Characterization of microRNAs in serum： a novel class of biomarkers for diagnosis of cancer and other diseases

Dysregulated expression of microRNAs （miRNAs） in various tissues has been associated with a variety of diseases, including cancers. Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Two non-small cell lung cancer-specific serum miRNAs obtained by Solexa were further validated in an independent trial of 75 healthy donors and 152 cancer patients, using quantitative reverse transcription polymerase chain reaction assays. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.

Identification of mouse liver mitochondria-associated miRNAs and their potential biological functions

Dear Editor, By analyzing the genome-wide microRNA (miRNA) expression profile using microRNA microarray and stemloop miRNA qPCR assay, we report that unique miRNAs are enriched in mitochondria and these mitochondriaassociated miRNAs may be involved in the regulation of gene expression of mitochondria and other general cellular processes such as apoptosis, proliferation and differentiation.

Identification and characterization of microRNAs in raw milk during different periods of lactation, commercial fluid, and powdered milk products

Recent baby formula milk powder contamination incidents have shown that the classic markers or standards in milk quality control are insufficient in identifying ＂manipulated＂ poor-quality milk. In the present study, we demonstrated for the first time that cow milk contains large amounts of microRNAs （miRNAs） and that the unique expression profile of milk-specific miRNAs can serve as a novel indicator and possible new standard for the quafity control of raw milk and milk-related commercial products, such as fluid milk and powdered formula milk. First, using Solexa sequencing, we systematically screened miRNA expression in raw milk and identified a total of 245 miR- NAs in raw milk. Unlike other classic biomarkers whose expression levels are nearly identical at different periods of lactation, individual miRNAs can be significantly altered during lactation process, implicating that miRNAs may be a more accurate indicator to reflect the quality alteration of milk. Second, using TaqMan probe-based miRNA quantitative RT-PCR, we further identified seven miRNAs that have a relatively consistent expression throughout the lactation process, and more importantly, the expression profile of these seven milk-specific miRNAs can serve as an ideal biomarker for discriminating poor-quality or ＂manipulated＂ milk from pure raw milk, as well as for the quality control of commercial milk products, such as fluid milk and powdered formula milk. Together, our findings provide a basis for understanding the physiological role of milk miRNAs and a new potential standard for determining the quality of raw milk or milk-related commercial products.

PGC-la induces apoptosis in human epithelial ovarian cancer cells through a PPARy-dependent pathway

Peroxisome proliferator-activated receptor gamma （PPAR）,） coactivator-1 alpha （PGC-1α） coactivates multiple transcription factors and regulates several metabolic processes. The current study investigated the role of PGC-1α in the induction of apoptosis in human epithelial ovarian cancer cells. The PGC-1α mRNA level between human ovaries and human ovarian epithelial tumors was examined by quantitative RT-PCR. Less PGC- 1α expression was found in the surface epithelium of malignant tumors compared with normal ovaries. Overexpression of PGC-1α in human epithelial ovarian cancer cell line Ho-8910 induced cell apoptosis through the coordinated regulation of Bcl-2 and Bax expression. Microarray analyses confirmed that PGC-1α dramatically affected the apoptosis-related genes in Ho-8910 cells. Mitochondrial functional assay showed that the induction of apoptosis was through the terminal stage by the release of cytochrome c. Furthermore, PG-C- 1 α-induced apoptosis was partially, but not completely, blocked by PPAR）, antagonist （GW9662）, and suppression of PPAR）, expression by siRNA also inhibited PGC-1α-induced apoptosis in Ho-8910 cells. These data suggested that PGC-1α exerted its effect through a PPARγ-dependent pathway. Our findings indicated that PGC-1α was involved in the apoptotic signal transduction pathways and downregulation of PGC-1α may be a key point in promoting epithelial ovarian cancer growth and progression.

Hypoxia induces PGC-la expression and mitochondrial biogenesis in the myocardium of TOF patients

PGC-1α, a potent transcriptional coactivator, is the major regulator of mitochondrial biogenesis and activity in the cardiac muscle. The dysregulation of PGC-la and its target genes has been reported to be associated with congenital and acquired heart diseases. By examining myocardium samples from patients with Tetralogy of Fallot, we show here that PGC-1α expression levels are markedly increased in patients compared with healthy controls and positively correlated with the severity of cyanosis. Furthermore, hypoxia significantly induced the expression of PGC-1α and mitochondrial biogenesis in cultured cardiac myocytes. Mechanistic studies suggest that hypoxia-induced PGC-1α expression is regulated through the AMPK signaling pathway. Together, our data indicate that hypoxia can stimulate the expression of PGC-1α and mitochondrial biogenesis in the cardiac myocytes, and this process might provide a potential adaptive mechanism for cardiac myocytes to increase ATP output and minimize hypoxic damage to the heart.

Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

A quantitative real time reverse-transcription polymerase chain reaction （qRT-PCR） assay with specific primers recommended by the World Health Organization （WHO） has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin （HA） genes of the pandemic HlN1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic HIN1/2009 virus infection.

Horizontal transfer of microRNAs: molecular mechanisms and clinical applications

A new class of RNA regulatory genes known as microRNAs(miRNAs)has been found to introduce a whole new layer of gene regulation in eukaryotes.The intensive studies of the past several years have demonstrated that miRNAs are not only found intracellularly,but are also detectable outside cells,including in various body fluids(e.g.serum,plasma,saliva,urine and milk).This phenomenon raises questions about the biological function of such extracellular miRNAs.Substantial amounts of extracellular miRNAs are enclosed in small membranous vesicles(e.g.exosomes,shedding vesicles and apoptotic bodies)or packaged with RNA-binding proteins(e.g.high-density lipoprotein,Argonaute 2 and nucleophosmin 1).These miRNAs may function as secreted signaling molecules to influence the recipient cell phenotypes.Furthermore,secreted extracellular miRNAs may reflect molecular changes in the cells from which they are derived and can therefore potentially serve as diagnostic indicators of disease.Several studies also point to the potential application of siRNA/miRNA delivery as a new therapeutic strategy for treating diseases.In this review,we summarize what is known about the mechanism of miRNA secretion.In addition,we describe the pathophysiological roles of secreted miRNAs and their clinical potential as diagnostic biomarkers and therapeutic drugs.We believe that miRNA transfer between cells will have a significant impact on biological research in the coming years.

Immune modulatory function of abundant immune-related microRNAs in microvesicles from bovine colostrum

Colostrum provides essential nutrients and immunologically active factors that are beneficial to newborns.Our previous work demonstrated that milk contains large amounts of miRNA that is largely stored in milk-derived microvesicles(MVs).In the present study,we found that the MVs from colostrum contain signifi cantly higher levels of several immune-related miRNAs.We hypothesized that the colostrum MVs may transfer the immune-related miR-NAs into cells,which contribute to its immune modulatory feature.We isolated colostrum MVs by ultracentrifugation and demonstrated several immune modulation features associated with miRNAs.We also provide evidence that the physical structure of milk-derived MVs is essential for transfer miRNAs and following immune modulation effect.Moreover,we found that colostrum powder-derived MVs also contains higher levels of immune-related miRNAs that display similar immune modulation effects.Taken together,these results show that MV-containing immunerelated miRNAs may be a novel mechanism by which co-lostrum modulates body immune response.

The protective role of myeloid-derived suppressor cells in concanavalin A-induced hepatic injury

The mechanism underlying T cell-mediated fulminant hepatitis is not fully understood. In this study, we investigated whether myeloid derived suppressor cells （MDSCs） could prevent the concanavalin A （ConA）- induced hepatitis through suppressing T cell proliferation. We observed an increase in the frequencies of MDSCs in mouse spleen and liver at early stage of ConA treatment, implicating that the MDSCs might be involved in the initial resistance of mice against ConA- mediated inflammation. Subpopulation analysis showed that the MDSCs in liver of ConA-induced mice were mainly granulocytic MDSCs. Adoptive transfer of the bone marrow-derived MDSCs into ConA-treated mice showed that the MDSCs migrated into the liver and spleen where they suppressed T cell proliferation through ROS pathway. In addition, the frequencies of MDSCs in mice were also significantly increased by the treatment with immune suppressor glucocorticoids. Transfer of MDSCs into the regulatory T cell （Treg）- depleted mice showed that the protective effect of MDSCs on ConA-induced hepatitis is Treg-independent. In conclusion, our results demonstrate that MDSCs possess a direct protective role in T cell-mediated hepatitis, and increasing the frequency of MDSCs by either adoptive transfer or glucocorticoid treatment represents a potential cell-based therapeutic strategy for the acute inflammatory disease.

