应用DNA芯片鉴定HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制

高危型人乳头瘤病毒（human papillomavirus,HPV）的E6基因在宫颈癌的发生中起关键作用,特异siRNA能有效抑制宫颈癌HeLa 细胞内HPV18 E6基因的表达,诱导肿瘤细胞凋亡.为进一步探讨HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制,针对HPV18-E6基因设计siRNA序列,利用人源U6启动子为模板,经PCR表达框架法体外扩增,转染宫颈癌HeLa细胞抑制HPV18 -E6基因表达,从而诱导肿瘤细胞凋亡.对转染前后HeLa细胞总RNA样品进行荧光标记后,与Agilent Human 1A寡核苷酸芯片杂交、扫描、数据分析及标准化处理,确定表达差异的基因并经荧光定量PCR对部分基因进行验证,结合PANTHER数据分析系统,将这些基因按照生物学功能进行归类,查阅GenBank数据库及相关文献,对其结果进行深入分析及讨论.在检测的18 716个基因和EST中,共筛出差异表达基因359个,其中307个基因表达上调,52个基因表达下调,主要包括细胞周期相关基因CCNG1、p21;凋亡相关基因CASP4、CASP6、IGFBP3、DFFA;泛素蛋白酶解途径相关基因E6-AP、UBE2C;角化细胞分化相关基因KRT4、KRT6E、KRT18;抑癌基因RECK、VHL等.研究结果表明,HPV18 -E6基因抑制引起的细胞凋亡效应主要是通过P53信号途径和泛素蛋白酶解信号途径调节细胞周期相关基因和凋亡相关基因的表达,从而抑制HeLa细胞增殖、促进细胞凋亡.同时,抑癌基因的激活,角化细胞分化和免疫相关基因的表达上调,都说明了E6抑制后肿瘤细胞恶性转化程度的下降.     

Specific gene silencing using small interfering RNAs in fish embryos

Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. Here we demonstrate a siRNA-mediated gene silencing technique in rainbow trout embryos. We found that siRNAs effectively suppressed the transient expression of episomally located foreign GFP genes at an early developmental stage and inhibited the expression of GFP genes in stable transgenic trout embryos. Similar gene silencing was observed with an siRNA against the endogenous tyrosinase A gene. siRNAs interfered with the expression of maternally inherited mRNA. siRNAs did not affect non-relevant gene expression and siRNAs with a 4 base mismatch did not affect target gene expression. siRNA gene silencing is therefore highly sequence-specific. Our findings are the first evidence that siRNA-mediated gene silencing is effective in fish. This technique could be a powerful tool for studying gene function during embryonic development in aquacultural fish species, zebrafish, and medaka.     

BRD7调控Rb/E2F通路的分子机制研究

BRD7是采用cDNA代表性差异分析法克隆的一个新的Bromodomain基因,过表达BRD7可抑制鼻咽癌细胞的生长和细胞周期进程,同时发现BRD7基因可以调控Rb/E2F通路的活性.该研究旨在进一步探讨BRD7调控Rb/E2F通路的分子机制.通过蛋白质印迹和RT-PCR实验方法发现,BRD7能够降低Rb的磷酸化水平,抑制cyclinD1、cyclinE的蛋白质表达,上调CDK4抑制子P19的mRNA表达,但对CDK4和CDK2的蛋白质表达没有明显影响;通过荧光素酶实验从转录调控水平进一步证实了BRD7能够明显抑制cyclinD1启动子活性;采用反义核酸技术抑制COS7细胞内源性BRD7的表达后,发现cyclinD1、cyclinE、磷酸化Rb的蛋白质表达水平上调,并且可以促进细胞生长.这些结果表明:BRD7参与调控Rb/E2F信号通路中重要靶分子的表达,抑制Rb/E2F通路的活性,从而阻止细胞周期G1-S期进程,抑制鼻咽癌细胞生长.     

Effect of siRNA nuclease stability on the in vitro and in vivo kinetics of siRNA-mediated gene silencing

Small interfering RNA (siRNA) molecules achieve sequence-specific gene silencing through the RNA interference (RNAi) mechanism. Here, live-cell and live-animal bioluminescent imaging (BLI) is used to directly compare luciferase knockdown by unmodified and nuclease-stabilized siRNAs in rapidly (HeLa) and slowly (CCD-1074Sk) dividing cells to reveal the impact of cell division and siRNA nuclease stability on the kinetics of siRNA-mediated gene silencing. Luciferase knockdown using unmodified siRNAs lasts approximately 1 week in HeLa cells and up to 1 month in CCD-1074Sk cells. There is a slight increase in the duration of luciferase knockdown by nuclease-stabilized siRNAs relative to unmodified siRNAs after cationic lipid transfection, but this difference is not observed after electroporation. In BALB/cJ mice, a fourfold increase in maximum luciferase knockdown is observed after hydrodynamic injection (HDI) of nuclease-stabilized siRNAs relative to unmodified siRNAs, yet the overall kinetics of the recovery after knockdown are nearly identical. By using a mathematical model of siRNA-mediated gene silencing, the trends observed in the experimental data can be duplicated by changing model parameters that affect the stability of the siRNAs before they reach the cytosolic compartment. Based on these findings, we hypothesize that the stabilization advantages of nuclease-stabilized siRNAs originate primarily from effects prior to and during internalization before the siRNAs can interact with the intracellular RNAi machinery.     

