Silencing suppressor activity of the Tobacco rattle virus-encoded 16-kDa protein and interference with endogenous small RNA-guided regulatory pathways

Higher plants use RNA silencing as a defense mechanism against viral infections, but viruses may encode suppressor proteins that counteract these defenses. Several virus-encoded suppressors also exert an inhibitory effect on endogenous small RNA regulatory pathways. Here we characterized the Tobacco rattle virus-encoded 16-kDa (TRV-16K) protein as a suppressor that blocked local RNA silencing induced by single (s)- and double-stranded (ds) RNA, indicating that TRV-16K interfered with a step in the silencing pathway downstream of dsRNA formation. The suppressor activity of TRV-16K was severely compromised by moderate to high dosages of dsRNA inducer. When silencing was locally triggered by ssRNA or low levels of dsRNA, silencing suppression by TRV-16K was associated with reduced accumulation of silencing-related siRNAs. TRV-16K also prevented partially cell-to-cell movement and systemic propagation of silencing but not transitive amplification of RNA silencing. We showed that neither TRV nor TRV-16K caused a global deregulation of the microRNA-regulatory pathway in Arabidopsis, suggesting that interference with microRNA biology was not a prerequisite for TRV, and probably many other plant viruses, to develop systemic infections in plants.     

Lack of association of polymorphism in miRNA-196a2 with Parkinson's disease risk in a Chinese population

MicroRNAs (miRNAs) are a new class of non-protein coding RNA molecules, which participate in diverse biological pathways. We hypothesized that miRNA-196a2 polymorphism is associated with the risk of Parkinson's disease (PD) in a Chinese population. In a case-control study of 549 PD patients and 736 control subjects frequency matched by age and gender, we genotyped the single-nucleotide polymorphism (SNP) rs11614913 (T>C) in miRNA-196a2, whose target mRNA was alpha-synuclein, and assessed its association with risk of PD by TaqMan Genotyping method. No association was found for the miR-196a2 rs11614913 CT/CC genotype (odds ratio (OR), 0.879, 95% confidence interval (CI), 0.681-1.135 for CT genotype; OR, 1.085, 95% CI, 0.793-1.484 for CC genotype) with risk of PD, compared with the TT genotype. These results suggest that SNP rs11614913 in miRNA-196a2 may not contribute to the susceptibility to PD.     

Changes in microRNA expression induced by rabies virus infection in mouse brains

MicroRNAs (miRNAs) are small RNA (≈22 nt) molecules expressed endogenously in cells. They are involved in the regulation of gene expression. Recently, evidence has shown that cellular miRNAs have key regulatory roles in virus-host interactions. The rabies virus (RABV) causes a fatal infection of the central nervous systems (CNS) of warm-blooded animals, yet its pathogenesis remains poorly understood. To gain more insight into the pathogenesis of RABV, a miRNA microarray was performed as part of an investigation of changes in host miRNA expression in the brains of mice infected with RABV. The results showed that RABV infection induced modulation of the expression of sixteen miRNA molecules. These data were verified by real-time PCR. Functional analysis showed the differentially expressed miRNAs to be involved in many immune-related signaling pathways, such as the RIG-I-like receptor signaling pathway, JAK-STAT signaling pathway, chemokine signaling pathway, T-cell receptor signaling pathway, MAPK signaling pathway, leukocyte transendothelial migration, and natural killer cell mediated cytotoxicity. The predicted expression levels of the target genes of these modulated miRNAs correlated with measurements of gene expression measured by DNA microarray and qRT-PCR.     

MicroRNA在感染性疾病相关肿瘤中作用的进展

MicroRNA（miRNA）是一类近年来新发现的非编码小RNA分子,广泛存在于动物、植物和病毒中。通过与靶mRNA3＇端非翻译区完全或不完全互补配对,调控靶基因的表达或翻译,从而参与调控个体发育、细胞凋亡、增殖及分化等生命活动,其表达失调会导致疾病的发生。本文就miRNA在感染性疾病相关肿瘤中的作用研究进行简要综述。     

维生素D受体特异性siRNA表达载体的构建

目的通过构建维生素D受体（VDR）基因特异性siRNA（small interfering RNA）真核表达载体,检测其对血管内皮细胞（EC）中VDR基因的沉默作用,为进一步探讨VDR与心血管疾病发生发展关系的研究奠定基础。方法构建小鼠VDR特异性siRNA表达载体并测序,进行人EC/鼠EC的培养,进行siRNA转染试验,倒置荧光显微镜下观察细胞转染效率,Western blot进行鉴定。结果针对VDR基因的4个不同位点,设计、构建了4个siRNA干扰载体,经测序,序列全部正确。成功完成了人EC/鼠EC的培养及转染,荧光显微镜观察发现转染的重组质粒在EC内出现不同的转染效率,1号质粒的转染效率约为65%,2号质粒的转染效率约为50%,3、4号质粒的转染效率比较低。取转染效率较高的1、2号质粒做进一步转染干预,转染72h后,采用Western blot方法检测人EC/鼠EC内VDR蛋白质表达,与空白对照组比较1,、2号质粒在人EC中对VDR的表达无明显抑制作用,而在鼠EC中则有明显抑制作用,其中以1号质粒抑制效果最为明显,目的蛋白IOD/内参蛋白IOD比值发现两干扰组在人EC中对VDR的表达无明显抑制作用,而在鼠EC中则有明显抑制作用,其中以1号干扰质粒抑制效果最明显,抑制率达到了70%。结论成功完成了人EC/鼠EC的培养、转染及转染干预,并行Western blot鉴定,发现抑制效率明显的为鼠EC 1号质粒组。表明针对鼠VDR基因的siRNA表达载体有很强的特异性,并且对VDR基因干扰的位点不同表现的抑制效应不一。     

