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.

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.

Identification of Ebola virus microRNAs and their putative pathological function

Ebola virus(EBOV),a member of the filovirus family,is an enveloped negative-sense RNA virus that causes lethal infections in humans and primates.Recently,more than 1000 people have been killed by the Ebola virus disease in Africa,yet no specific treatment or diagnostic tests for EBOV are available.In this study,we identified two putative viral microRNA precursors(pre-miRNAs)and three putative mature microRNAs(miRNAs)derived from the EBOV genome.The production of the EBOV miRNAs was further validated in HEK293T cells transfected with a pcDNA6.2-GW/EmGFP-EBOV-pre-miRNA plasmid,indicating that EBOV miRNAs can be produced through the cellular miRNA processing machinery.We also predicted the potential target genes of these EBOV miRNAs and their possible biological functions.Overall,this study reports for the first time that EBOV may produce miRNAs,which could serve as non-invasive biomarkers for the diagnosis and prognosis of EBOV infection and as therapeutic targets for Ebola viral infection treatment.

MicroRNA-200a is up-regulated in aged rats with erectile dysfunction and could attenuate endothelial function via SIRT1 inhibition

MiR-200a was shown to be upregulated in the corpus cavernosum （CC） of rats with aging-related erectile dysfunction （A-ED） in our previous study. Among its target genes, SIRT1 was also reported as a protective factor in erectile function by our groups previously. Thus, miR-200a might attenuate the erectile function in A-ED via SIRT1 inhibition. In the present study, three animal groups were included： aged rats with ED （group AE, n = 8）, aged rats with normal erectile function （group AN, n = 8）, and young rats as normal controls （group YN, n = 8）. CCs from each group were collected for histological and molecular measurements to validate the dysregulation of miR-200a and SIRT1. After that, the cavernous endothelial cells （CECs） from CC of aged rats with normal erectile function were transfected with miR-200a in vitro. Then the expression of SIRT1 and molecules within the eNOS/NO/PKG pathway were measured to investigate whether the transfection could imitate the attenuated process of erectile function in the aged. As a result, miR-200a was upregulated while the SIRT1, the levels of eNOS and cGMP were all downregulated in the CCs from AE group. After transfection in vitro, the miR-200a was upregulated while the SIRT1 and levels of eNOS and cGMP were obviously downregulated. Finally, based on the results of our previous study, we further verify that up-regulation of miR-200a could participate in the mechanisms of A-ED via SIRT1 inhibition, and mainly attenuate endothelial function via influencing the eNOS/NO/PKG pathway.

A Single Central Pattern Generator for the Control of a Locomotor Rolling Wave in Mollusc Aplysia

Locomotion in mollusc Aplysia is implemented by a pedal rolling wave,a type of axial locomotion.Well-studied examples of axial locomotion(pedal waves in Drosophila larvae and body waves in leech,lamprey,and fish)are generated in a segmented nervous system via activation of multiple coupled central pattern generators(CPGs).Pedal waves in molluscs,however,are generated by a single pedal ganglion,and it is unknown whether there are single or multiple CPGs that generate rhythmic activity and phase shifts between different body parts.During locomotion in intact Aplysia,bursting activity in the parapedal commissural nerve(PPCN)was found to occur during tail contraction.A cluster of 20 to 30 P1 root neurons(P1Ns)on the ventral surface of the pedal ganglion,active during the pedal wave,were identified.Computational cluster analysis revealed that there are 2 phases to the motor program:phase I(centered around 168°)and phase II(centered around 357°).PPCN activity occurs during phase II.The majority of P1Ns are motoneurons.Coactive P1Ns tend to be electrically coupled.Two classes of pedal interneurons(PIs)were characterized.Class 1(PI1 and PI2)is active during phase I.Their axons make a loop within the pedal ganglion and contribute to locomotor pattern generation.They are electrically coupled to P1Ns that fire during phase I.Class 2(PI3)is active during phase II and innervates the contralateral pedal ganglion.PI3 may contribute to bilateral coordination.Overall,our findings support the idea that Aplysia pedal waves are generated by a single CPG.

Microvesicle-delivery miR-150 promotes tumorigenesis by up-regulating VEGF,and the neutralization of miR-150 attenuate tumor development

Tumor-associated macrophages(TAMs)mostly exhibit M2-like(alternatively activated)properties and play posi-tive roles in angiogenesis and tumorigenesis.Vascular en-dothelial growth factor(VEGF)is a key angiogenic factor.During tumor development,TAMs secrete VEGF and other factors to promote angiogenesis;thus,anti-treatment against TAMs and VEGF can repress cancer development,which has been demonstrated in clinical trials and on an experimental level.In the present work,we show that miR-150 is an oncomir because of its promotional effect on VEGF.MiR-150 targets TAMs to up-regulate their secre-tion of VEGF in vitro.With the utilization of cell-derived vesicles,named microvesicles(MVs),we transferred an-tisense RNA targeted to miR-150 into mice and found that the neutralization of miR-150 down-regulates miR-150 and VEGF levels in vivo and attenuates angiogenesis.There-fore,we proposed the therapeutic potential of neutralizing miR-150 to treat cancer and demonstrated a novel,natu-ral,microvesicle-based method for the transfer of nucleic acids.

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.

