these, expression of miR-1260, -26a, -335*, -576-3p, -628-3p and -664 were consistently dysregulated in both whole blood and H1N1 infected cells. Potential host and viral gene targets were identified and the impact of microRNA dysregulation on the host proteome was studied. Consequences of their altered expression were extrapolated to changes in the host proteome expression. These highly dysregulated microRNAs may have crucial roles in influenza pathogenesis and are potential biomarkers of influenza.     

Human-specific regulation of alpha 2-6-linked sialic acids

Many microbial pathogens and toxins recognize animal cells via cell surface sialic acids (Sias) that are alpha 2-3- or alpha 2-8-linked to the underlying glycan chain. Human influenza A/B viruses are unusual in preferring alpha 2-6-linked Sias, undergoing a switch from alpha 2-3 linkage preference during adaptation from animals to humans. This correlates with the expression of alpha 2-6-linked Sias on ciliated human airway epithelial target cells and of alpha 2-3-linked Sias on secreted soluble airway mucins, which are unable to inhibit virus binding. Given several known differences in Sia biology between humans and apes, we asked whether this pattern of airway epithelial Sia linkages is also human-specific. Indeed, we show that since the last common ancestor with apes, humans underwent a concerted bidirectional switch in alpha 2-6-linked Sia expression between airway epithelial cell surfaces and secreted mucins. This can explain why the chimpanzee appears relatively resistant to experimental infection with human Influenza viruses. Other tissues showed additional examples of human-specific increases or decreases in alpha 2-6-linked Sia expression and only one example of a change specific to certain great apes. Furthermore, while human and great ape leukocytes both express alpha 2-6-linked Sias, only human erythrocytes have markedly up-regulated expression. These cell type-specific changes in alpha 2-6-Sia expression during human evolution represent another example of a human-specific change in Sia biology. Because the data set involves multiple great apes, we can also conclude that Sia linkage expression patterns can be conserved during millions of years of evolution within some vertebrate taxa while undergoing sudden major changes in other closely related ones.     

MicroRNA expression and regulation in human, chimpanzee, and macaque brains

Among other factors, changes in gene expression on the human evolutionary lineage have been suggested to play an important role in the establishment of human-specific phenotypes. However, the molecular mechanisms underlying these expression changes are largely unknown. Here, we have explored the role of microRNA (miRNA) in the regulation of gene expression divergence among adult humans, chimpanzees, and rhesus macaques, in two brain regions: prefrontal cortex and cerebellum. Using a combination of high-throughput sequencing, miRNA microarrays, and Q-PCR, we have shown that up to 11% of the 325 expressed miRNA diverged significantly between humans and chimpanzees and up to 31% between humans and macaques. Measuring mRNA and protein expression in human and chimpanzee brains, we found a significant inverse relationship between the miRNA and the target genes expression divergence, explaining 2%-4% of mRNA and 4%-6% of protein expression differences. Notably, miRNA showing human-specific expression localize in neurons and target genes that are involved in neural functions. Enrichment in neural functions, as well as miRNA-driven regulation on the human evolutionary lineage, was further confirmed by experimental validation of predicted miRNA targets in two neuroblastoma cell lines. Finally, we identified a signature of positive selection in the upstream region of one of the five miRNA with human-specific expression, miR-34c-5p. This suggests that miR-34c-5p expression change took place after the split of the human and the Neanderthal lineages and had adaptive significance. Taken together these results indicate that changes in miRNA expression might have contributed to evolution of human cognitive functions.     