miR-106a抑制胶质瘤细胞增殖并诱导凋亡

目的胶质瘤是最常见的原发性中枢神经系统肿瘤,具有较高的病死率。现有的研究表明miR-106a在多种肿瘤中表达上调,并发挥着致癌性作用,但miR-106a在胶质瘤中的表达和作用却并不清楚。方法不同级别胶质瘤组织和胶质瘤细胞系(T98G,SHG44,U87,U251,U373)内的miR-106a水平通过实时定量聚合酶链式反应(qRT-PCR)测定。转染miRNA寡聚核苷酸后的U87和U251细胞的增殖能力和细胞周期分别采用四甲基偶氮唑盐(MTT)比色法和流式细胞仪测定,并且通过膜联蛋白/碘化丙啶(annexin V/PI)双染法测定了miR-106a对胶质瘤细胞的凋亡影响。结果miR-106a在胶质瘤组织和胶质瘤细胞系内表达水平相对正常脑组织显著下降,并且miR-106a表达水平与胶质瘤病理级别负相关。胶质瘤细胞转染miR-106a后,可检测到过表达的miR-106a可显著抑制胶质瘤细胞的增殖能力,阻滞细胞周期于G0/G1期,同时诱导胶质瘤细胞凋亡增加。结论 miR-106a可阻滞胶质瘤细胞细胞周期、抑制增殖和诱导凋亡。     

微小RNA与子宫颈癌

子宫颈癌是妇科最常见的恶性肿瘤之一。微小RNA(miRNA)是一类小分子非编码调控的单链RNA。近年来,关于miRNA与子宫颈癌关系的研究成为热点。大量的研究表明,miRNA在细胞增殖、凋亡和肿瘤形成等方面广泛而精确地调控蛋白质的合成。miRNA的异常表达被认为与宫颈癌的发生与发展密切相关。现就miRNA与子宫颈癌的发生、发展及治疗和预后等方面予以综述。     

MicroRNA在肺癌中的研究进展

MicroRNA(miRNA)是一类非编码RNA,在转录水平负性调控基因表达,miRNAs几乎参与调节细胞的各个生物过程。在肿瘤组织中,miRNAs表达异常。MiRNAs既可起癌基因功能,又能作为抑癌因子,参与肿瘤的发生及发展。在肺癌组织中miRNAs有特定的表达谱,参与调节肿瘤血管生成等多个过程,并可作为生物标志物用以早期诊断,靶向治疗及临床预后。作为一类新的分子靶标,miRNA为肺癌的诊断和治疗提供了新的方向。     

A delivery system specifically approaching bone resorption surfaces to facilitate therapeutic modulation of microRNAs in osteoclasts

Dysregulated microRNAs in osteoclasts could cause many skeletal diseases. The therapeutic manipulation of these pathogenic microRNAs necessitates novel, efficient delivery systems to facilitate microRNAs modulators targeting osteoclasts with minimal off-target effects. Bone resorption surfaces characterized by highly crystallized hydroxyapatite are dominantly occupied by osteoclasts. Considering that the eight repeating sequences of aspartate (D-Asp₈) could preferably bind to highly crystallized hydroxyapatite, we developed a targeting system by conjugating D-Asp₈ peptide with liposome for delivering microRNA modulators specifically to bone resorption surfaces and subsequently encapsulated antagomir-148a (a microRNA modulator suppressing the osteoclastogenic miR-148a), i.e. (D-Asp₈)-liposome-antagomir-148a. Our results demonstrated that D-Asp₈ could facilitate the enrichment of antagomir-148a and the subsequent down-regulation of miR-148a in osteoclasts in vivo, resulting in reduced bone resorption and attenuated deterioration of trabecular architecture in osteoporotic mice. Mechanistically, the osteoclast-targeted delivery depended on the interaction between bone resorption surfaces and D-Asp₈. No detectable liver and kidney toxicity was found in mice after single/multiple dose(s) treatment of (D-Asp₈)-liposome-antagomir-148a. These results indicated that (D-Asp₈)-liposome as a promising osteoclast-targeting delivery system could facilitate clinical translation of microRNA modulators in treating those osteoclast-dysfunction-induced skeletal diseases.