降解组测序技术在植物miRNA研究中的应用

目前, 利用芯片技术和miRNA测序可快速、准确地检测到物种中所含有的miRNA。随着越来越多的miRNA被发现, miRNA靶基因的确定已成为研究miRNA生物学功能的关键。传统的miRNA靶基因的寻找主要依赖生物信息学预测、AGO蛋白免疫共沉淀和荧光素酶法等。随着高通量测序技术的持续革新, 出现了一种新的miRNA靶基因的检测方法, 即降解组测序(degradome sequencing)法, 该方法拥有高通量测序技术、生物信息学分析和RACE验证三者的优势, 并已成功应用于拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)和小立碗藓(Physcomitrella patens)等模式植物miRNA靶基因的检测。基于已发表的相关文献和联川生物降解组测序平台, 该文对降解组测序技术应用于植物miRNA靶基因的研究进展及其实验原理进行了综述, 同时对运用该技术可进行的更深入研究进行了讨论。     

Potent effect of target structure on microRNA function

MicroRNAs (miRNAs) are small noncoding RNAs that repress protein synthesis by binding to target messenger RNAs. We investigated the effect of target secondary structure on the efficacy of repression by miRNAs. Using structures predicted by the Sfold program, we model the interaction between an miRNA and a target as a two-step hybridization reaction: nucleation at an accessible target site followed by hybrid elongation to disrupt local target secondary structure and form the complete miRNA-target duplex. This model accurately accounts for the sensitivity to repression by let-7 of various mutant forms of the Caenorhabditis elegans lin-41 3' untranslated region and for other experimentally tested miRNA-target interactions in C. elegans and Drosophila melanogaster. These findings indicate a potent effect of target structure on target recognition by miRNAs and establish a structure-based framework for genome-wide identification of animal miRNA targets.     

MicroRNA-target gene responses to root knot nematode (Meloidogyne incognita) infection in cotton (Gossypium hirsutum L.)

MicroRNAs (miRNAs) are a large class of small regulatory RNA molecules, however no study has been performed to elucidate the role of miRNAs in cotton (Gossypium hirsutum) response to the root knot nematode (RKN, Meloidogyne incognita) infection. We selected 28 miRNAs and 8 miRNA target genes to investigate the miRNA-target gene response to M. incognita infection. Our results show that RKN infection significantly affected the expression of several miRNAs and their targeted genes. After 10 days of RKN infection, expression fold changes on miRNA expressions ranged from down-regulated by 33% to upregulated by 406%; meanwhile the expression levels of miRNA target genes were 45.8% to 231%. Three miRNA-target pairs, miR159-MYB, miR319-TCP4 and miR167-ARF8, showed inverse expression patterns between gene targets and their corresponded miRNAs, suggesting miRNA-mediated gene regulation in cotton roots in response to RKN infection.     

A wide repertoire of miRNA binding sites: prediction and functional implications

MOTIVATION: Over the past decade, deciphering the roles of microRNAs (miRNAs) has relied heavily upon the identification of their targets. Most of the targets that were computationally and experimentally characterized were evolutionarily conserved 'seed' targets, containing a perfect 6-8 nt match between the miRNA 5(')-region and the messenger RNA (mRNA). Gradually, it has become evident that other types of miRNA binding can confer target regulation, but their characterization has been lagging behind. RESULTS: Here, we complement the putative evolutionarily-conserved seed-containing targets by a wide repertoire of putative targets exhibiting a variety of miRNA binding patterns, predicted by our algorithm RepTar. These include non-conserved sites, 'seed' binding sites with G:U-wobbles within the seed, '3(') compensatory' sites and 'centered' sites. Apart from the centered sites, we demonstrate the functionality of these sites and characterize the target profile of a miRNA by the types of binding sites predicted in its target 3(') UTRs. We find that different miRNAs have individual target profiles, with some more inclined to seed binding and others more inclined to binding through 3(') compensatory sites. This diversity in targeting patterns is also evident within several miRNA families (defined by common seed sequences), leading to divergence in the target sets of members of the same family. The prediction of non-conventional miRNA targets is also beneficial in the search for targets of the non-conserved viral miRNAs. Analyzing the cellular targets of viral miRNAs, we show that viral miRNAs use various binding patterns to exploit cellular miRNA binding sites and suggest roles for these targets in virus-host interactions.     

