BmNPV-miR-415 up-regulates the expression of TOR2 via Bmo-miR-5738

MicroRNAs (miRNAs) have emerged as key players in host–pathogen interaction and many virus-encoded miRNAs have been identified (computationally and/or experimentally) in a variety of organisms. A novel Bombyx mori nucleopolyhedrosis virus (BmNPV)-encoded miRNA miR-415 was previously identified through high-throughput sequencing. In this study, a BmNPV-miR-415 expression vector was constructed and transfected into BmN cells. The differentially expressed protein target of rapamycin isoform 2 (TOR2) was observed through two-dimensional gel electrophoresis and mass spectrometry. Results showed that TOR2 is not directly a target gene of BmNPV-miR-415, but its expression is up-regulated by BmNPV-miR-415 via Bmo-miR-5738, which could be induced by BmNPV.     

Differential expression of microRNAs in mouse embryonic bladder

MicroRNAs (miRNAs) are involved in several biological processes including development, differentiation and proliferation. Analysis of miRNA expression patterns in the process of embryogenesis may have substantial value in determining the mechanism of embryonic bladder development as well as for eventual therapeutic intervention. The miRNA expression profiles are distinct among the cellular types and embryonic stages as demonstrated by microarray technology and validated by quantitative real-time RT-PCR approach. Remarkably, the miRNA expression patterns suggested that unique miRNAs from epithelial and submucosal areas are responsible for mesenchymal cellular differentiation, especially regarding bladder smooth muscle cells. Our data show that miRNA expression patterns are unique in particular cell types of mouse bladder at specific developmental stages, reflecting the apparent lineage and differentiation status within the embryonic bladder. The identification of unique miRNAs expression before and after smooth muscle differentiation in site-specific area of the bladder indicates their roles in embryogenesis and may aid in future clinical intervention.     

miR-30 family promotes migratory and invasive abilities in CD133<Superscript>+</Superscript> pancreatic cancer stem-like cells

Pancreatic cancer is a deadly disease with a poor prognosis. Recently, miRNAs have been reported to be abnormally expressed in several cancers and play a role in cancer development and progression. However, the role of miRNA in cancer stem cells remains unclear. Therefore, our aim was to investigate the role of miRNA in the CD133⁺ pancreatic cancer cell line Capan-1M9 because CD133 is a putative marker of pancreatic cancer stem cells. Using miRNA microarray, we found that the expression level of the miR-30 family decreased in CD133 genetic knockdown shCD133 Capan-1M9 cells. We focused on miR-30a, -30b, and -30c in the miR-30 family and created pancreatic cancer cell sublines, each transfected with these miRNAs. High expression of miR-30a, -30b, or -30c had no effect on cell proliferation and sphere forming. In contrast, these sublines were resistant to gemcitabine, which is a standard anticancer drug for pancreatic cancer, and in addition, promoted migration and invasion. Moreover, mesenchymal markers were up-regulated by these miRNAs, suggesting that mesenchymal phenotype is associated with an increase in migration and invasion. Thus, our study demonstrated that high expression of the miR-30 family modulated by CD133 promotes migratory and invasive abilities in CD133⁺ pancreatic cancer cells. These findings suggest that targeted therapies to the miR-30 family contribute to the development of novel therapies for CD133⁺ pancreatic cancer stem cells.     

Snai1 miRNA干扰质粒构建及胃癌细胞系稳定株筛选

目的：设计并构建针对Snai1的微小干扰核糖核酸（miRNA）,最终鉴定出有效干扰质粒并筛选稳定转染的胃癌细胞株SGC-7901。方法：设计并构建4对Snai1的pcDNATM6.2-GW/EmGFPmiR microRNA及1对无效对照microRNA干扰质粒。将干扰质粒用罗氏BD转染试剂转染胃癌细胞株SGC-7901,通过倒置荧光显微镜观察绿色荧光确定转染效率。分别用不同浓度l的杀稻瘟菌素作用于SGC-7901细胞,得到杀稻瘟菌素对SGC-7901细胞的筛选浓度。Westernblot检测4对干扰质粒、阴性对照质粒对snai1蛋白水平表达的影响。结果：测序表明,Snai1干扰序列及读码框完全正确,干扰质粒瞬时转染的SGC-7901细胞系在倒置荧光显微镜下观察绿色荧光达85%以上。杀稻瘟菌素对于SGC-7901细胞的筛选浓度为5μg/ml。Westernblot结果显示,干扰序列Mi-1对Snai1有较强的干扰效果。结论：成功构建了Snai1干扰真核表达载体,同时筛选出有效干扰质粒及稳定转染株,为进一步研究Snai1在胃癌中的作用奠定了基础。     

Identification of potential microRNA markers related to Crimean-Congo hemorrhagic fever disease

