Targeted silencing of heparanase gene by small interfering RNA inhibits invasiveness and metastasis of osteosarcoma cells

The effects of targeted silencing of heparanase gene by small interfering RNA(siRNA) on invasiveness and metastasis of osteosarcoma cells(MG63 cells) were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA(pGenesil-shRNA) was constructed successfully.MG63 cells were randomly allocated into 3 groups:blank group,empty vector(pGenesil) transfected group and expression vector(pGenesil-shRNA) transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63 cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell prolifera-tion was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels of heparanase were down-regulated by 76.1%(P<0.01) and 75.3%(P<0.01) respectively in the pGenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.     

Neocortical disruption and behavioral impairments in rats following in utero RNAi of candidate dyslexia risk gene Kiaa0319

Within the last decade several genes have been identified as candidate risk genes for developmental dyslexia. Recent research using animal models and embryonic RNA interference (RNAi) has shown that a subset of the candidate dyslexia risk genes--DYX1C1, ROBO1, DCDC2, KIAA0319--regulate critical parameters of neocortical development, such as neuronal migration. For example, embryonic disruption of the rodent homolog of DYX1C1 disrupts neuronal migration and produces deficits in rapid auditory processing (RAP) and working memory--phenotypes that have been reported to be associated with developmental dyslexia. In the current study we used a modified prepulse inhibition paradigm to assess acoustic discrimination abilities of male Wistar rats following in utero RNA interference targeting Kiaa0319. We also assessed spatial learning and working memory using a Morris water maze (MWM) and a radial arm water maze. We found that embryonic interference with this gene resulted in disrupted migration of neocortical neurons leading to formation of heterotopia in white matter, and to formation of hippocampal dysplasia in a subset of animals. These animals displayed deficits in processing complex acoustic stimuli, and those with hippocampal malformations exhibited impaired spatial learning abilities. No significant impairment in working memory was detected in the Kiaa0319 RNAi treated animals. Taken together, these results suggest that Kiaa0319 plays a role in neuronal migration during embryonic development, and that early interference with this gene results in an array of behavioral deficits including impairments in rapid auditory processing and simple spatial learning.     

肿瘤坏死因子α主要通过其受体1作用于鼠肾上腺皮质Y1细胞皮质醇的表达

背景和目的：全身炎症反应综合征时肿瘤坏死因子α（TNF-α）在导致肾上腺相对不足中起着重要的作用。为探讨TNF-α是通过其哪个受体来抑制皮质醇释放,我们研究肾上腺皮质Y1细胞TNF-α受体表达情况及其受体阻断与皮质醇释放间的关系。 方法: 应用RT-PCR和免疫细胞化学方法来评估Y1细胞中的TNF受体的表达。 合成与TNF受体1（TNF-R1）DNA片段相对应的shRNA序列,并克隆于表达载体pcDNA™6.2-GW/EmGFP中,然后转染Y1细胞。采用实时定量PCR（Q-PCR）检测转染shRNA或 mock-shRNA Y1细胞TNF-R1表达的阻断率,同时对转染的Y1细胞加入TNF-α 和 ACTH,检测Y1细胞皮质醇的表达水平。 结果： RT-PCR和免疫细胞化学方法,在肾上腺皮质Y1细胞检测到TNF-R1的表达,而检测不到TNF-R2的表达。shRNA 转染的Y1细胞中TNF-R1 mRNA表达明显低于mock shRNA转染的Y1细胞（P<0.05）。通过shRNA阻断TNF-R1表达,TNF-α就不能抑制ACTH促皮质醇的合成,shRNA 转染Y1细胞的皮质醇表达水平明显高于mock-shRNA Y1细胞(P<0.05)。 结论：我们的结果显示,在肾上腺皮质中,TNF-α抑制ACTH促皮质醇的合成是通过TNF-R1受体。     

慢病毒载体介导shRNA的研究进展

RNA干扰（RNAi）通过双链RNA的介导,特异性阻止相关序列的表达,从而导致转录后水平的基因沉默.RNAi广泛存在于真核生物中.慢病毒载体是理想的真核细胞基因转移工具,广泛应用于相关的RNAi研究领域,如抗病毒研究、癌症及其治疗、基因治疗.现已发现,慢病毒载体能够介导组织、时间特异的RNAi,在疾病的基因靶向治疗上必有广阔前景.     