Identification and expression analysis of miRNAs in germination and seedling growth of Tibetan hulless barley

Germination and seedling growth are crucial for plant development and agricultural production. While, the regulatory mechanisms during these processes in Tibetan hulless barley (Hordeum vulgare L. var. nudum) are not well understood. Given the regulatory roles of microRNAs (miRNAs) in crop plants and the irreplaceability of barley in the highland area of China, we herein presented a genome-wide survey of miRNAs to reveal a potential regulatory network in the early developmental stages of two Tibetan hulless barleys, from which a total of 156 miRNAs was identified including 35 known and 121 novel ones. Six of the identified novel miRNAs were further experimentally validated. According to the evolutionary analysis, miR156, miR166, miR168, and miR171 were conserved across Tibetan hulless barleys and eight other seed plants. Expression profiles of ten known miRNAs showed that they were involved in phytohormone signaling, carbohydrate and lipid metabolism, as well as juvenile-adult transition during barley development. Moreover, a total of 1280 genes targeted by 101 miRNAs were predicted from both barley libraries. Three genes (PLN03212, MATE eukaryotic, and GRAS) were validated via the RNA ligase-mediated 5′-rapid amplification of cDNA ends (RLM-5' RACE) to be the targets of hvu-miR159a, hvu-miR166a, and hvu-miR171-3p, respectively. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of putative targets, the most abundant pathways were related to “metabolism”.These results revealed that miRNA-target pairs participating in the regulation of multigene expression and the embryonic development of Tibetan hulless barleys were controlled by complex mechanisms involving the concordant expression of different miRNAs and feedback loops among miRNAs as well as their targets. The study provides insight into the regulatory network of barley miRNAs for better understanding of miRNA functions during germination and seedling growth.     

Functional Profiling of Precursor MicroRNAs Identifies MicroRNAs Essential for Glioma Proliferation

Cancer initiation and progression involve microRNAs that can function like tumor suppressors and oncogenes. The functional significance of most miRNAs is currently unknown. To determine systematically which microRNAs are essential for glioma growth, we screened a precursor microRNA library in three human glioblastoma and one astroglial cell line model systems. The most prominent and consistent cell proliferation–reducing hits were validated in secondary screening with an additional apoptosis endpoint. The functional screening data were integrated in the miRNA expression data to find underexpressed true functional tumor suppressor miRNAs. In addition, we used miRNA-target gene predictions and combined siRNA functional screening data to find the most probable miRNA-target gene pairs with a similar functional effect on proliferation. Nine novel functional miRNAs (hsa-miR-129, -136, -145, -155, -181b, -342-5p, -342-3p, -376a/b) in GBM cell lines were validated for their importance in glioma cell growth, and similar effects for six target genes (ROCK1, RHOA, MET, CSF1R, EIF2AK1, FGF7) of these miRNAs were shown functionally. The clinical significance of the functional hits was validated in miRNA expression data from the TCGA glioblastoma multiforme (GBM) tumor cohort. Five tumor suppressor miRNAs (hsa-miR-136, -145, -342, -129, -376a) showed significant underexpression in clinical GBM tumor samples from the TCGA GBM cohort further supporting the role of these miRNAs in vivo. Most importantly, higher hsa-miR-145 expression in GBM tumors yielded significantly better survival (p<0.005) in a subset of patients thus validating it as a genuine tumor suppressor miRNA. This systematic functional profiling provides important new knowledge about functionally relevant miRNAs in GBM biology and may offer new targets for treating glioma.     

Detecting microRNAs of high influence on protein functional interaction networks: a prostate cancer case study

ABSTRACT: BACKGROUND: The use of biological molecular network information for diagnostic and prognostic purposes and elucidation of molecular disease mechanism is a key objective in systems biomedicine. The network of regulatory miRNA-target and functional protein interactions is a rich source of information to elucidate the function and the prognostic value of miRNAs in cancer. The objective of this study is to identify miRNAs that have high influence on target protein complexes in prostate cancer as a case study. This could provide biomarkers or therapeutic targets relevant for prostate cancer treatment. RESULTS: Our findings demonstrate that a miRNA's functional role can be explained by its target protein connectivity within a physical and functional interaction network. To detect miRNAs with high influence on target protein modules, we integrated miRNA and mRNA expression profiles with a sequence based miRNA-target network and human functional and physical protein interactions (FPI). miRNAs with high influence on target protein complexes play a role in prostate cancer progression and are promising diagnostic or prognostic biomarkers. We uncovered several miRNA-regulated protein modules which were enriched in focal adhesion and prostate cancer genes. Several miRNAs such as miR-96, miR-182, and miR-143 demonstrated high influence on their target protein complexes and could explain most of the gene expression changes in our analyzed prostate cancer data set. CONCLUSIONS: We describe a novel method to identify active miRNA-target modules relevant to prostate cancer progression and outcome. miRNAs with high influence on protein networks are valuable biomarkers that can be used in clinical investigations for prostate cancer treatment.