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by the arbovirus Crimean-Congo hemorrhagic fever virus (CCHFV). The CCHFV has a single-stranded RNA genome of negative sense. MicroRNAs (miRNAs) are key players in virus-host interactions and viral pathogenesis. We investigated the miRNA gene expression profiles in patients with CCHF using microarray for the first time in the world. Microarray analysis was performed using mirBase Ver 21 (Agilent Technologies, Santa Clara, CA). All statistical analyses were performed across the case-control, fatal-control, and fatal-nonfatal case groups using Genespring (Ver 3.0). Fifteen miRNAs were statistical significant in patients with CCHF compared with the controls (5 were upregulated, 10 were downregulated). Seventy-five and sixty-six miRNAs are in fatal compared with control and nonfatal case, respectively (fold change ([FC] ≥50) were statistically significant. In this study, the target genes of important miRNAs were identified and Gene Ontology analyses were performed across all groups. As a result of this study, we propose that the detection of miRNAs in patients with CCHF will allow the determination of therapeutic targets in diseases. CCHF is an important public health problem that can often be fatal. In this study, we investigated miRNA expression in case-control, fatal-control, and fatal-nonfatal case groups. Significant miRNAs associated with fatality were detected in CCHF. This study will serve as a source of data for the development of an antagomir-based therapy against CCHF using miRNAs in the future.     

MiR-133b Is Down-Regulated in Human Osteosarcoma and Inhibits Osteosarcoma Cells Proliferation,Migration and Invasion,and Promotes Apoptosis

MicroRNAs (miRNAs) decrease the expression of specific target oncogenes or tumor suppressor genes and thereby play crucial roles in tumorigenesis and tumor growth. To date, the potential miRNAs regulating osteosarcoma growth and progression are not fully identified yet. In this study, the miRNA microarray assay and hierarchical clustering analysis were performed in human osteosarcoma samples. In comparison with normal human skeletal muscle, 43 miRNAs were significantly differentially expressed in human osteosarcomas (fold change ≥2 and p≤0.05). Among these miRNAs, miR-133a and miR-133b expression was decreased by 135 folds and 47 folds respectively and the decreased expression was confirmed in both frozen and paraffin-embedded osteosarcoma samples. The miR-133b precursor expression vector was then transfected into osteosarcoma cell lines U2-OS and MG-63, and the stable transfectants were selected by puromycin. We found that stable over-expression of miR-133b in osteosarcoma cell lines U2-OS and MG-63 inhibited cell proliferation, invasion and migration, and induced apoptosis. Further, over-expression of miR-133b decreased the expression of predicted target genes BCL2L2, MCL-1, IGF1R and MET, as well as the expression of phospho-Akt and FAK. This study provides a new insight into miRNAs dysregulation in osteosarcoma, and indicates that miR-133b may play as a tumor suppressor gene in osteosarcoma.     

Blockade of invasion and metastasis of breast cancer cells via targeting CXCR4 with an artificial microRNA

miRNAs have been shown to function as regulatory molecules and to play an important role in cancer progression. Very little is currently known about the increasing invasion and metastasis of breast cancer due to the loss of expressive levels of certain miRNAs in breast tumor cells. In order to determine whether the CXCR4/SDF-1 pathway is regulated by expression of miRNAs, we designed and synthesized pre-miRNA against CXCR4. This double-stranded miRNA gene was ligated with a miR-155-based Block-iT Pol II miR RNAi Expression Vector (Invitrogen). Expression levels of CXCR4 in CXCR4-miRNA-transfected breast tumor cells had significantly declined. These cells exhibited reduced migration and invasion in vitro. Furthermore, they formed fewer lung metastases in vivo compared to ctrl-miRNA-transfected cells. These data support the conclusion that miRNA against CXCR4 can serve as an alterative means of therapy to lower CXCR4 expression and to block the invasion and metastasis of breast cancer cells.     

Identification and characterization of miRNAome in tobacco (Nicotiana tabacum) by deep sequencing combined with microarray

Tobacco is one of the most important economic and agricultural crops worldwide. miRNAs have been increasingly acknowledged for their important roles in different biological processes of tobacco. However, few miRNAs have been identified so far in tobacco impeding the development of new tobacco strains with better properties. In this study, high-throughput sequencing technology was employed to identify novel tobacco miRNAs. A total of 84 potential miRNAs were obtained in tobacco, including 33 conserved and 51 novel miRNAs. Tissue-specific and topping-related miRNAs were identified. A tobacco miRNA microarray was also constructed to investigate miRNA expression patterns in different tissues, and their expression patterns were further validated by qRT-PCR and Northern Blot. Finally, the potential targets of these miRNAs were predicted based on a sequence homology search. Thus, in the current study, we have performed the comprehensive analysis of tobacco miRNAs, including their identification, expression pattern and target prediction. Our study opens a new avenue for further elucidation for their roles underlying the regulation of diversity of physiological processes.     

