Ror1-Ror2 complexes modulate synapse formation in hippocampal neurons

Ror1 and Ror2, a small family of tyrosine kinase receptors, have been implicated in multiple aspects of brain development in C. elegans and X. laevis. More recently, we have shown that these receptors modulate the rate of neurite elongation in cultured rat hippocampal neurons. However, no information is available regarding a potential role of these receptors in other developmental milestones in mammalian central neurons. Neither is the identity known of the Ror ligand(s) and/or the signal transduction pathway(s) in which they participate. Here we report that the down regulation of either Ror1 or Ror2 led to a significant decrease in synapse formation in cultured hippocampal neurons. Simultaneous targeting of Ror proteins, however, did not result in an additive phenotype. Our results also indicated that Ror1 and Ror2 physically interact in the mouse brain, suggesting that they might function as heterodimers in central neurons. In addition, these Ror complexes interacted with Wnt-5a mediating its effects on synaptogenesis. Together, these data suggest that Ror proteins play a key role in Wnt-5a-activated signaling pathways leading to synapse formation in the mammalian CNS.     

RNAi干扰对K562细胞表达谱的影响

目的 联合运用RNAi技术和cDNA表达谱芯片研究干扰c-mye对K562细胞表达谱的影响。方法 经过RT-PCR和流武细胞仪检测。筛选出干扰效果最好的siRNAs,经Lipofectamine^TM 2000脂质体法转染K562细胞,提取总RNA,逆转录为cDNA,荧光标记后与K562表达谱芯片杂交。结果 经扫描、分析杂交结果,有455个基因表达下调,有12个基因表达上调。结论 基因芯片和RNAi是分析基因功能的有力工具,也是发现新的肿瘤治疗靶标的有效方法。     

Downregulation of alpha-fetoprotein siRNA inhibits proliferation of SMMC-7721 cells

AIM: To study the function of α-fetoprotein (AFP) in SMMC-7721 hepatoma cells. METHODS: A hairpin siRNA expressing plasmid pSilencer3.0-H1-afp was constructed and transfected into SMMC-7721 cells with Lipofectamine 2000. The expression of AFP was monitored by real-time RT-PCR and immunoassays, its effect on SMMC-7721 cell proliferation and cell death was detected by MTT and fluorescenceactivated cell sorter (FACS). RESULTS: The AFP-siRNA expressing plasmid downregulated the expression of AFP obviously (about 34%), and inhibited SMMC-7721 cell proliferation, but did not induce apoptosis. CONCLUSION: Downregulation of AFP siRNA inhibits proliferation of SMMC-7721 cells, but cannot cause apoptosis.     

Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs

In response to infection, invertebrates process replicating viral RNA genomes into siRNAs of discrete sizes to guide virus clearance by RNA interference. Here, we show that viral siRNAs sequenced from fruit fly, mosquito, and nematode cells were all overlapping in sequence, suggesting a possibility of using siRNAs for viral genome assembly and virus discovery. To test this idea, we examined contigs assembled from published small RNA libraries and discovered five previously undescribed viruses from cultured Drosophila cells and adult mosquitoes, including three with a positive-strand RNA genome and two with a dsRNA genome. Notably, four of the identified viruses exhibited only low sequence similarities to known viruses, such that none could be assigned into an existing virus genus. We also report detection of virus-derived PIWI-interacting RNAs (piRNAs) in Drosophila melanogaster that have not been previously described in any other host species and demonstrate viral genome assembly from viral piRNAs in the absence of viral siRNAs. Thus, this study provides a powerful culture-independent approach for virus discovery in invertebrates by assembling viral genomes directly from host immune response products without prior virus enrichment or amplification. We propose that invertebrate viruses discovered by this approach may include previously undescribed human and vertebrate viral pathogens that are transmitted by arthropod vectors.     

Mu opioid receptor knockdown in the substantia nigra/ventral tegmental area by synthetic small interfering RNA blocks the rewarding and locomotor effects of heroin

Mu opioid receptors (MOP-r) play an important role in the rewarding and locomotor stimulatory effects of heroin. The aim of the current study was to determine whether infusion of small interfering RNAs (siRNA) targeting MOP-r into the midbrain could knock down MOP-r mRNA and affect heroin-induced locomotor activity or heroin-induced conditioned place preference. Ten-week-old male C57BL/6J mice were surgically implanted bilaterally with guide cannulae directed between the substantia nigra and ventral tegmental area. After 4 days' recovery, mice were infused bilaterally with siRNAs that target the MOP-r (2 mMx0.75 microl/side/day for 3 days) or control siRNA. Seven days after the last infusion, a procedure for conditioned place preference was begun with four heroin (3 mg/kg i.p.) administration sessions alternating with four saline sessions. While heroin induced an increase in locomotor activity in all groups, siRNAs targeting specific regions of MOP-r significantly attenuated this effect. Of particular interest, mice infused with specific siRNAs targeting the MOP-r failed to develop and express conditioned place preference to heroin, or showed a significantly attenuated preference. These alterations in reward-related behaviors are likely due to the reduction in MOP-r mRNA and protein, shown in separate studies by in situ hybridization and autoradiography using the same MOP-r- siRNA infusions. Taken together, these studies demonstrate the utility of siRNA in the neurobiological study of specific components of the reward system and should contribute to the study of other complex behaviors.     

In Vivo RNAi Efficacy of Palmitic Acid‐Conjugated Dicer‐Substrate siRNA in a Subcutaneous Tumor Mouse Model

Short interfering RNAs are used in RNA interference technology and are powerful tools for target gene silencing in a sequence‐specific manner. In this study, we synthesized Dicer‐substrate siRNAs consisting of 27‐nt double‐stranded RNAs conjugated with palmitic acid at the 5′‐end of the sense strand and investigated their RNA interference efficacies in vitro and in vivo. The palmitic acid‐conjugated 27‐nt DsiRNAs (C16‐Dsi27RNAs) were prepared by our simple synthesis strategy and achieved a good yield. C16‐Dsi27RNAs showed enhanced in vitro RNA interference potency compared with not only non‐modified Dsi27RNAs but also cholesterol‐conjugated Dsi27RNAs against both an exogenous enhanced green fluorescent protein and the endogenous vascular endothelial growth factor gene in a human scirrhous‐type gastric cancer cell line that stably expressed the enhanced green fluorescent protein gene (GCIY‐eGFP). Additionally, C16‐Dsi27RNAs had potent gene silencing activity against both enhanced green fluorescent protein and vascular endothelial growth factor as target genes in a subcutaneous tumor mouse model generated from GCIY‐eGFP cells administered by intratumoral injection. These results suggest that the C16‐Dsi27RNAs will be useful next‐generation RNA interference molecules that can overcome the problems associated with RNA interference technology.     

RNA干预与microRNA

RNA干预 (RNA mediatedinterference,RNAi)已逐渐成为基因功能研究的重要工具。RNAi主要通过双链RNA的介导 ,特异性地降解相应序列的靶mRNA ,从而阻断相应基因表达的转录后水平的基因沉默 (genesilencing)。microRNAs(miRNAs)是近年才研究发现的一类新型的小分子RNAs。随着对miRNAs进一步研究 ,表明miRNAs具有组织特异性 ,并在生物生长发育阶段中起到重要的调控作用 ,但具体机制还在进一步研究中。随着对microRNAs功能及RNAi作用机理的深入研究 ,人们期望在不久的将来能从基因水平上控制人类疾病的发生发展