Identification of let-7a-2-3p or/and miR-188-5p as Prognostic Biomarkers in Cytogenetically Normal Acute Myeloid Leukemia

Cytogenetically normal acute myeloid leukemia (CN-AML) is the largest and most heterogeneous AML subgroup. It lacks sensitive and specific biomarkers. Emerging evidences have suggested that microRNAs are involved in the pathogenesis of various leukemias. This paper evaluated the association between microRNA expression and prognostic outcome for CN-AML, based on the RNA/microRNA sequencing data of CN-AML patients. High let-7a-2-3p expression and low miR-188-5p expression were identified to be significantly associated with longer overall survival (OS) and event free survival (EFS) for CN-AML, independently or in a combined way. Their prognostic values were further confirmed in European Leukemia Net (ELN) genetic categories. Also, in multivariable analysis with other known risk factors, high let-7a-2-3p and low miR-188-5p expression remained to be associated with longer OS and EFS. In addition, the prognostic value of these two microRNAs was confirmed in patients who received hematopoietic stem cell transplantation (HSCT). To gain more biological insights of the underlying mechanisms, we derived the genome-wide differential gene/microRNA signatures associated with the expression of let-7a-2-3p and miR-188-5p. Several common microRNA signatures indicating favorable outcome in previous studies were up-regulated in both high let-7a-2-3p expressers and low miR-188-5p expressers, including miR-135a, miR-335 and miR-125b and all members of miR-181 family. Additionally, common up-regulated genes included FOSB, IGJ, SNORD50A and ZNF502, and FOSB was a known favorable signature in AML. These common signatures further confirmed the underlying common mechanisms for these two microRNAs value as favorable prognostic biomarkers. We concluded that high let-7a-2-3p and low miR-188-5p expression could be potentially used as favorably prognostic biomarkers independently or in a combined way in CN-AML patients, whether they received HSCT or not.     

Three Drosophila Hox complex microRNAs do not have major effects on expression of evolutionarily conserved Hox gene targets during embryogenesis

The discovery of microRNAs has resulted in a major expansion of the number of molecules known to be involved in gene regulation. Elucidating the functions of animal microRNAs has posed a significant challenge as their target interactions with messenger RNAs do not adhere to simple rules. Of the thousands of known animal microRNAs, relatively few microRNA:messenger RNA regulatory interactions have been biologically validated in an normal organismal context. Here we present evidence that three microRNAs from the Hox complex in Drosophila (miR-10-5p, miR-10-3p, miR-iab-4-5p) do not have significant effects during embryogenesis on the expression of Hox genes that contain high confidence microRNAs target sites in the 3' untranslated regions of their messenger RNAs. This is significant, in that it suggests that many predicted microRNA-target interactions may not be biologically relevant, or that the outcomes of these interactions may be so subtle that mutants may only show phenotypes in specific contexts, such as in environmental stress conditions, or in combinations with other microRNA mutations.     

MicroRNA Profiling of Primary Cutaneous Large B-Cell Lymphomas

Aberrant expression of microRNAs is widely accepted to be pathogenetically involved in nodal diffuse large B-cell lymphomas (DLBCLs). However, the microRNAs profiles of primary cutaneous large B-cell lymphomas (PCLBCLs) are not yet described. Its two main subtypes, i.e., primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) and primary cutaneous follicle center lymphoma (PCFCL) are characterized by an activated B-cell (ABC)-genotype and a germinal center B-cell (GCB)-genotype, respectively. We performed high-throughput sequencing analysis on frozen tumor biopsies from 19 cases of PCFCL and PCLBCL-LT to establish microRNA profiles. Cluster analysis of the complete microRNome could not distinguish between the two subtypes, but 16 single microRNAs were found to be differentially expressed. Single microRNA RT-qPCR was conducted on formalin-fixed paraffin-embedded tumor biopsies of 20 additional cases, confirming higher expression of miR-9-5p, miR-31-5p, miR-129-2-3p and miR-214-3p in PCFCL as compared to PCLBCL-LT. MicroRNAs previously described to be higher expressed in ABC-type as compared to GCB-type nodal DLBCL were not differentially expressed between PCFCL and PCLBCL-LT. In conclusion, PCFCL and PCLBCL-LT differ in their microRNA profiles. In contrast to their gene expression profile, they only show slight resemblance with the microRNA profiles found in GCB- and ABC-type nodal DLBCL.     