带有丝素重链信号肽序列的家蚕丝胶蛋白启动子驱动DsRed的瞬时分泌表达

为了探讨家蚕Bombyx mori丝素蛋白重链信号肽序列在中部丝腺组织中是否具有功能活性,根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,构建了带有丝素重链基因fib-H信号肽的家蚕丝胶-1(ser-1)启动子(ser-HS),用ser-HS驱动DsRed基因构建了分泌型瞬时表达载体pSK-SerHS-DsRed-polyA。转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed。家蚕注射载体后,可在中部丝腺腔中检测到红色荧光,表明瞬时表达的DsRed已分泌到丝腺腔内。据此提出克隆的fib-H信号肽序列在家蚕中部丝腺组织中具有信号肽的功能。     

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.     

miRNA-1和miRNA-133过表达对L6细胞增殖与分化的影响

目的观察大鼠微小RNA-1（miR-1）、微小RNA-133（miR-133）过表达对L6成肌细胞增殖分化的影响。方法分别构建miR-1、miR-133的重组慢病毒载体,并进行测序鉴定,稳定转染L6细胞后,用RT-PCR Taqman探针的方法检测miR-1、miR-133的表达水平;细胞计数实验（CCK-8试剂盒）评价miR-1、miR-133过表达后对L6细胞增殖的影响。诱导稳定转染后L6成肌细胞进行分化,观察miR-1、miR-133过表达后对L6细胞分化的影响。以Western blot法检测miR-1、miR-133过表达后,α肌动蛋白（skeletalα-actin）表达水平的变化。采用Kruskal-Wallis H检验、重复测量和单因素方差分析进行比较。结果miR-1慢病毒载体经酶切和测序鉴定序列准确,转染48 h后L6细胞miR-1组（4.292±0.50）比control组（0.231±0.86）、miR-133组（0.205±0.48）比control组（3.564±0.45）表达均显著上调（P〈0.001）;细胞计数和细胞分化实验显示,培养120 h后,过表达miR-1的L6细胞α肌动蛋白（skeletalα-actin）比对照组（0.415±0.02）表达显著升（0.676±0.02,F=222.144,P〈0.001）,分化明显加快,但增殖无明显变化;而过表达miR-133的L6细胞增殖明显加快,α肌动蛋白表达呈下降趋势（0.363±0.02,F=2385.643,P〈0.001）,分化受到抑制。结论 miR-1、miR-133慢病毒表达载体稳定转染L6成肌细胞后高效表达miR-1和miR-133,miR-1可促进L6细胞分化,miR-133能促进L6细胞增殖但抑制其分化。     

成簇pri-miRNAs表达载体的构建及表达验证

目的:旨在建立一个多重表达任意miRNA的方案,实现多个pri-miRNA表达阅读框的串联,用于miRNA簇或者miRNA家族的功能研究。方法:通过改造CRISPR/Cas9系统共表达载体42230,插入EGFP编码序列以及单个pri-miRNA序列,实现单个pri-miRNA表达载体可视化的构建。在单个pri-miRNA表达载体的基础上,通过同源重组的方式,插入下一个pri-miRNA序列,实现多个pri-miRNA表达阅读框的串联表达。结果:以miR29家族的三个miRNA为例,验证了在pri-miRNA表达阅读框串联方面的可行性;在多重的pri-miRNA表达载体中,miRNA成熟体验证结果表明,pri-miR29a和pri-miR29b能够在哺乳动物细胞中被加工成熟,其成熟体明显地过表达。在转染的293A细胞中,miR29家族的靶基因PTEN的表达水平显著降低。结论:所构建的pri-miRNA多重表达方案,能够实现多个外源miRNA在同一细胞中的共表达,对于探索miRNA家族和miRNA簇的拮抗或协同调控功能起到重要的推进作用。     

Effects of upregulated expression of microRNA-16 on biological properties of culture-activated hepatic stellate cells

In our previous studies, we identified miR-16 as being downregulated during activation of hepatic stellate cells (HSCs) by microarray hybridization. However, the roles and related mechanisms of miR-16 in HSCs are not understood. In this study, The miRNA RNAi technique was used to analyze the effects of miR-16 on biological properties of HSCs in vitro. The lentiviral vector encoding miR-16 was constructed and transfected. Furthermore, the expression level of miR-16 was measured by real-time PCR. Cellular growth and proliferation capacity were assayed using the cell counting kit-8 (CCK-8). The apoptosis rate and cell-cycle distribution were measured by flow cytometry. Cell morphological characteristics were identified by phase-contrast microscopy, fluorescence microscopy and electron microscopy. The underlying mechanisms related to the changes in biological properties were assessed. The identity of the recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. Virus titer was 10⁸ > ifu/m. Restoring the intracellular miRNAs by miR-16 administration greatly reduced the expression levels of cyclin D1 (CD1). Cell-cycle arrest and typical features of apoptosis were detected in activated HSCs treated with pLV-miR-16. Our results indicate that transduction of miR-16 offers a feasible approach to significantly inhibit HSC proliferation and increase the apoptosis index. Thus, targeted transfer of miR-16 into HSC may be useful for the treatment of hepatic fibrosis.