Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing comple...Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA- targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA- specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells’ antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).展开更多
为了探讨靶向人端粒酶反转录酶(hTERT)基因的小干扰RNA(s iRNA)表达载体是否具有抑制H eL a细胞端粒酶活性的能力,人工合成2条64个核苷酸(n t)的片段,其中19n t与hTERT基因1789-1807位碱基同源,将其退火、连接到质粒pSU PER中,构建pSU P...为了探讨靶向人端粒酶反转录酶(hTERT)基因的小干扰RNA(s iRNA)表达载体是否具有抑制H eL a细胞端粒酶活性的能力,人工合成2条64个核苷酸(n t)的片段,其中19n t与hTERT基因1789-1807位碱基同源,将其退火、连接到质粒pSU PER中,构建pSU P-hTE。在pSU P-hTE基础上,把该质粒的启动子和64n t插入片段酶切、克隆到质粒pEGFP-C 1中生成pEGFP-hTE,从而获得新霉素抗性筛选质粒。将pEGFP-hTE用脂质体转染H eL a细胞,G 418筛选后获得抗性克隆并将其收获、传代,用不同方法检测hTERT的mRNA和蛋白表达水平、H eL a细胞的端粒酶活性以及细胞的增殖能力。结果显示,pEGFP-hTE转染的H eL a细胞与对照组比较,hTERT的mRNA水平下降及蛋白表达减少、细胞端粒酶活性降低38%,但细胞增殖能力没有明显改变。以上结果表明,pEGFP-hTE能通过RNA干扰(RNA i)途径特异性抑制H eL a细胞hTERT基因的表达,从而有效抑制细胞端粒酶活性,这可能将为肿瘤生物治疗提供一条新的途径。展开更多
文摘Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA- targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA- specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells’ antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).
文摘为了探讨靶向人端粒酶反转录酶(hTERT)基因的小干扰RNA(s iRNA)表达载体是否具有抑制H eL a细胞端粒酶活性的能力,人工合成2条64个核苷酸(n t)的片段,其中19n t与hTERT基因1789-1807位碱基同源,将其退火、连接到质粒pSU PER中,构建pSU P-hTE。在pSU P-hTE基础上,把该质粒的启动子和64n t插入片段酶切、克隆到质粒pEGFP-C 1中生成pEGFP-hTE,从而获得新霉素抗性筛选质粒。将pEGFP-hTE用脂质体转染H eL a细胞,G 418筛选后获得抗性克隆并将其收获、传代,用不同方法检测hTERT的mRNA和蛋白表达水平、H eL a细胞的端粒酶活性以及细胞的增殖能力。结果显示,pEGFP-hTE转染的H eL a细胞与对照组比较,hTERT的mRNA水平下降及蛋白表达减少、细胞端粒酶活性降低38%,但细胞增殖能力没有明显改变。以上结果表明,pEGFP-hTE能通过RNA干扰(RNA i)途径特异性抑制H eL a细胞hTERT基因的表达,从而有效抑制细胞端粒酶活性,这可能将为肿瘤生物治疗提供一条新的途径。