Nuclear microRNAs and their unconventional role in regulating non-coding RNAs

MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that are involved in post-transcriptional gene regulation. It has long been assumed that miRNAs exert their roles only in the cytoplasm, where they recognize their target protein-coding messenger RNAs (mRNAs), and result in translational repression or target mRNA degradation. Recent studies, however, have revealed that mature miRNAs can also be transported from the cytoplasm to the nucleus and that these nuclear miRNAs can function in an unconventional manner to regulate the biogenesis and functions of ncRNAs (including miRNAs and long ncRNAs), adding a new layer of complexity to our understanding of gene regulation. In this review, we summarize recent literature on the working model of these unconventional miRNAs and speculate on their biological significance. We have every reason to believe that these novel models of miRNA function will become a major research topic in gene regulation in eukaryotes.

miR-10a inhibits cell proliferation and promotes cell apoptosis by targeting BCL6 in diffuse large B-cell lymphoma

The BCL6 （B-Cell Lymphoma 6） gene is a proto-onco- gene that is often expressed in diffuse large B-ceU lymphomas （DLBCLs）. BCL6 loss of function can kill DLBCL cells, demonstrating that BCL6 is necessary for the survival of DLBCL cells and could be a therapeutic target. In this study, we found that BCL6 protein levels were consistently upregulated in DLBCL tissues, whereas its mRNA levels varied randomly in tissues, suggesting that a post-transcriptional mechanism was involved in BCL6 regulation. We used bioinformatics analysis to search for miRNAs, which potentially target BCL6, and identified specific targeting sites for miR-10a in the 3＇-untranslated region （3＇-UTR） of BCL6. We fur- ther identified an inverse correlation between miR-10a levels and BCL6 protein levels, but not mRNA levels, in DLBCL tumor tissue samples. By overexpressing or knocking down miR-10a in DLBCL cells, we experi- mentally validated that miR-10a directly recognizes the 3＇-UTR of the BCL6 transcript and regulated BCL6expression. Furthermore, we demonstrated that nega- tively regulating BCL6 by miR-10a suppressed the pro- liferation and promoted apoptosis of DLBCL cells.

VlicroRNA-495 induces breast cancer ce migration by targeting JAM-A

MicroRNAs （miRNAs） are small, non-coding RNAs that function as post-transcriptional regulators of gene expression. The deregulated expression of miRNAs is associated with a variety of diseases, including breast cancer. In the present study, we found that miR-495 was markedly up-regulated in clinical breast cancer samples by quantitative real time-PCR （qRT-PCR）. Junctional adhesion molecule A （JAM-A） was predicted to be a potential target of miR-495 by bioinformatics analysis and was subsequently verified by luciferase assay and Western blotting. JAM-A was found to be negatively correlated with the migration of breast cancer cells through loss-of-function and gain-offunction assays, and the inhibition of JAM-A by miR- 495 promoted the migration of MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of JAM-A could restore miR-495-induced breast cancer cell migration. Taken together, our findings suggest that miR-4g5 could facilitate breast cancer progression through the repression of JAM-A, making this miRNA a potential therapeutic target.

Accurate quantification of 3'-terminal 2'-O-methylated small RNAs by utilizing oxidative deep sequencing and stem-loop RT-qPCR

The continuing discoveries of novel classes of RNA modifications in various organisms have raised the need for improving sensitive,convenient,and reliable methods for quantifying RNA modifications.In particular,a subset of small RNAs,including microRNAs(miRNAs)and Piwi-interacting RNAs(piRNAs),are modified at their 3'-terminal nucleotides via 2'-0-methylation.However,quantifying the levels of these small RNAs is difficult because 2'-0-methylation at the RNA 3'-terminus inhibits the activity of polyadenylate polymerase and T4 RNA ligase.These two enzymes are indispensable for RNA labeling or ligation in conventional miRNA quantification assays.In this study,we profiled 3'-terminal 2'-0-methyl plant miRNAs in the livers of rice-fed mice by oxidative deep sequencing and detected increasing amounts of plant miRNAs with prolonged oxidation treatment.We further compared the efficiency of stem-loop and poly(A)-tailed RT-qPCR in quantifying plant miRNAs in animal tissues and identified stem-loop RT-qPCR as the only suitable approach.Likewise,stem-loop RT-qPCR was superior to poly(A)-tailed RT-qPCR in quantifying 3'-terminal 2'-0-methyl piRNAs in human seminal plasma.In summary,this study established a standard procedure for quantifying the levels of 3'-terminal 2'-0-methyl miRNAs in plants and piRNAs.Accurate measurement of the 3'-terminal 2'-0-methylation of small RNAs has profound implications for understanding their pathophysiologic roles in biological systems.