Effect of miR-143-3p on C2C12 myoblast differentiation

MicroRNAs are a class of 18–22 nucleotide non-coding RNAs that modulate gene expression by associating with the 3′ untranslated regions of mRNAs. A large number of microRNAs are involved in the regulation of myoblast differentiation, many of which remain undiscovered. In this study, we found that miR-143-3p was upregulated during C2C12 myoblast differentiation and over-expression of miR-143-3p significantly inhibited the relative expression levels of MyoD, MyoG, myf5, and MyHC genes, especially in the later stages of differentiation. In addition, miR-143-3p inhibited expression of genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and β-catenin. These results indicate that miR-143-3p represents a new myogenic differentiation-associated microRNA that can inhibit C2C12 myoblast differentiation, especially in the later stages of differentiation.     

Characterization of human-specific DNA sequences obtained by genome subtraction

Recent studies on molecular evolution using nucleotide sequence data to clarify phylogenetic relationships among humans and the African great apes, have revealed that humans are more closely related to chimpanzees than to gorillas. However, the genetic basis of human uniqueness remains unclear. This is because phylogenetic studies have merely evaluated the degree of similarity by calculating the accumulation of nucleotide substitutions that have occurred in neutral DNA regions commonly present in all the species examined. In contrast, the genome subtraction method recently developed by us has revealed dissimilarity even among the genomes of the most closely related species. Here we describe the characteristics of the DNA sequences obtained by genome subtraction between humans and chimpanzees.     

The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition

Background

MicroRNAs are modifiers of gene expression, acting to reduce translation through either translational repression or mRNA cleavage. Recently, it has been shown that some microRNAs can act to promote or suppress cell transformation, with miR-17-92 described as the first oncogenic microRNA. The association of miR-17-92 encoded microRNAs with a surprisingly broad range of cancers not only underlines the clinical significance of this locus, but also suggests that miR-17-92 may regulate fundamental biological processes, and for these reasons miR-17-92 has been considered as a therapeutic target.

Results

In this study, we show that miR-17-92 is a cell cycle regulated locus, and ectopic expression of a single microRNA (miR-17-5p) is sufficient to drive a proliferative signal in HEK293T cells. For the first time, we reveal the mechanism behind this response - miR-17-5p acts specifically at the G1/S-phase cell cycle boundary, by targeting more than 20 genes involved in the transition between these phases. While both pro- and anti-proliferative genes are targeted by miR-17-5p, pro-proliferative mRNAs are specifically up-regulated by secondary and/or tertiary effects in HEK293T cells.

Conclusion

The miR-17-5p microRNA is able to act as both an oncogene and a tumor suppressor in different cellular contexts; our model of competing positive and negative signals can explain both of these activities. The coordinated suppression of proliferation-inhibitors allows miR-17-5p to efficiently de-couple negative regulators of the MAPK (mitogen activated protein kinase) signaling cascade, promoting growth in HEK293T cells. Additionally, we have demonstrated the utility of a systems biology approach as a unique and rapid approach to uncover microRNA function.     

MicroRNA and target protein patterns reveal physiopathological features of glioma subtypes

Gliomas such as oligodendrogliomas (ODG) and glioblastomas (GBM) are brain tumours with different clinical outcomes. Histology-based classification of these tumour types is often difficult. Therefore the first aim of this study was to gain microRNA data that can be used as reliable signatures of oligodendrogliomas and glioblastomas. We investigated the levels of 282 microRNAs using membrane-array hybridisation and real-time PCR in ODG, GBM and control brain tissues. In comparison to these control tissues, 26 deregulated microRNAs were identified in tumours and the tissue levels of seven microRNAs (miR-21, miR-128, miR-132, miR-134, miR-155, miR-210 and miR-409-5p) appropriately discriminated oligodendrogliomas from glioblastomas. Genomic, epigenomic and host gene expression studies were conducted to investigate the mechanisms involved in these deregulations. Another aim of this study was to better understand glioma physiopathology looking for targets of deregulated microRNAs. We discovered that some targets of these microRNAs such as STAT3, PTBP1 or SIRT1 are differentially expressed in gliomas consistent with deregulation of microRNA expression. Moreover, MDH1, the target of several deregulated microRNAs, is repressed in glioblastomas, making an intramitochondrial-NAD reduction mediated by the mitochondrial aspartate-malate shuttle unlikely. Understanding the connections between microRNAs and bioenergetic pathways in gliomas may lead to identification of novel therapeutic targets.     

