Protein arginine methyltransferase 5-mediated epigenetic silencing of IRX1 contributes to tumorigenicity and metastasis of gastric cancer

IRX1 is originally characterized as a tumor suppressor gene of gastric cancer (GC) by our group based on serially original studies. However, the molecular regulatory mechanisms of IRX1 are not clear yet. Here, we identified protein arginine methyltransferase 5 (PRMT5) as a major upstream regulator of IRX1 for determining GC progression. Expression of PRMT5 was significantly increased in human GC tissues (433 out of 602 cases, 71.93%) compared with normal gastric mucosa, and exhibited diagnostic and prognostic potential. Overexpression of PRMT5 promoted tumorigenicity and metastasis of GC cells, while knockdown of PRMT5 abrogated tumorigenicity and metastasis of GC cells in vitro and in vivo. By co-immunoprecipitation and chromatin immunoprecipitation assays, we proved that PRMT5 elevated methylation levels of tumor suppressor IRX1 promoter via recruiting DNMT3A at promoter region. Knockdown of PRMT5 in SGC7901 and NCI-N87 cells decreased the recruitment of DNMT3A at IRX1 promoter, and reduced the methylation level of IRX1 promoter, then re-activated IRX1 expression. Whereas, overexpression of PRMT5 could epigenetically suppress IRX1 expression. Overall, PRMT5 promoted tumorigenicity and metastasis of gastric cancer cells via epigenetic silencing of IRX1. Targeting PRMT5 in GC might inhibit the malignant characters of GC and drawing a novel therapeutic potential.     

Expression in yeast of antisense RNA to ADE1 mRNA

The utility of antisense RNA as a means of regulating gene expression in yeast has been explored by inserting into a high copy number yeast expression vector an ADE1 gene fragment in such an orientation so as to produce antisense RNA in vivo which could hybridize to natural ADE1 mRNA. Northern blotting analysis of total cellular RNA extracted from transformed yeast cells confirmed the presence of high levels of antisense RNA to ADE1 mRNA within cells. However the high level of expression of antisense RNA did not result in production of Ade- cells.     

Targeting of dicer-2 and RNA by a viral RNA silencing suppressor in Drosophila cells

RNA interference (RNAi) is a eukaryotic gene-silencing mechanism that functions in antiviral immunity in diverse organisms. To combat RNAi-mediated immunity, viruses encode viral suppressors of RNA silencing (VSRs) that target RNA and protein components in the RNAi machinery. Although the endonuclease Dicer plays key roles in RNAi immunity, little is known about how VSRs target Dicer. Here, we show that the B2 protein from Wuhan nodavirus (WhNV), the counterpart of Flock House virus (FHV), suppresses Drosophila melanogaster RNAi by directly interacting with Dicer-2 (Dcr-2) and sequestering double-stranded RNA (dsRNA) and small interfering RNA (siRNA). Further investigations reveal that WhNV B2 binds to the RNase III and Piwi-Argonaut-Zwille (PAZ) domains of Dcr-2 via its C-terminal region, thereby blocking the activities of Dcr-2 in processing dsRNA and incorporating siRNA into the RNA-induced silencing complex (RISC). Moreover, we uncover an interrelationship among diverse activities of WhNV B2, showing that RNA binding enhances the B2-Dcr-2 interaction by promoting B2 homodimerization. Taken together, our findings establish a model of suppression of Drosophila RNAi by WhNV B2 targeting both Dcr-2 and RNA and provide evidence that an interrelationship exists among diverse activities of VSRs to antagonize RNAi.     

Synergism between DNA methylation and macroH2A1 occupancy in epigenetic silencing of the tumor suppressor gene p16(CDKN2A)

Promoter hypermethylation and heterochromatinization is a frequent event leading to gene inactivation and tumorigenesis. At the molecular level, inactivation of tumor suppressor genes in cancer has many similarities to the inactive X chromosome in female cells and is defined and maintained by DNA methylation and characteristic histone modifications. In addition, the inactive-X is marked by the histone macroH2A, a variant of H2A with a large non-histone region of unknown function. Studying tumor suppressor genes (TSGs) silenced in cancer cell lines, we find that when active, these promoters are associated with H2A.Z but become enriched for macroH2A1 once silenced. Knockdown of macroH2A1 was not sufficient for reactivation of silenced genes. However, when combined with DNA demethylation, macroH2A1 deficiency significantly enhanced reactivation of the tumor suppressor genes p16, MLH1 and Timp3 and inhibited cell proliferation. Our findings link macroH2A1 to heterochromatin of epigenetically silenced cancer genes and indicate synergism between macroH2A1 and DNA methylation in maintenance of the silenced state.     

Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless

Endeavours to obtain elevated and prolonged levels of foreign gene expression in plants are often hampered by the onset of RNA silencing that negatively affects target gene expression. Plant virus-encoded suppressors of RNA silencing are useful tools for counteracting silencing but their wide applicability in transgenic plants is limited because their expression often causes harmful developmental effects. We hypothesized that a previously characterized tombusvirus P19 mutant (P19/R43W), typified by reduced symptomatic effects while maintaining the ability to sequester short-interfering RNAs, could be used to suppress virus-induced RNA silencing without the concomitant developmental effects. To investigate this, transient expression in Nicotiana benthamiana was used to evaluate the ability of P19/R43W to enhance heterologous gene expression. Although less potent than wt-P19, P19/R43W was an effective suppressor when used to enhance protein expression from either a traditional T-DNA expression cassette or using the CPMV-HT expression system. Stable transformation of N.?benthamiana yielded plants that expressed detectable levels of P19/R43W that was functional as a suppressor. Transgenic co-expression of green fluorescent protein (GFP) and P19/R43W also showed elevated accumulation of GFP compared with the levels found in the absence of a suppressor. In all cases, transgenic expression of P19/R43W caused no or minimal morphological defects and plants produced normal-looking flowers and fertile seed. We conclude that the expression of P19/R43W is developmentally harmless to plants while providing a suitable platform for transient or transgenic overexpression of value-added genes in plants with reduced hindrance by RNA silencing.     

Expression of the class II tumor suppressor gene RIG1 is directly regulated by p53 tumor suppressor in cancer cell lines

Recent studies indicated that the RIG1 (RARRES3/TIG3) plays an important role in cell proliferation, differentiation, and apoptosis. However, the regulatory mechanism of RIG1 gene expression has not been clearly elucidated. In this study, we identified a functional p53 response element (p53RE) in the RIG1 gene promoter. Transfection studies revealed that the RIG1 promoter activity was greatly enhanced by wild type but not mutated p53 protein. Sequence specific mutation of the p53RE abolished p53-mediated transactivation. Specific binding of p53 protein to the rig-p53RE was demonstrated using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. Further studies confirmed that the expression of RIG1 mRNA and protein is enhanced through increased p53 protein in HepG2 or in H24-H1299 cells. In conclusion, our results indicated that RIG1 gene is a downstream target of p53 in cancer cell lines.     

Expression of a transgene encoded on a non-viral episomal vector is not subject to epigenetic silencing by cytosine methylation

Currently available vectors for mammalian cells suffer from a number of limitations which make them only partially useful for genetic modification of eukaryotic cells and organisms and for gene therapy. While integration of a vector can lead to unpredictable interactions with the host genome and silencing of the integrated transgene, most non-integrating vectors mediate only transient expression of a transgene. All available vector types can lead to transformation of the recipient cell and many of them can cause serious immunological side effects in the organism. The ideal vector has to be free of these side effects and should allow long-term expression of a transgene in the absence of selection. In this report we describe a novel non-viral episomal expression system fulfilling these criteria. The gene encoding the truncated rat NGF-receptor gene under the control of the CMV-promoter was inserted into a vector construct containing a scaffold/matrix attached region (S/MAR). This vector was then transfected into CHO cells and human HaCat cells. We show that this vector replicates episomally in these cells and is mitotically stable in the abscence of selection over more than 100 generations. Moreover, we provide the first experimental data that the CMV-promoter in an episome is not subject to silencing by cytosine methylation, thus allowing long-term expression of the transgene in the absence of selection.     

抑癌候选基因NDRG2在甲状腺癌组织中的表达及其意义

目的:研究抑癌候选基因NDRG2在人类甲状腺癌组织及其癌旁组织中的表达情况.方法:收集30例甲状腺癌组织及其癌旁组织,提取总RNA,应用半定量RT-PCR方法检测NDRG2 mRNA的表达水平.分别提取30例组织的总蛋白,应用蛋白印迹技术检测其NDRG2的蛋白表达水平.结果:RT-PCR结果显示,30例甲状腺癌组织中,有25例NDRG2的mRNA水平明显降低,蛋白印迹结果显示,30例甲状腺癌组织中发现25例NDRG2的蛋白水平明显下降,与RT-PCR检测结果一致.结论:NDRG2在甲状腺癌组织中呈低表达,提示其可能对甲状腺癌的发生或发展有重要作用影响.