MicroRNAs Are Involved in the Regulation of Ovary Development in the Pathogenic Blood Fluke Schistosoma japonicum.

Schistosomes, blood flukes, are an important global public health concern. Paired adult female schistosomes produce large numbers of eggs that are primarily responsible for the disease pathology and critical for dissemination. Consequently, understanding schistosome sexual maturation and egg production may open novel perspectives for intervening with these processes to prevent clinical symptoms and to interrupt the life-cycle of these blood-flukes. microRNAs (miRNAs) are key regulators of many biological processes including development, cell proliferation, metabolism, and signal transduction. Here, we report on the identification of Schistosoma japonicum miRNAs using small RNA deep sequencing in the key stages of male-female pairing, gametogenesis, and egg production. We identified 38 miRNAs, including 10 previously unknown miRNAs. Eighteen of the miRNAs were differentially expressed between male and female schistosomes and during different stages of sexual maturation. We identified 30 potential target genes for 16 of the S. japonicum miRNAs using antibody-based pull-down assays and bioinformatic analyses. We further validated some of these target genes using either in vitro luciferase assays or in vivo miRNA suppression experiments. Notably, suppression of the female enriched miRNAs bantam and miR-31 led to morphological alteration of ovaries in female schistosomes. These findings uncover key roles for specific miRNAs in schistosome sexual maturation and egg production.

miR-290 contributes to the low abundance of cyclin D1 protein in mouse embryonic stem cells.

Mouse miR-290 cluster miRNAs are expressed specifically in early embryos and embryonic germ cells. These miRNAs play critical roles in the maintenance of pluripotency and self-renewal. Here, we showed that Cyclin D1 is a direct target gene of miR-290 cluster miRNAs. Negative relationships between the expression of Cyclin D1 protein and miR-290 cluster miRNAs in pluripotent and non-pluripotent cells, as well as in differentiating CGR8 cells were observed. Inhibition of miR-290 cluster miRNAs could arrest cells at the G1 phase and slow down the cell proliferation in CGR8 mouse stem cells. Since miR-290 cluster miRNAs are the most dominant stem-cell-specific miRNAs, our results revealed an important cause for the absence of Cyclin D1 in mouse embryonic stem cells.

MicroRNA-34a inhibits the proliferation and promotes the apoptosis of non-small cell lung cancer H1299 cell line by targeting TGFßR2.

MicroRNAs (MiRNAs) are small non-coding RNA molecules which act as important regulators of post-transcriptional gene expression by binding 3'-untranslated region (3'-UTR) of target messenger RNA (mRNA). In this study, we analyzed miRNA-34a (miR-34a) as a tumor suppressor in non-small cell lung cancer (NSCLC) H1299 cell line. The expression level of miR-34a in four different NSCLC cell lines, H1299, A549, SPCA-1, and HCC827, was significantly lower than that in the non-tumorigenic bronchial epithelium cell line BEAS-2B. In human NSCLC tissues, miR-34a expression level was also significantly decreased in pT2-4 compared with the pT1 group. Moreover, miR-34a mimic could inhibit the proliferation and triggered apoptosis in H1299 cells. Luciferase assays revealed that miR-34a inhibited TGFßR2 expression by targeting one binding site in the 3'-UTR of TGFßR2 mRNA. Quantitative real-time PCR (qRT-PCR) and Western blot assays verified that miR-34a reduced TGFßR2 expression at both mRNA and protein levels. Furthermore, downregulation of TGFßR2 by siRNA showed the same effects on the proliferation and apoptosis as miR-34a mimic in H1299 cells. Our results demonstrated that miR-34a could inhibit the proliferation and promote the apoptosis of H1299 cells partially through the downregulation of its target gene TGFßR2.

Identification of differentially expressed miRNAs in mouse spinal cord development.

