应用RNA干扰抑制端粒酶逆转录酶表达诱导HepG2细胞凋亡

人端粒酶由RNA亚单位（hTR）和蛋白组分构成。蛋白组分包括端粒酶逆转录酶催化亚基（hTERT）和端粒酶相关蛋白1（hTEPl）。hTERT表达与端粒酶活性密切相关，是细胞永生化过程中端粒酶活化的限速步骤。抑制hTERT的表达可快速诱导细胞凋亡，其机制不是通过端粒缩短所致。表明hTERT除了其蛋白催化活性外，尚可与其他分子相互作用诱导细胞凋亡。     

靶向多药耐药基因mdr1 RNA干扰载体的构建及鉴定

目的 构建靶向人多药耐药基因mdr1的microRNA(miRNA)干扰表达载体并进行鉴定. 方法 设计并合成4对编码mdr1 pre-miRNA的单链寡核苷酸,退火成双链后将其连接入miRNA干扰载体pcDNA6.2-GW/EmGFP-miR,构建成重组质粒,酶切并测序鉴定其正确性. 结果 获得的重组质粒经酶切测序证实其插入片段大小与预期相符,序列完全正确. 结论 成功构建了4个靶向mdr1的miRNA干扰载体.     

STAT3靶向RNAi腺病毒表达载体的构建与鉴定

目的 观察针对STAT3基因的RNAi腺病毒表达载体对大鼠神经胶质瘤细胞的干扰效果,为探索肿瘤基因治疗的新途径奠定基础.方法设计针对STAT3编码区的短发夹结构的两条DNA序列,经退火成互补双链,克隆到PGEM-T中,构建重组体,再对重组质粒进行酶切鉴定,DNA测序分析后再克隆到穿梭质粒PDC316中,通过Lipofectamine2000的介导和腺病毒包装质粒PBHG共转染至人胚肾HEK293细胞株中,经同源重组后获得重组腺病毒rAD-STAT3,应用PCR鉴定重组腺病毒,空斑传代纯化病毒并反复冻融扩增病毒,以50%组织培养感染剂量法(TCID50)测定病毒滴度.以阴性为对照,转染到大鼠神经胶质瘤(C6)细胞,Western印迹法检测STAT3蛋白质表达量的改变,RT-PCR法观察STAT3基因表达改变.结果酶切鉴定和测序分析均提示STAT3靶向RNAi病毒表达载体构建成功,阳性病毒载体转染后C6细胞STAT3基因表达及蛋白质表达均较阴性转染的对照组显著下降.结论 STAT3靶向RNAi腺病毒表达载体成功构建,并能有效抑制C6细胞中STAT3基因表达.     

Lentivirus-mediated RNA interference targeting enhancer of zeste homolog 2 inhibits hepatocellular carcinoma growth through down-regulation of stathmin

Enhancer of zeste homolog 2 (EZH2) has been shown to be overexpressed in hepatocellular (HCC). We investigated the potential role of EZH2 in HCC tumorigenesis and examined the usefulness of RNA interference (RNAi) targeting EZH2 as a form of HCC treatment. Lentivirus-mediated RNAi was employed to knock-down EZH2 expression in human hepatoma cells to study the function of EZH2 in tumorigenesis and evaluate the treatment efficacy. Lentivirus-mediated RNAi effectively reduced EZH2 expression. Suppression of EZH2 in HCC cells significantly reduced their growth rate in vitro and markedly diminished their tumorigenicity in vivo. Moreover, in a mice model of established large-sized HCC, we showed that intratumor injection of lentiviral (Lenti)-shRNA (short hairpin RNA) or siRNA (small interfering RNA) targeting EZH2 produced significant tumor regression. To understand its molecular mechanism of action, we employed proteomic profiling technique and found that stathmin 1 is the downstream target of EZH2, as Lenti-shEZH2 treatment decreased stathmin protein expression, and ectopic overexpression of stathmin prevented Lenti-shEZH2 mediated tumor growth inhibition. CONCLUSION: Results from our study suggested for the first time that EZH2 plays a key role in HCC tumorigenesis, and is a novel therapeutic target for HCC.     

