Expression of Gab2 in human osteosarcoma U2-OS cells

AIM: To investigate the expression of Grb2-associated binding protein 2 (Gab2) in human osteosarcoma cells and its relationship with the invasion and metastases of human osteosarcoma cells. METHODS: The technique of small RNA interference was used to transfect human osteosarcoma U2-OS cell lines. Western blotting and RT-PCR were used to detect the protein and mRNA expression of Gab2 in transfected U2-OS cells. After transfection, through chemotaxis and invasion assays in vitro, the cell migration and invasion abilities were detected. RESULTS: After transfection, the expression of Gab2 at mRNA and protein levels in Gab2 siRNA transfected cells (SiGab2/U2-OS) was lower than that in scrambled siRNA transfected cells (Scr/U2-OS) and U2-OS cells. After stimulation with epidermal growth factor (EGF) at concentration of 10 μg/L, the migration SiGab2/U2-OS cells was significantly less than Scr/U2-OS cells and U2-OS cells (P<0.01). The number of invasion cells of SiGab2/U2-OS group was significantly lower than the other 2 control groups (P<0.01). CONCLUSION: Inhibition of Gab2 expression obviously attenuates the migration and invasion abilities of human osteosarcoma U2-OS cell line.     

Effect of long non-coding RNA MEG3 on invasion and migration of colorectal cancer cells

AIM: To investigate the expression of long non-coding RNA maternally expressed gene 3(MEG3) in colorectal cancer(CRC) cells, and to observe the effect of MEG3 on the invasion and migration of CRC cells. METHODS: The levels of MEG3 in human normal colon cell NCM460 and CRC cells SW48 and LoVo were detected by real- time PCR. MEG3 was over-expressed by plasmid transfection, and the effects of MEG3 on the invasion and migration of SW48 and LoVo cells were analyzed by Transwell assay and wound healing assay. The expression of matrix metalloproteinase(MMP) family proteins was determined by Western blotting. RESULTS: The level of MEG3 was down-regulated in CRC cells compared with normal colon cell NCM460. The invasion and migration of CRC cells were reduced after MEG3 over-expression. Transwell invasion and migration assays showed that the numbers of transmembrane SW48 and LoVo cells were smaller in MEG3 over-expression group than control group(CONCLUSION: The expression of MEG3 is down-regulated in CRC cells. Over-expression of MEG3 inhibits the invasion and migration of CRC cells. TIMP-2, MMP-2 and MMP-9 might play an important role in this regulation.     

Ⅰ型鸭肝炎病毒RNA聚合酶基因的克隆及原核表达

根据Ⅰ型鸭肝炎病毒（Duck hepatitis virus,DHV-Ⅰ）ZJ-Ⅴ株基因组序列（GenBank登录号：EF382778）,设计了一对扩增RNA聚合酶基因（RNA-dependent RNA polymerase,RdRp）的特异性引物,以ZJ-Ⅴ株基因组为模板,扩增DHV-Ⅰ的RdRp基因。运用DNAStar软件对ZJ-Ⅴ株病毒与GenBank上已发表的其他不同DHV毒株的RdRp基因序列进行同源性比较和遗传进化分析,结果表明,ZJ-Ⅴ株与A型DHV-ⅠR85952株（DQ226541）的同源性为97.1%,亲缘关系最近,与2株台湾新型（B型）DHV之间为76.9%和77.0%,与C-GY株和4株韩国新型（C型）DHV株之间为76.2%~76.5%,亲缘关系较远。将RdRp基因克隆至原核表达载体pET-32a（＋）,获得重组表达质粒pET-RdRp,转化宿主菌E.coli BL21（DE3）,用IPTG进行诱导表达。SDS-PAGE分析结果显示RdRp蛋白分子量约为65 kDa,Western blot检测证明RdRp蛋白能与Ⅰ型鸭病毒性肝炎阳性血清产生特异性免疫反应,具有良好的免疫学活性。本文为进一步研究DHV-Ⅰ的复制机理奠定了基础。     

猪源隐孢子虫卵囊的分离及基因型鉴定

应用RT-PCR方法对南京、上海和合肥猪源隐孢子虫卵囊SSU rRNA部分序列进行扩增,产物测序后提交GenBank,收录号为DQ855266、DQ855267;用BLAST和DNAStar软件与GenBank参考序列进行比较,分析其同源性,绘制系统发育进化树,结合卵囊形态学观察和对小鼠、大鼠、兔、山羊和鸡的传染性试验确定隐孢子虫种类或基因型。结果表明,3地区猪源隐孢子虫分离株与微小隐孢子虫(C.parvum)同源性达94%~100%,与C.parvummouse型有99.8%~100%的同源性,并处于进化树的同一分支。因此,3地区猪源隐孢子虫是C.parvummouse型,提示猪和鼠之间存在交叉传播的可能。     

RNAi基因沉默技术及其在植物抗病性改良中应用的策略

RNAi (RNA interference) 是一种由dsRNA参与、对靶基因表达进行干扰或沉默的现象。由此发展起来的RNAi基因沉默技术已成为当今植物基因功能研究和遗传改良的一个重要手段。该技术已经在靶向病原物(真菌、细菌、病毒和线虫)基因沉默方面得到了广泛的应用,并且产生了一批抗病性增强的转基因植物。人工设计和合成的amiRNAs和ata siRNAs的成功研发加快了RNAi技术的应用。本文对RNAi基因沉默机制、RNAi技术研发进展及其在植物抗病性遗传改良中的应用进行综述,并对其应用策略进行探讨。     

RNAi及沉默通路调控研究进展

RNA干扰（RNA interference,RNAi）是由双链RNA（double-stranded RNA,dsRNA）介导的细胞内双链mRNA特异性降解的现象,属于转录后的基因沉默机制。迄今为止,RNAi已经是一种广泛应用于研究基因功能的最新和最有效的方法,并且在治疗人类和动物疾病中也有很大潜力。作者主要综述了RNAi的过程、小RNA的生物发生学及沉默通路的保护和调控。     

RNA干扰技术在动物繁殖中的应用

RNA干扰(RNA interference,RNAi)是一种由双链RNA诱发的转录后水平的基因沉默现象,是近几年发展起来的基因表达调节新机制,广泛地存在于真菌、植物和动物中。到目前为止,已有很多试验证明,RNAi作为一种基因功能研究的新手段,在动物繁殖研究中发挥了极其高效的作用。作者主要介绍了RNAi的研究历史、分子机制、作用特点、生物学意义及在动物繁殖中的应用。     

ES-D3细胞nanog基因干扰载体的构建 及其干涉效果*

选择ES细胞nanog基因中4个区域合成寡聚核苷酸,构建了4个含有小鼠U6启动子的siRNA（小分子干涉RNA）表达载体并瞬时转染小鼠ES-D3细胞.半定量RT-PCR分析显示,所构建的4个siRNA表达载体中有3个可以在细胞内转录出siRNA或shRNA（短发夹RNA）,诱发RNA干涉（RNAinterference,RNAi）,能显著抑制目的基因nanog的表达;同时发现,干扰的ES-D3细胞生长48 h后,集落形态与未干扰细胞相比差别不大,但隆起不如后者明显,形态也不如后者规则,生长速度也较慢,AKP染色阳性,与未转染细胞相比没有差别.