MicroRNAs (miRNAs) are a class of non-coding, regulatory small RNAs of ∼22 nt. It was implicated that these small RNAs play critical roles in various important biological processes. During development, some miRNAs are specifically expressed in individual tissues and at particular developmental stages. Many miRNAs show distinct expression patterns in the development of central nervous system, including spinal cord. In this study, we first reported the miRNAs expression in the development of mouse spinal cord. Differentially expressed miRNAs in embryonic (day 13.5) and neonatal mice spinal cords were identified. The predicted target genes of the differentially expressed miRNAs were subject to gene ontology and KEGG pathway analysis, and several nervous development-related pathways were enriched, implying that these miRNAs may be involved in these pathways that regulate mouse spinal cord development.

MiR-181a-5p inhibits cell proliferation and migration by targeting Kras in non-small cell lung cancer A549 cells.

MicroRNAs play important roles in carcinogenesis and tumor progress. Lung cancer is the leading cause of cancer mortality worldwide. In this study, the function of miR-181a-5p was investigated in non-small-cell lung cancer (NSCLC). Results showed that miR-181a-5p was significantly decreased in NSCLC tissues and cell lines. The proliferation and migration of A549 cells transfected with miR-181a-5p mimic was significantly inhibited. Luciferase activity assay results demonstrated that two binding sites of Kras could be directly targeted by miR-181a-5p. Furthermore, Kras was down-regulated by miR-181a-5p at both transcriptional and translational levels. SiRNA-mediated Kras down-regulation could mimic the effects of miR-181a-5p mimic in A549 cells. Our findings suggest that miR-181a-5p plays a potential role in tumor suppression by partially targeting Kras and has the potential therapeutic application in NSCLC patients.

Genome-wide mapping of miRNAs expressed in embryonic stem cells and pluripotent stem cells generated by different reprogramming strategies.

BACKGROUND: Reprogrammed cells, including induced pluripotent stem cells (iPSCs) and nuclear transfer embryonic stem cells (NT-ESCs), are similar in many respects to natural embryonic stem cells (ESCs). However, previous studies have demonstrated that iPSCs retain a gene expression signature that is unique from that of ESCs, including differences in microRNA (miRNA) expression, while NT-ESCs are more faithfully reprogrammed cells and have better developmental potential compared with iPSCs. RESULTS: We focused on miRNA expression and explored the difference between ESCs and reprogrammed cells, especially ESCs and NT-ESCs. We also compared the distinct expression patterns among iPSCs, NT-ESCs and NT-iPSCs. The results demonstrated that reprogrammed cells (iPSCs and NT-ESCs) have unique miRNA expression patterns compared with ESCs. The comparison of differently reprogrammed cells (NT-ESCs, NT-iPSCs and iPSCs) suggests that several miRNAs have key roles in the distinct developmental potential of reprogrammed cells. CONCLUSIONS: Our data suggest that miRNAs play a part in the difference between ESCs and reprogrammed cells, as well as between MEFs and pluripotent cells. The variation of miRNA expression in reprogrammed cells derived using different reprogramming strategies suggests different characteristics induced by nuclear transfer and iPSC generation, as well as different developmental potential among NT-ESCs, iPSCs and NT-iPSCs.

Deep sequencing-based identification of pathogen-specific microRNAs in the plasma of rabbits infected with Schistosoma japonicum.

Circulating microRNAs (miRNAs) have received considerable attention as a novel class of biomarkers for the diagnosis of cancer and as signalling molecules in mediating intercellular communication. Schistosomes, the causative agents of schistosomiasis, live in the blood vessels of a mammalian host in the adult stage. In the present study, we characterized schistosome-specific small RNA populations in the plasma of rabbits infected with Schistosoma japonicum (S. japonicum) using a deep sequencing method and then identified five schistosome-specific miRNAs, including four known miRNAs (Bantam, miR-3479, miR-10 and miR-3096), and one novel miRNA (miR-0001, miRBase ID: sja-miR-8185). Four of the five schistosome-specific miRNAs were also detected by real-time RT-PCR in the plasma of S. japonicum-infected mice. In addition, our study indicated that schistosome Argonaute 2/3 may be an excretory-secretory (ES) protein. In summary, our findings are expected to provide useful information for further development of novel biomarkers for the diagnosis of schistosomiasis and also for deeper understanding of the mechanism of host-parasite interaction.