RNA干扰在白血病基因治疗中的应用前景

RNA干扰(RNAi)作为一种进化保守的转录后基因沉默机制,是指双链RNA分子(dsRNA)被切割成21～23个核苷酸的小干扰RNA(siRNA),最终使其同源的mRNA特异性降解.RNAi现广泛用于多个生物体的功能基因组学研究,以及在基因表达异常导致的多种疾病的临床前模型中进行干预治疗.大约50%的白血病伴有异常基因的表达,利用RNAi肿瘤特异性基因靶向治疗为治疗白血病提供了一条新途径.随着siRNA在活体中的稳定性和转染效率的提高、非特异性作用的减少,正迅速应用于临床中.     

Cx43基因shRNA慢病毒载体的构建及其对大鼠心肌细胞Cx43基因的作用

目的构建缝隙连接蛋白(Cx)43基因shRNA慢病毒载体,并检测其对大鼠心肌细胞Cx43基因的作用。方法针对Cx43基因序列,设计RNA干扰靶点序列,合成靶序列的双链DNA,接入pGCL-GFP载体,挑选阳性克隆行PCR鉴定及测序。用pHelper1.0和pHelper2.0质粒转染293T细胞,包装产生具备感染能力的慢病毒。以293T细胞中绿色荧光蛋白的细胞数量计算病毒滴度;以最适感染复数感染大鼠心肌细胞,通过荧光显微镜观察感染效率;应用real-time PCR和Western blot法检测大鼠心肌细胞Cx43 mRNA及Cx43蛋白表达,并评价其抑制效果。结果经PCR鉴定和测序证实,Cx43慢病毒载体构建正确,其病毒滴度为8×108TU/mL、转染效率为82.59%。荧光定量real-time PCR法检测Cx43 mRNA的抑制率为96.10%,Western blot法检测Cx43蛋白的抑制率为77.16%。结论成功构建了Cx43基因shRNA慢病毒载体,其能显著抑制大鼠心肌细胞Cx43基因的表达。     

Accessibility of DNA in Chromatin to DNA Polymerase and RNA Polymerase

The accessibility of DNA in chromatin to both exogenous DNA polymerase and RNA polymerase is slight when compared to isolated DNA. DNA in extracted chromatin is somewhat more accessible to these enzymes than is DNA in the chromatin of isolated nuclei; and the DNA template of chromatin is more accessible to DNA polymerase than to RNA polymerase. In these experiments we have given much attention to the technique of scintillation counting, since artifacts arising in this procedure can lead to erroneous conclusions.     

聚合酶RNA干扰对小鼠体内乙型肝炎病毒的抑制作用

目的:利用流体动力学法构建小鼠HBV感染模型,观察pSiHBV/P在体内对HBV的抑制作用.方法:以流体动力学法构建小鼠HBV感染模型,将pSiHBV/P与pHBV1.3尾静脉共注射Balb/c小鼠.转染后第6天,用酶联免疫分析法检测小鼠血清乙肝表面抗原(HBsAg),用荧光定量PCR检测血清HBV DNA的水平,用免疫组织化学方法检测肝内HBsAg、乙肝病毒核心抗原(HBcAg)的表达,RT-PCR法半定量检测肝内HBV 3.5 kb mRNA、S-mRNA及0.7 kb mRNA的水平.结果:感染组和无关干扰组血清HBsAg表达量平均值分别为9.11±3.34和8.19±5.41,pSiHBV/P干扰组平均值1.94±1.78;pSiHBV/P干扰组血清中HBV DNA的含量较感染组明显下降(2.91±0.55 vs 4.32±0.57,P<0.05),抑制率为33%;干扰组肝细胞中HBsAg和HBcAg阳性细胞数较对照组明显减少;干扰组3.5 kbmRNA、S-mRNA的水平较感染组和无关干扰组均有明显下降(0.48±0.19 vs 1.06±0.40,0.88±0.54;0.46±0.18 vs 1.05±0.38.0.90±0.51,P<0.05).结论:RNAi技术在体内能高效、特异地抑制小鼠体内HBV.     