Importance of DNA methylation in the inheritance of radiation-induced aberrant expression of microRNA

We studied microRNA gene expression in HeLa cells following exposure for 6 h and 8 days to Co⁶⁰ gamma rays at a dose of 4 Gy using an approach of large-scale parallel DNA sequencing. We identified 12 microRNAs with aberrant expression which were maintained in cell generations. The analysis of radiation-induced aberrant expression of pre-microRNAs made it possible to assess the importance of nuclear and cytoplasmic stages of microRNA biogenesis for preservation of its aberrant expression. On cell treatment by 5-azacytidine, aberrant expression was maintained only in two microRNAs: miR-21-3p and miR-422a, which demonstrated an increase in expression. Radiation-induced decrease in expression in ten examined microRNAs was dependent on DNA demethylation. At the same time, expression in a microRNA set, which demonstrated inheritable alteration of the expression after gamma-radiation exposure in the untreated cells, was not dependent or was weakly dependent on DNA methylation. The obtained results suggest that ionizing radiation induces aberrant DNA methylation, which affects inherited expression changes in microRNAs in cell generations after exposure to the mutagen.     

Cardiovascular and Cerebrovascular Disease Associated microRNAs Are Dysregulated in Placental Tissues Affected with Gestational Hypertension,Preeclampsia and Intrauterine Growth Restriction

Aims

To demonstrate that pregnancy-related complications are associated with alterations in cardiovascular and cerebrovascular microRNA expression. Gene expression of 32 microRNAs (miR-1-3p, miR-16-5p, miR-17-5p, miR-20a-5p, miR-20b-5p, miR-21-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-29a-3p, miR-33a-5p, miR-92a-3p, miR-100-5p, miR-103a-3p, miR-122-5p, miR-125b-5p, miR-126-3p, miR-130b-3p, miR-133a-3p, miR-143-3p, miR-145-5p, miR-146a-5p, miR-155-5p, miR-181a-5p, miR-195-5p, miR-199a-5p, miR-208a-3p, miR-210-3p, miR-221-3p, miR-342-3p, miR-499a-5p, and miR-574-3p) was assessed in placental tissues, compared between groups (35 gestational hypertension, 80 preeclampsia, 35 intrauterine growth restriction and 20 normal pregnancies) and correlated with the severity of the disease with respect to clinical signs, delivery date, and Doppler ultrasound parameters. Initially, selection and validation of endogenous controls for microRNA expression studies in placental tissues affected by pregnancy-related complications have been carried out.

Results

The expression profile of microRNAs was different between pregnancy-related complications and controls. The up-regulation of miR-499a-5p was a common phenomenon shared between gestational hypertension, preeclampsia, and intrauterine growth restriction. Preeclamptic pregnancies delivering after 34 weeks of gestation and IUGR with abnormal values of flow rate in the umbilical artery demonstrated up-regulation of miR-1-3b. Preeclampsia and IUGR requiring termination of gestation before 34 weeks of gestation were associated with down-regulation of miR-26a-5p, miR-103a-3p and miR-145-5p. On the other hand, some of microRNAs (miR-16-5p, miR-100-5p, miR-122-5p, miR-125b-5p, miR-126-3p, miR-143-3p, miR-195-5p, miR-199a-5p, miR-221-3p, miR-342-3p, and miR-574-3p) were only down-regulated or showed a trend to down-regulation just in intrauterine growth restriction pregnancies requiring the delivery before 34 weeks of gestation.

Conclusion

Epigenetic changes induced by pregnancy-related complications in placental tissue may cause later onset of cardiovascular and cerebrovascular diseases in offspring.