Identification of microRNA-like RNAs in Ophiocordyceps sinensis

Ophiocordyceps sinensis is well known as a traditional Chinese medicine and has widely been used for over 2,000 years to stimulate immune system, decrease blood pressure and to inhibit tumor growth. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, miRNAs in fungi were less studied until the discovery of microRNA-like RNA(milRNA). High-throughput sequencing and bioinformatics approaches were used to identify conserved and novel milRNAs in O. sinensis. 40 conserved milRNAs were identified, while 23 pre-miRNA candidates encoding 31 novel milRNAs were predicted. Furthermore, the potential target genes of milRNAs in human were predicted and gene ontology analysis was applied to these genes. Enrichment analysis of GO-represented biological process showed that target genes of both conserved and novel milRNAs are involved in development, metabolic and immune processes, indicating the potential roles of milRNAs of O. sinensis in pharmacological effects as health food and traditional Chinese medicine. This study is the first report on genome-wide analysis of milRNAs in O. sinensis and it provides a useful resource to further study the potential roles of milRNAs as active components of O. sinensis in health food or traditional Chinese medicine.

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.

Serum mitochondrial tsRNA serves as a novel biomarker for hepatocarcinoma diagnosis

Hepatocellular carcinoma(HCC),which makes up the majority of liver cancer,is induced by the infection of hepatitis B/C virus.Biomarkers are needed to facilitate the early detection of HCC,which is often diagnosed too late for effective therapy.The tRNA-derived small RNAs(tsRNAs)play vital roles in tumorigenesis and are stable in circulation.However,the diagnostic values and biological functions of circulating tsRNAs,especially for HCC,are still unknown.In this study,we first utilized RNA sequencing followed by quantitative reverse-transcription PCR to analyze tsRNA signatures in HCC serum.We identified tRF-Gln-TTG-006,which was remarkably upregulated in HCC serum(training cohort:24 HCC patients vs.24 healthy controls).In the validation stage,we found that tRF-Gln-TTG-006 signature could distinguish HCC cases from healthy subjects with high sensitivity(80.4%)and specificity(79.4%)even in the early stage(Stage I:sensitivity,79.0%;specificity,74.8%;155 healthy controls vs.153 HCC patients from two cohorts).Moreover,in vitro studies indicated that circulating tRF-Gln-TTG-006 was released from tumor cells,and its biological function was predicted by bioinformatics assay and validated by colony formation and apoptosis assays.In summary,our study demonstrated that serum tsRNA signature may serve as a novel biomarker of HCC.

Spectrin:Structure, function and disease

Spectrin is a large,cytoskeletal,and heterodimeric protein composed of modular structure of and subunits,it typically contains 106 contiguous amino acid sequence motifs called"spectrin repeats".Spectrin is crucial for maintaining the stability and structure of the cell membrane and the shape of a cell.Moreover,it contributes to diverse cell functions such as cell adhesion,cell spreading,and the cell cycle.Mutations of spectrin lead to various human diseases such as hereditary hemolytic anemia,type 5 spinocerebellar ataxia,cancer,as well as others.This review focuses on recent advances in determining the structure and function of spectrin as well as its role in disease.

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.

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.

Decreased inhibition of exosomal miRNAs on SARS-CoV-2 replication underlies poor outcomes in elderly people and diabetic patients

Elderly people and patients with comorbidities are at higher risk of COVID-19 infection,resulting in severe complications and high mortality.However,the underlying mechanisms are unclear.In this study,we investigate whether miRNAs in serum exosomes can exert antiviral functions and affect the response to COVID-19 in the elderly and people with diabetes.

MiRNA-203 suppresses tumor cell proliferation, migration and invasion by targeting Slug in gastric cancer

Snail, a family of zinc finger transcription factors, plays an important role in morphogenesis and embryogenesis. Snail zinc finger family 2 （SNAI2 or Slug） has been demonstrated to regulate carcinogenesis of several human cancers including breast, prostate, head, neck,

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.

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.

miR-181b functions as an oncomiR in colorectal cancer by targeting PDCDZ

Programmed cell death 4 （PDCD4） is a RNA-binding protein that acts as a tumor suppressor in many cancer types, including colorectal cancer （CRC）. During CRC carcinogenesis, PDCD4 protein levels remarkably decrease, but the underlying molecular mechanism for decreased PDCD4 expression is not fully understood. In this study, we performed bioinformatics analysis to identify miRNAs that potentially target PDCD4. We demonstrated miR-181b as a direct regulator of PDCD4. We further showed that activation of IL6/STAT3 signaling pathway increased miR-181b expression and conse- quently resulted in downregulation of PDCD4 in CRC cells. In addition, we investigated the biological effects of PDCD4 inhibition by miR-181b both in vitro and in vivo and found that miR-181b could promote cell proliferation and migration and suppress apoptosis in CRC cells and accelerate tumor growth in xenograft mice, potentially through targeting PDCD4. Taken toge- ther, this study highlights an oncomiR role for miR-181b in regulating PDCD4 in CRC and suggests that miR-181b may be a novel molecular therapeutic target for CRC.

Secreted miR-34a in astrocytic shedding vesicles enhanced the vulnerability of dopaminergic neurons to neurotoxins by targeting Bcl-2

MicroRNAs （miRNAs） are a class of noncoding RNAs that regulates target gene expression at posttranscrip- tional level, leading to further biological functions. We have demonstrated that microvesicles （MVs） can deliver miRNAs into target cells as a novel way of intercellular communication. It is reported that in central nervous system, glial cells release MVs, which modulate neu-ronal function in normal condition. To elucidate the potential role of glial MVs in disease, we evaluated the effects of secreted astrocytic MVs on stress condition. Our results demonstrated that after Lipopolysaccharide （LPS） stimulation, astrocytes released shedding vesi- cles （SVs） that enhanced vulnerability of dopaminergic neurons to neurotoxin. Further investigation showed that increased astrocytic miR-34a in SVs was involved in this progress via targeting anti-apoptotic protein Bcl-2 in dopaminergic neurons. We also found that inhibition of astrocytic miR-34a after LPS stimulation can postpone dopaminergic neuron loss under neurotoxin stress. These data revealed a novel mechanism underlying astrocyte-neuron interaction in disease.

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.