EBV promotes human CD8 NKT cell development.

The reports on the origin of human CD8(+) Valpha24(+) T-cell receptor (TCR) natural killer T (NKT) cells are controversial. The underlying mechanism that controls human CD4 versus CD8 NKT cell development is not well-characterized. In the present study, we have studied total 177 eligible patients and subjects including 128 healthy latent Epstein-Barr-virus(EBV)-infected subjects, 17 newly-onset acute infectious mononucleosis patients, 16 newly-diagnosed EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. We have established human-thymus/liver-SCID chimera, reaggregated thymic organ culture, and fetal thymic organ culture. We here show that the average frequency of total and CD8(+) NKT cells in PBMCs from 128 healthy latent EBV-infected subjects is significantly higher than in 17 acute EBV infectious mononucleosis patients, 16 EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. However, the frequency of total and CD8(+) NKT cells is remarkably increased in the acute EBV infectious mononucleosis patients at year 1 post-onset. EBV-challenge promotes CD8(+) NKT cell development in the thymus of human-thymus/liver-SCID chimeras. The frequency of total (3% of thymic cells) and CD8(+) NKT cells ( approximately 25% of NKT cells) is significantly increased in EBV-challenged chimeras, compared to those in the unchallenged chimeras (<0.01% of thymic cells, CD8(+) NKT cells undetectable, respectively). The EBV-induced increase in thymic NKT cells is also reflected in the periphery, where there is an increase in total and CD8(+) NKT cells in liver and peripheral blood in EBV-challenged chimeras. EBV-induced thymic CD8(+) NKT cells display an activated memory phenotype (CD69(+)CD45RO(hi)CD161(+)CD62L(lo)). After EBV-challenge, a proportion of NKT precursors diverges from DP thymocytes, develops and differentiates into mature CD8(+) NKT cells in thymus in EBV-challenged human-thymus/liver-SCID chimeras or reaggregated thymic organ cultures. Thymic antigen-presenting EBV-infected dendritic cells are required for this process. IL-7, produced mainly by thymic dendritic cells, is a major and essential factor for CD8(+) NKT cell differentiation in EBV-challenged human-thymus/liver-SCID chimeras and fetal thymic organ cultures. Additionally, these EBV-induced CD8(+) NKT cells produce remarkably more perforin than that in counterpart CD4(+) NKT cells, and predominately express CD8alphaalpha homodimer in their co-receptor. Thus, upon interaction with certain viruses, CD8 lineage-specific NKT cells are developed, differentiated and matured intrathymically, a finding with potential therapeutic importance against viral infections and tumors.

A simple and fast method for profiling microRNA expression from low-input total RNA by microarray.

Analysis of mature microRNA (miRNA) expression is important to understand its physiological functions and pathological implications. Microarray is a powerful technology to profile global miRNA expression. In this study, we developed a rapid miRNA labeling method by which miRNA was directly labeled in total RNA for microarray detection. This method consists of RNA tailing by poly(A) polymerase, reverse transcription, and template-switching by moloney murine leukemia virus (MMLV) reverse transcriptase. After these reactions, the small RNA cDNA was ready for labeling for microarray detection. The whole process of prearray sample preparation was dramatically shortened to 2 h. Furthermore, this method allows very limited starting total RNA (100 ng) for microarray analysis. Our data showed that the results from our method were highly consistent with that of real-time polymerase chain reaction (PCR).

MicroRNAs: potentially important regulators for schistosome development and therapeutic targets against schistosomiasis.

MicroRNAs (miRNAs) are small, endogenous non-coding RNA molecules that regulate gene expression post-transcriptionally by targeting the 3' untranslated region (3' UTR) of messenger RNAs. Since the discovery of the first miRNA in Caenorhabditis elegans, important regulatory roles for miRNAs in many key biological processes including development, cell proliferation, cell differentiation and apoptosis of many organisms have been described. Hundreds of miRNAs have been identified in various multicellular organisms and many are evolutionarily conserved. Schistosomes are multi-cellular eukaryotes with a complex life-cycle that require genes to be expressed and regulated precisely. Recently, miRNAs have been identified in two major schistosome species, Schistosoma japonicum and S. mansoni. These miRNAs are likely to play critical roles in schistosome development and gene regulation. Here, we review recent studies on schistosome miRNAs and discuss the potential roles of miRNAs in schistosome development and gene regulation. We also summarize the current status for targeting miRNAs and the potential of this approach for therapy against schistosomiasis.