Non-Nucleotide RNA-Dependent RNA Polymerase Inhibitor That Blocks SARS-CoV-2 Replication

SARS-CoV-2 has caused an extensive pandemic of COVID-19 all around the world. Key viral enzymes are suitable molecular targets for the development of new antivirals against SARS-CoV-2 which could represent potential treatments of the corresponding disease. With respect to its essential role in the replication of viral RNA, RNA-dependent RNA polymerase (RdRp) is one of the prime targets. HeE1-2Tyr and related derivatives were originally discovered as inhibitors of the RdRp of flaviviruses. Here, we present that these pyridobenzothiazole derivatives also significantly inhibit SARS-CoV-2 RdRp, as demonstrated using both polymerase- and cell-based antiviral assays.     

siRNA靶向沉默Smad7对PC12细胞氧糖剥夺敏感性的影响

目的探讨siRNA靶向沉默Smad7基因对PC12细胞氧糖剥夺(OGD)损伤的影响及其相关机制。方法体外建立PC12细胞OGD模型,利用RNA干扰技术,沉默Smad7基因表达,实时RT-PCR及Western印迹检测Smad7基因的表达,Hoechst33342染色检测细胞凋亡情况,实时RT-PCR检测Smad3基因mRNA的表达变化。结果 siRNA转染下调Smad7基因mRNA及蛋白的表达,靶向沉默Smad7联合OGD 16 h组细胞凋亡较OGD 16 h组明显减少;Smad7低表达的PC12细胞内Smad3 mRNA的表达明显增加。结论 Smad7基因沉默降低PC12细胞对OGD损伤的敏感性,提高Smad3基因在转录水平上的表达。     

RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects >270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both a messenger RNA and replication template, making it an attractive target for the study of RNA interference. Double-stranded small interfering RNA (siRNA) molecules designed to target the HCV genome were introduced through electroporation into a human hepatoma cell line (Huh-7) that contained an HCV subgenomic replicon. Two siRNAs dramatically reduced virus-specific protein expression and RNA synthesis to levels that were 90% less than those seen in cells treated with negative control siRNAs. These same siRNAs protected naive Huh-7 cells from challenge with HCV replicon RNA. Treatment of cells with synthetic siRNA was effective >72 h, but the duration of RNA interference could be extended beyond 3 weeks through stable expression of complementary strands of the interfering RNA by using a bicistronic expression vector. These results suggest that a gene-therapeutic approach with siRNA could ultimately be used to treat HCV.     

Isolation of a Viral Polypeptide Associated with Poliovirus RNA Polymerase

Poliovirus-infected HeLa cells were labeled with radioactive methionine or phenylalanine and subjected to a new purification procedure for the viral induced RNA polymerase activity. Detergent-solubilized polymerase activity was purified by precipitation with 2 M LiCl and sedimentation through sucrose gradients. Approximately 0.001% of the incorporated amino acid radio-activity sediments with the peak of polymerase activity. Gradient fractions comprising the polymerase activity peak were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to contain predominantly one virus-specific polypeptide. Polyacrylamide gel electrophoresis also reveals that this purified polypeptide migrates with a 58,000 molecular weight noncapsid polio-virus polypeptide.     

RNA干扰技术及其在2型糖尿病中的应用

RNA干扰(RNAi)技术是基因分析和基因治疗的重要手段,它在2型糖尿病中的应用涉及功能基因组研究、2型糖尿病发病机制及其心血管并发症研究。RNAi具有高效特异性的特点。然而仍需克服如何避免RNAi衰减现象,如何合理调节多基因表达等困难。     

小干扰RNA沉默垂体瘤转化基因1对肝癌细胞增殖和凋亡水平的影响

肝癌是全世界范围的高发恶性肿瘤,在亚洲的发病率、病死率都很高。肝癌的分子学发病机制尚不完全清楚,包括基因结构和功能的缺陷、癌基因的激活、抑癌基因的失活、细胞周期紊乱、染色体的非整倍体出现以及细胞凋亡途径受阻等。垂体瘤转化基因(PTTG)1是细胞周期调节蛋白,能抑制姐妹染色单体分离,导致染色体不稳定,形成非整倍体。在成人大多数正常组织中PTTG1表达较弱,甚至检测不到,而在垂体肿瘤等多种肿瘤细胞中表达明显升高,PTTG1基因可能是一种潜在的抗癌基因治疗靶点[1]。本研究用小干扰RNA (siRNA)降低肝癌细胞株HepG2、SMMC-7721细胞中PTTG1的表达,检测PTTG1表达下调对肝癌细胞增殖及凋亡的影响,探讨PTTG1对肝癌细胞生物学行为的影响以及作为肝癌治疗靶基因的可能性。