C-erbB-2 shRNA对小鼠肺腺癌细胞化疗敏感性的影响及其机制

目的：探讨人表皮生长因子受体2短发夹RNA(C-erbB-2 shRNA)对小鼠肺腺癌Lewis细胞化疗敏感性的影响及其机制，为非小细胞肺癌尤其是腺癌的治疗提供新的思路。方法：常规培养小鼠肺腺癌Lewis细胞，分为未转染组、pGPU6/RFP/Neo-shNC组和pGPU6/RFP/Neo-erbB-2组；应用Lipofectamine 2000将质粒快速转染各组Lewis细胞；应用RT-PCR法检测各组细胞C-erbB-2 mRNA表达水平；应用Western blottting法检测各组细胞C-erbB-2蛋白表达水平。将Lewis细胞分为未转染对照组、pGPU6/RFP/Neo-shNC组、卡铂组、pGPU6/RFP/Neo-erbB-2组、pGPU6/RFP/Neo-erbB-2+卡铂组和pGPU6/RFP/Neo-shNC + 卡铂组；应用流式细胞术检测各组细胞凋亡率；应用Western blotting法检测各组细胞Bcl-2和Bax蛋白表达水平。结果：pGPU6/RFP/Neo-erbB-2组C-erbB-2 mRNA和蛋白表达水平均低于未转染组和pGPU6/RFP/Neo-shNC组；pGPU6/RFP/Neo-erbB-2+卡铂组细胞凋亡率高于其他各组（P<0.01）；与其他各组比较，pGPU6/RFP/Neo-erbB-2 + 卡铂组Bcl-2蛋白表达水平下降、Bax蛋白表达水平增加。结论：C-erbB-2 shRNA能增强小鼠肺腺癌Lewis细胞对卡铂的敏感性，其机制可能是通过上调Bax蛋白、下调Bcl-2蛋白表达，进而增强卡铂诱导的细胞凋亡。     

食管鳞癌ECA109细胞中Survivin通过ERK信号通路调控c-myc基因表达的机制

目的：探讨在食管鳞癌ECA109细胞中Survivin对c-myc基因的调控作用。方法：将食管鳞癌ECA109细胞分为Survivin shRNA干扰组、阴性质粒对照组（Control shRNA）和空白细胞株（未加任何处理的食管癌ECA109细胞）,将2μg Survivin shRNA质粒、2μg Control shRNA质粒分别转染ECA109细胞,48 h后收集各组细胞,采用RT-PCR法检测各组Survivin、c-myc mRNA的表达,Western blot法检测Survivin、c-myc蛋白及p-ERK蛋白的表达;采用ERK、p38、JNK、JAK/STA3、PI3K/Akt信号传导通路的特异性抑制剂分别阻断ECA109细胞中关键激酶的表达,RT-PCR法检测c-myc mRNA的表达。结果：与阴性质粒对照组和空白细胞组比较,Survivin shRNA组Survivin表达下调,c-myc mRNA及蛋白的表达降低,差异均具有统计学意义（P均 < 0.05）;采用ERK、p38、JNK、JAK/STA3、PI3K/Akt信号通路抑制剂分别作用于食管癌ECA109细胞,PD98059（ERK信号通路阻断剂）组c-myc mRNA及蛋白表达降低（P < 0.05）;Survivin shRNA干扰沉默Survivin基因后ERK磷酸化蛋白表达降低（P < 0.05）。结论：Survivin对c-myc具有正向调控作用,可能通过ERK信号传导通路调控c-myc的表达。     

RNA干扰体外抑制乙型肝炎病毒抗原表达的实验研究

目的 针对乙型肝炎病毒(HBV)prec/c区构建shRNA表达载体,观察shRNA表达载体对HBV抗原表达的抑制作用,为运用RNA干扰(RNAi)技术治疗乙型肝炎奠定基础.方法 运用基因重组技术构建shRNA表达载体,经酶切,测序鉴定确认后,与1.5倍HBV真核表达质粒pHBV1.5共转染Hela细胞,并于转染后72 h,用微粒子酶免疫实验(MEIA)分别检测细胞上清液和细胞裂解液中HBsAg和HBeAg表达水平;用半定量PCR检测prec/c mRNA的转录情况;分析shRNA表达载体对HBe/HBs抗原表达的抑制情况.结果 成功构建了针对HBV prec/c区的shRNA表达载体psiHBV4、psiHBV5、psiHBV6及无关shRNA表达载体psiC;筛选到对HBeAg有明显的抑制作用的psiHBV4载体,同时其对HBsAg的表达有促进作用;psiHBV5和psiHBV6对HBeAg的抑制作用相对较弱,其对HBsAg的表达也有不同程度的促进作用;无关干扰对照psiC对HBV两种抗原的表达均无显著抑制作用.结论 成功构建带U6启动子的shRNA表达载体并初步筛选到可显著抑制HBeAg表达的psiHBV4;本研究设计的3个靶向序列中,只有一个序列有大约70%的抑制率,而另外两个序列的抑制率相对较小,说明RNA干扰作用有较强的序列依赖性;无关干扰序列psiC几乎无干扰作用,说明siRNA产生的干扰作用具有高度的特异性.以上结果为应用RNAi治疗乙型肝炎奠定了一定的基础.     

Inhibition of reporter gene and Iridovirus-tiger frog virus in fish cell by RNA interference

We describe the specific silencing of reporter gene lacZ in FHM cells (muscle cells of fathead minnow, a fish cell line) by either expressing small hairpin RNAs (shRNAs) from plasmids or transfecting small interfering RNAs (siRNAs) transcribed in vitro. Two types of dsRNAs could inhibit reporter gene expression, and siRNAs were more effective, while both of them worked very well in HeLa cells. siRNAs were tested for silencing expression of the major capsid protein (MCP) encoded by tiger frog virus (TFV), an iridovirus causing severe disease in fish. siRNAs targeting mcp gene effectively inhibited TFV replication in fish cells as demonstrated by reduced mcp RNA level, postponed emergence of cytopathogenic effect, as well as reduced TFV titer and particles in cells. The results suggest that the siRNA method suppressed TFV efficiently in fish cells, providing a potential approach to the therapy of aquaculture viral diseases.