Simple and sensitive microRNA labeling by terminal deoxynucleotidyl transferase.

MicroRNAs (miRNAs) constitute a critically important class of non-translated, small RNAs, which post-transcriptionally regulate gene expression via one of the multiple mechanisms. To profile miRNA expression, microarrays have been extensively applied to the high-throughput detection of miRNAs. Here, we described a novel 3'-end miRNA-labeling method for microarray detection by terminal-deoxynucleotidyl transferase (TdT). TdT can catalyze the formation of polynucleotides at RNA receptor molecule with deoxycytidine triphosphate (dCTP). Using this activity, miRNA was successfully labeled by adding fluorescence dCTPs to its 3'-end. This labeling method was very simple and sensitive. The TdT-labeling method can detect as little as 0.04 fmol of synthetic small RNA, and produce precise and accurate measurements that span a linear dynamic range from 0.04 to 5 fmol of synthetic small RNA. The high consistency of miRNA expression data between our TdT method and real-time polymerase chain reaction analysis indicated the reliability and accuracy of the TdT method. Taken together, these results emphasize the immense potential application of the TdT-labeling method for sensitive and high-throughput microarray analysis of miRNA expression.

MiR-200a is involved in rat epididymal development by targeting ß-catenin mRNA.

The expression of 350 microRNAs (miRNAs) in epididymis of rat from postnatal development to adult (from postnatal days 7-70) was profiled with home-made miRNA microarray. Among them, 48 miRNAs changed significantly, in which the expression of miR-200a increased obviously with time, in a good agreement with that obtained from northern blot analysis. The real-time quantitative-polymerase chain reaction result indicated that temporal expression of rat ß-catenin was exactly inversed to that of miR-200a during rat epididymal development, implying that miR-200a might also target ß-catenin mRNA in rat epididymis as reported by Saydam et al. in humans. The bioinformatic analysis indicated that 3' untranslated region of rat ß-catenin mRNA did contain a putative binding site for miR-200a. Meanwhile, it was found that the sequence of this binding site was different from that of human ß-catenin mRNA with a deletion of two adjacent nucleotides (U and C). But the results of luciferase targeting assay in HEK 293T cells and the overexpression of miR-200a in rat NRK cells demonstrated that miR-200a did target rat ß-catenin mRNA and cause the suppression of its expression. All these results show that miR-200a should be involved in rat epididymal development by targeting ß-catenin mRNA of rat and suppressing its expression.

Alleviating allergic airway diseases by means of short-term administration of IL-2 and dexamethasone.

BACKGROUND: IL-2 combined with dexamethasone can upregulate regulatory T (Treg) cells, but the mechanism is still under exploration. OBJECTIVE: Although previous studies focused on upregulating Treg cells in normal mice, here we investigated whether the IL-2 and dexamethasone combination treatment can upregulate Treg cells in pathological conditions, specifically in alleviating allergic airway disease. We also examined the potential pathway involved in Treg cell upregulation by IL-2 and dexamethasone. METHODS: We evaluated the dose of IL-2 and dexamethasone required to upregulate Treg cells in vivo and in vitro. We also tested IL-2 and dexamethasone in the intervention of allergic airway disease in a murine model. RESULTS: We found that administration of 400,000 IU of IL-2 and 0.1 mg of dexamethasone per mouse was effective in upregulating Treg cells, as well as in alleviating allergic airway disease in an established animal model, but this phenomenon disappeared after anti-CD25 antibody administration. We discovered that an in vitro low dose of IL-2 can protect Treg cells did not protect CD4(+)CD25(-) cells from dexamethasone-induced apoptosis by affecting forkhead box O3a phosphorylation through the Akt and serum and glucocorticoid-induced protein kinase pathways. CONCLUSIONS: IL-2/dexamethasone treatment can alleviate existing allergic airway diseases by upregulating Treg cells in vivo. A low dose of IL-2 (10(-9) to 10(-11) mol/L) can protect Treg cells but not CD4(+)CD25(-) cells from dexamethasone-induced apoptosis in vitro, thereby explaining a possible mechanism of increased proportion of Treg cells.

