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Does any one have a target gene knockdown with negative control siRNA? Hi, I have siRNA for gene A and looking for it's effects on gene B. My siRNA has knock down almost 80% of the trageted gene and affected the...
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Integrative Genomic Analyses Identify BRF2 as a Novel Lineage-Specific Oncoge... Related Articles

Integrative Genomic Analyses Identify BRF2 as a Novel Lineage-Specific Oncogene in Lung Squamous Cell Carcinoma.

PLoS Med. 2010;7(7):e1000315

Authors: Lockwood WW, Chari R, Coe BP, Thu KL, Garnis C, Malloff CA, Campbell J, Williams AC, Hwang D, Zhu CQ, Buys TP, Yee J, English JC, Macaulay C, Tsao MS, Gazdar AF, Minna JD, Lam S, Lam WL

BACKGROUND: Traditionally, non-small cell lung cancer is treated as a single disease entity in terms of systemic therapy. Emerging evidence suggests the major subtypes-adenocarcinoma (AC) and squamous cell carcinoma (SqCC)-respond differently to therapy. Identification of the molecular differences between these tumor types will have a significant impact in designing novel therapies that can improve the treatment outcome. METHODS AND FINDINGS: We used an integrative genomics approach, combing high-resolution comparative genomic hybridization and gene expression microarray profiles, to compare AC and SqCC tumors in order to uncover alterations at the DNA level, with corresponding gene transcription changes, which are selected for during development of lung cancer subtypes. Through the analysis of multiple independent cohorts of clinical tumor samples (>330), normal lung tissues and bronchial epithelial cells obtained by bronchial brushing in smokers without lung cancer, we identified the overexpression of BRF2, a gene on Chromosome 8p12, which is specific for development of SqCC of lung. Genetic activation of BRF2, which encodes a RNA polymerase III (Pol III) transcription initiation factor, was found to be associated with increased expression of small nuclear RNAs (snRNAs) that are involved in processes essential for cell growth, such as RNA splicing. Ectopic expression of BRF2 in human bronchial epithelial cells induced a transformed phenotype and demonstrates downstream oncogenic effects, whereas RNA interference (RNAi)-mediated knockdown suppressed growth and colony formation of SqCC cells overexpressing BRF2, but not AC cells. Frequent activation of BRF2 in >35% preinvasive bronchial carcinoma in situ, as well as in dysplastic lesions, provides evidence that BRF2 expression is an early event in cancer development of this cell lineage. CONCLUSIONS: This is the first study, to our knowledge, to show that the focal amplification of a gene in Chromosome 8p12, plays a key role in squamous cell lineage specificity of the disease. Our data suggest that genetic activation of BRF2 represents a unique mechanism of SqCC lung tumorigenesis through the increase of Pol III-mediated transcription. It can serve as a marker for lung SqCC and may provide a novel target for therapy. Please see later in the article for the Editors' Summary.

PMID: 20668658 [PubMed - in process]

Identification of Roles for Peptide: N-Glycanase and Endo-beta-N-Acetylglucosaminidase (Engase1p) during Protein N-Glycosylation in Human HepG2 Cells.

PLoS One. 2010;5(7):e11734

Authors: Chantret I, Fasseu M, Zaoui K, Le Bizec C, Sadou Yayé H, Dupré T, Moore SE

BACKGROUND: During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo beta-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells. METHODS/PRINCIPAL FINDINGS: During metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Delta deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man(8)GlcNAc(2), corresponding to approximately 70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc(1)Man(9)GlcNAc(2) and Man(9)GlcNAc(2) that corresponds to approximately 30% of total fOS. CONCLUSIONS/SIGNIFICANCE: As the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either LLO or glycoproteins during protein N-glycosylation. The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming.

PMID: 20668520 [PubMed - in process]

Characterization of the human HSC20, an unusual DnaJ type III protein, involved in iron sulfur cluster biogenesis.

Hum Mol Genet. 2010 Jul 28;

Authors: Uhrigshardt H, Singh A, Kovtunovich G, Ghosh M, Rouault TA

The importance of mitochondrial iron-sulfur cluster (ISC) biogenesis for human health has been well established, but the roles of some components of this critical pathway still remain uncharacterized in mammals. Among them is hHSC20, the putative human homologue of the specialized DnaJ-type co-chaperones, which are crucial for bacterial and fungal ISC assembly. Here, we show here that the human HSC20 protein can complement for its counterpart in yeast, Jac1p, and interacts with its proposed human partners, hISCU and hHSPA9. hHSC20 is expressed in various human tissues, and localizes mainly to the mitochondria in HeLa cells. However, small amounts were also detected extramitochondrially. RNAi-mediated depletion of hHSC20 specifically reduced the activities of both mitochondrial and cytosolic ISC-containing enzymes. Recovery of inactivated ISC enzymes was markedly delayed after oxidative insult of hHSC20-deficient cells. Conversely, over-expression of hHSC20 substantially protected cells from oxidative insults. These results imply that hHSC20 is an integral component of the human ISC biosynthetic machinery that is particularly important in the assembly of iron-sulfur clusters under conditions of oxidative stress. A cysteine rich N-terminal domain, which clearly distinguishes hHSC20 from the specialized DnaJ-type III proteins of fungi and most bacteria, was found to be important for the integrity and function of the human co-chaperone.

PMID: 20668094 [PubMed - as supplied by publisher]

EZH2 supports ovarian carcinoma cell invasion and/or metastasis via regulation of TGF-{beta}1 and is a predictor of outcome in ovarian carcinoma patients.

Carcinogenesis. 2010 Jul 28;

Authors: Rao ZY, Cai MY, Yang GF, He LR, Mai SJ, Hua WF, Liao YJ, Deng HX, Chen YC, Guan XY, Zeng YX, Kung HF, Xie D

It was suggested that the enhancer of zeste homolog 2 (EZH2) gene is a putative candidate oncogene in several types of human cancer. The potential oncogenic role of EZH2 and its clinical/prognostic significance, however, in ovarian carcinoma are unclear. In this study, EZH2 expression was examined by immunohistochemistry in cohorts of normal and tumorous ovarian tissues. High expression of EZH2 was examined in none of the normal ovaries, in 3% of the cystadenomas, in 23% of the borderline tumors and in 50% of the ovarian carcinomas, respectively. In the ovarian carcinomas, high expression of EZH2 was positively correlated with an ascending histological grade and/or advanced stage of the disease (P<0.05). Moreover, high expression of EZH2 in ovarian carcinoma was determined to be a strong and an independent predictor of short overall survival (P<0.05). In ovarian carcinoma HO-8910 and UACC-326 cell lines, EZH2 knockdown by RNAi led to a G1 phase cell cycle arrest, reduced cell growth/proliferation and inhibited cell migration and/or invasion in vitro. In addition, EZH2 knockdown was found to reduce TGF-beta1 expression and increase E-cadherin expression either in the transcript or protein levels. Furthermore, a significant positive correlation between overexpression of EZH2 and TGF-beta1 in ovarian carcinoma tissues was observed (p<0.001). These findings suggest a potential important role of EZH2 in the control of cell migration and/or invasion via the regulation of TGF-beta1 expression, and the high expression of EZH2, as detected by immunohistochemistry, is an independent molecular marker for shortened survival time of patients with ovarian carcinoma.

PMID: 20668008 [PubMed - as supplied by publisher]