Wnt/β-catenin signaling pathway may regulate cell cycle and expression of cyclin A and cyclin E protein in hepatocellular carcinoma cells

Wnt/β-catenin signaling pathway and cell cycle play the key roles during the genesis and development of hepatocellular carcinoma (HCC). The cytoplasmic protein β-catenin is a multifunctional protein and a central molecule in the Wnt signaling pathway. Cell cycle is regulated by a a series of regulatory factors. Current researches indicated that expression of cyclin D1 and c-myc decreased after silencing β-catenin gene in HCC, but it is unclear if other cyclins are affected. To determine the relation, small interference RNA(siRNA) against β-catenin was transfected into HCC cell line HepG2, and cell cycle and cyclin A and cyclin E protein expression were detected. We demonstrated that cell cycle was arrested in G0/G1 at 72 h after the transfection and with the time passing, the cell cycle began to transfer from G0/G1 to G2/M through S and had a trend to revert at 96 h. In addition, β-catenin protein expression was decreased at both 72 and 96 h, although the level was slightly higher at 96 h than that at 72 h. However, cyclin A and cyclin E protein expression increased at 72 h and decreased at 96 h. These findings suggest that silencing β-catenin gene may induce the changes of cell cycle and cyclin A and cyclin E expression. Wnt/β-catenin signaling pathway probably takes part in the genesis and development of HCC through regulating cell cycle and the expression of cyclin A and cyclin E.     

siRNA-mediated NCAM1 gene silencing suppresses oxidative stress in pre-eclampsia by inhibiting the p38MAPK signaling pathway

Pre-eclampsia (PE), whose pathophysiology and etiology remain undefined, represents a leading consequence of fetal and maternal mortality and morbidity. Oxidative stress (OS) is recognized to involve in this disorder. In this study, we hypothesized that neural cell adhesion molecule 1 (NCAM1) gene silencing would suppress the OS in the pregnancy complicated by PE. Initially, clinical samples were collected for determination of NCAM1 expression in placental tissues and levels of OS products in blood. To assess the regulatory mechanism of NCAM1 knockdown on OS, we used small interfering RNA (siRNA) to silence NCAM1 expression in human umbilical vein endothelial cells (HUVECs). Next, cells were treated with or without hypoxia/reoxygenation to observe the level changes of OS products and p38 mitogen-activated protein kinase (p38MAPK) pathway-related genes. Finally, an evaluation of HUVEC migration and invasion abilities was conducted by wound-healing and transwell assays. Placenta of pregnancy with PE presented significantly increased NCAM1 expression in comparison to placenta of normal pregnancy. Meanwhile, enhanced OS in blood of pregnant women with PE was observed relative to women with normal pregnancy. siRNA-mediated knockdown of NCAM1 gene could inhibit the p38MAPK signaling pathway, repress OS, and promote cell migration and invasion in HUVECs, indicating that NCAM1 inhibition could reduce the influence of PE. Importantly, blocking the p38MAPK signaling pathway reversed the inhibitory role of NCAM1 gene silencing on PE. Collectively, this study defines potential role of NCAM1 gene silencing as a therapeutic target in PE through inhibiting OS and enhancing HUVEC migration and invasion by disrupting the p38MAPK signaling pathway.     

Transgenic small interfering RNA halts amyotrophic lateral sclerosis in a mouse model

Many autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with copper/zinc superoxide dismutase (SOD1) mutation may be induced by missense point mutations that result in the production of proteins with toxic properties. Reduction in the encoding of proteins from such mutated genes can therefore be expected to improve the disease phenotype. The duplex of 21-nucleotide RNA, known as small interfering RNA (siRNA), has recently emerged as a powerful gene silencing tool. We made transgenic (Tg) mice with modified siRNA, which had multiple mismatch alternations within the sense strand, to prevent the "shutdown phenomenon" of transgenic siRNA. Consequently, the in vivo knockdown effect of siRNA on SOD1 expression did not diminish over four generations. When we crossed these anti-SOD1 siRNA Tg mice with SOD1G93A Tg mice, a model for ALS, siRNA prevented the development of disease by inhibiting mutant G93A SOD1 production in the central nervous system. Our findings clearly proved the principle that siRNA-mediated gene silencing can stop the development of familial ALS with SOD1 mutation.