Selective secretion of microRNA in CNS system

EXTRACELLULAR miRNAs ARE PRESENT IN VARIOUS BODY FLUIDS MicroRNAs(miRNAs)are a diverse class of endogenous small non-coding RNAs,which posttranscriptionally regulate gene expression by interacting with their binding sites in target mRNAs.Recently,several studies have demonstrated that miRNAs are also detectable outside cells,and these miRNAs may be called extracellular miRNAs.Extracellular miRNAs are found in various body fluids,including plasma and serum(Chen et al.,2008),saliva(Park et al.,2009),amniotic fl uid(Weber et al.,2010),Bronchial lavage(Molina-Pinelo et al.,2012;Weber et al.,2010),sputum(Xie et al.,2010),tears and urine(Weber et al.,2010),bile(Shigehara et al.,2011),seminal plasma(Wang et al.,2011),breast milk(Zhou et al.,2012),peritoneal fluid(Chen et al.,2012a).Finding the extracellular miRNAs extended the research field of miRNAs,and got a lot of fruitful research fi ndings.

MicroRNAs in Drug-induced Liver Injury

Drug-induced liver injury (DILI) is a leading cause of acute liver failure,and a major reason for the recall of marketed drugs.Detection of potential liver injury is a challenge for clinical management and preclinical drug safety studies,as well as a great obstacle to the development of new,effective and safe drugs.Currently,serum levels of alanine and aspartate aminotransferases are the gold standard for evaluating liver injury.However,these levels are assessed by nonspecific,insensitive,and non-predictive tests,and often result in false-positive results.Therefore,there is an urgent need for better DILI biomarkers to guide risk assessment and patient management.The discovery of microRNAs (miRNAs) as a new class of gene expression regulators has triggered an explosion of research,particularly on the measurement of miRNAs in various body fluids as biomarkers for many human diseases.The properties of miRNA-based biomarkers,such as tissue specificity and high stability and sensitivity,suggest they could be used as novel,minimally invasive and stable DILI biomarkers.In the current review,we summarize recent progress concerning the role of miRNAs in diagnosing and monitoring both clinical and preclinical DILI,and discuss the main advantages and challenges of miRNAs as novel DILI biomarkers.

MicroRNA-29a modulates axon branching by targeting doublecortin in primary neurons

MicroRNAs （miRNAs） are endogenously expressed small, non-coding transcripts that regulate protein expression. Substantial evidences suggest that miRNAs are enriched in central nervous system, where they are hypothesized to play pivotal roles during neural devel- opment. In the present study, we analyzed miRNAs expression in mice cerebral cortex and hippocampus at different developmental stages and found miR-29a increased dramatically at postnatal stages. In addition, we provided strong evidences that miR-29a is enriched in mature neurons both in vitro and in v/vo. Further investigation demonstrated that the activation of gluta- mate receptors induced endogenous miR-29a level in primary neurons. Moreover, we showed that miR-29a directly regulated its target protein Doublecortin （DCX） expression, which further modulated axon branching in primary culture. Together, our results suggested that miR-29a play an important role in neuronal development of mice cerebrum.

Gold glitters everywhere: nucleus microRNAs and their functions

As a highly conserved class of endogenous small(~22 nucleotides)non-coding RNAs,microRNAs(miRNAs)regulate a broad spectrum of biological processes.Increasing evidences suggested that miRNAs generally regulated gene expression at the posttranscriptional stage via inhibiting the translational process or degrading mRNA.Recent studies have also revealed that there is extensive amount of miRNA,as well as miRNA function-related proteins,in the cell nucleus.Although the molecular basis underneath the biogenesis and function of nucleus miRNAs remains largely unknown,the presence of various miRNAs and miRNA function-related proteins in the nucleus strongly argue that miRNAs may execute their role throughout the whole gene expression pathway.Here we review the recent advances in the researches about the nucleus miRNAs,including the biosynthesis pathways,biological functions and potential regulation machinery of nucleus miRNAs.

PKM2 controls the degranulation of secondary and tertiary granules in neutrophils by phosphorylating SNAP-23

Polymorphonuclear neutrophils(PMNs)possess multiple distinct types of cytoplasmic granules:secondary granules,tertiary granules,and azurophil granules[1].It has been suggested that PMN degranulation is controlled by exocytotic machinery comprising membrane-specific SNARE proteins,soluble N-ethylmaleimide sensitive factor(NSF)and soluble NSF-attaching proteins[2].