Evolution of MIR159/319 microRNA genes and their post-transcriptional regulatory link to siRNA pathways.

BACKGROUND: MicroRNAs (miRNAs) are prevalent and important endogenous gene regulators in eukaryotes. MiR159 and miR319 are highly conserved miRNAs essential for plant development and fertility. Despite high similarity in conservation pattern and mature miRNA sequences, miR159 and miR319 have distinct expression patterns, targets and functions. In addition, both MIR319 and MIR159 precursors produce multiple miRNAs in a phased loop-to-base manner. Thus, MIR159 and MIR319 appear to be related in origin and considerably diverged. However the phylogeny of MIR159 and MIR319 genes and why such unusual style of miRNA production has been conserved during evolution is not well understood. RESULTS: We reconstructed the phylogeny of MIR159/319 genes and analyzed their mature miRNA expression. The inferred phylogeny suggests that the MIR159/319 genes may have formed at least ten extant early-branching clades through gene duplication and loss. A series of duplications occurred in the common ancestor of seed plants leading to the original split of flowering plant MIR159 and MIR319. The results also indicate that the expression of MIR159/319 is regulated at post-transcriptional level to switch on the expression of alternative miRNAs during development in a highly spatio-temporal specific manner, and to selectively respond to the disruption of defensive siRNA pathways. Such intra-stem-loop regulation appears diverged across the early-branching clades of MIR159/319 genes. CONCLUSIONS: Our results support that the MIR159 and MIR319 genes evolve from a common ancestor, which is likely to be a phased stem-loop small RNA. Through duplication and loss of genes this miRNA gene family formed clades specific to moss, lycopods, gymnosperms and angiosperms including the two major clades of flowering plants containing the founding members of MIR319 and MIR159 genes in A.thaliana. Our analyses also suggest that some MIR159/319 have evolved into unusual miRNA genes that are regulated at post-transcriptional level to express multiple mature products with variable proportions under different circumstances. Moreover, our analyses reveal conserved regulatory link of MIR159/319 genes to siRNA pathway through post-transcriptional regulation.

Hybridization kinetics analysis of an oligonucleotide microarray for microRNA detection.

MicroRNA (miRNA) microarrays have been successfully used for profiling miRNA expression in many physiological processes such as development, differentiation, oncogenesis, and other disease processes. Detecting miRNA by miRNA microarray is actually based on nucleic acid hybridization between target molecules and their corresponding complementary probes. Due to the small size and high degree of similarity among miRNA sequences, the hybridization condition must be carefully optimized to get specific and reliable signals. Previously, we reported a microarray platform to detect miRNA expression. In this study, we evaluated the sensitivity and specificity of our microarray platform. After systematic analysis, we determined an optimized hybridization condition with high sensitivity and specificity for miRNA detection. Our results would be helpful for other hybridization-based miRNA detection methods, such as northern blot and nuclease protection assay.

Identification of targets of specific miRNAs by selective amplification of Ago2-associated mRNA.

To study the function of a miRNA, it is necessary to identify its target genes. The most common methods to reveal miRNA target genes rely on ectopically expressed tagged Ago2 and nonphysiological overexpression or inhibition of the miRNA of interest. To uncover the natural association between miRNAs and their target genes, we isolated endogenous Ago2 protein followed by a selective strategy, which only amplified target genes of the selected miRNA from the purified RNA-induced silencing complex by miRNA specific primers. This enabled us to identify the mRNAs regulated by miRNAs of interest. Our data demonstrated that this strategy is effective and highly credible. Moreover, our results showed the evidence of efficient miRNA target sites in 5' untranslated regions and open reading frames of target mRNAs.