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PubMed2006

RNA interference of sialidase improves glycoprotein sialic acid content consistency.

An important challenge facing therapeutic protein production in mammalian cell culture is the cleavage of terminal sialic acids on recombinant protein glycans by the glycosidase enzymes released by lysed cells into the supernatant. This undesired phenomenon results in a protein product which is rapidly cleared from the plasma by asialoglycoprotein receptors in the liver. In this study, RNA interference was utilized as a genetic approach to silence the activity of sialidase, a glycosidase responsible for cleaving terminal sialic acids on IFN-gamma produced by Chinese Hamster Ovary (CHO) cells. We first identified a 21-nt double stranded siRNA that reduced endogenous sialidase mRNA and protein activity levels. Potency of each siRNA sequences was compared using real time RT-PCR and a sialidase activity assay. We next integrated the siRNA sequence into CHO cells, allowing production and selection of stable cell lines. We isolated stable clones with sialidase activity reduced by over 60% as compared to the control cell line. Micellar electrokinetic chromatography (MEKC), thiobarbituric acid assay (TAA), and high performance anion exchange chromatography (HPAEC) coupled to amperometric detection were performed to analyze glycan site occupancy, sialic acid content, and distribution of asialo-/sialylated-glycan structures, respectively. Two of the stable clones successfully retained the full sialic acid content of the recombinant IFN-gamma, even upon cells' death. This was comparable to the case where a chemically synthesized sialidase inhibitor was used. These results demonstrated that RNA interference of sialidase can prevent the desialylation problem in glycoprotein production, resulting improved protein quality during the entire cell culture process.

Animals↗
PubMed2004

Gene silencing of selected calcium-signalling molecules in a Drosophila cell line using double-stranded RNA interference.

Using the Drosophila melanogaster S2 cell line, stably expressing a cloned muscarinic acetylcholine receptor (AChR), DM1, we have applied gene silencing by double-stranded RNA interference (RNAi) to knock down gene products involved in DM1-mediated calcium signalling. We have shown that RNAi knock down of either the inositol 1,4,5-trisphosphate receptor (Ins(1,4,5)P(3)R), or the SERCA calcium pump in the S2-DM1 cells blocks the increase in intracellular calcium concentration ([Ca(2+)](i)) resulting from activation of the DM1 receptor by 100 microM carbamylcholine (CCh). When RNAi designed to knock down the ryanodine receptor (RyR) was tested, there was no change in the calcium increase detected in response to CCh, consistent with a failure to detect RyRs in S2-DM1 cells using RT-PCR. A combination of RNAi and calcium imaging has provided a direct demonstration of key roles for the Ins(1,4,5)P(3)R and the SERCA pump in the response to DM1 receptor activation.Thus, we show that silencing of individual genes by RNAi in a well characterised Drosophila S2 cell line offers experimental opportunities for cell-signalling studies. Future investigations with RNAi libraries taking full advantage of the wealth of new information available from sequencing the Drosophila genome, may help identify novel components of cell-signalling pathways and functionally linked gene products.

Animals↗
PubMed2004

RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting.

We describe the successful use of RNA interference (RNAi) to investigate gene function in the human filarial parasite Onchocerca volvulus third-stage larvae (L3). We targeted two specific gene products, the O. volvulus cathepsin L (Ov-CPL) and cathepsin Z-like (Ov-CPZ) cysteine proteases, which were proposed to function during O. volvulus L3 molting. We show that fluorescent-labeled Cy3-dsRNA corresponding to cpl or cpz regions encoding the mature enzymes can enter the larvae. The molting rate of larvae treated overnight with 0.5 mg ml(-1) cpl was reduced by 92% and 86% in comparison to normal control worms. It appeared that although the larvae started the molting process the last stage of molting, ecdysis was inhibited. The effect was gene specific, as larvae that did not molt in the presence of cpl or cpz dsRNA expressed the other cysteine protease, CPZ and CPL, respectively. This was confirmed by immunoelectron microscopy using antibodies directed against each enzyme. Our present study validate conclusively that both enzymes are essential for the molting of O. volvulus L3 to fourth-stage larvae. We also confirmed that the activity of the enzymes is specific to the changes that occur during the molting process on days 1-3, when the separation between the cuticles is in progress. The development of RNAi in O. volvulus L3 could further help study many of the abundant L3 and molting L3 genes identified through the filarial genome project, many of which, although have no attributed function, were identified as vaccine candidates or potential drug targets.

Animals↗
PubMed2004

Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells.

We have used RNA interference (RNAi) to downregulate beta2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not beta2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

Antigens, Surface↗
PubMed2006

[Biological implications of the inhibition of survivin by RNA interference in human androgen-independent prostate carcinoma with highly metastatic potential].

OBJECTIVE: To determine the expression level of survivin in androgen-independent prostate carcinoma, and to investigate the biological role of survivin in invasion and metastasis of androgen-independent prostate carcinoma. METHODS: Highly metastatic prostatic cancer cell line PC-3M-1E8 was stably transfected with pSilencer plasmid targeting survivin expression by RNA interference. The biological effects were observed, including anchorage-independent growth, in vitro invasion by soft agar colony formation and Boyden chamber assay, and also in vivo tumorigenesis in nude mice. Cell cycle and apoptosis indices were evaluated by flow cytometry and Western blot analysis of bioactive fragments of caspase 3. RESULTS: The expression of survivin in transfected PC-3M-1E8 cells was markedly depressed at both mRNA and protein levels (about 78% to 80%) as compared with control. The growth of tumor cells was retarded by anchorage-independent growth assay. The survivin transfectants formed smaller and fewer colonies (14.33 +/- 3.51) than the negative (52.33 +/- 6.81) and blank controls (54.00 +/- 6.00). Inhibition of survivin expression was correlated with enhanced apoptosis of tumor cells (percentages of apoptotic cells of the negative control, blank control and experimental groups were 5.88 +/- 0.99, 6.97 +/- 1.60, 16.40 +/- 1.95 respectively), along with an increased expression of activated caspase 3, and cell cycle inhibition at G(0)/G(1) phase (the relative number of cells at G(0)/G(1) phase were 43.65 +/- 3.44, 43.59 +/- 1.83 and 52.71 +/- 1.10, respectively). In addition, multinucleated giant cells were observed along with a marked inhibition of invasion as reflected by fewer penetrating cells by Boyden chamber assay (46.07 +/- 9.97, 47.87 +/- 9.58 and 38.67 +/- 6.59, respectively). CONCLUSIONS: Survivin expression is high in androgen-independent prostate cancer cells and likely may be related to the apoptosis, growth and invasion of the tumor cells. Targeting the survivin pathway by RNA interference appears to be a promising approach for clinical treatment of androgen-independent prostate cancer.

Androgens↗
PubMed2006

RNA interference with measles virus N, P, and L mRNAs efficiently prevents and with matrix protein mRNA enhances viral transcription.

In contrast to studies with genetically modified viruses, RNA interference allows the analysis of virus infections with identical viruses and posttranscriptional ablation of individual gene functions. Using RNase III-generated multiple short interfering RNAs (siRNAs) against the six measles virus genes, we found efficient downregulation of viral gene expression in general with siRNAs against the nucleocapsid (N), phosphoprotein (P), and polymerase (L) mRNAs, the translation products of which form the ribonucleoprotein (RNP) complex. Silencing of the RNP mRNAs was highly efficient in reducing viral messenger and genomic RNAs. siRNAs against the mRNAs for the hemagglutinin (H) and fusion (F) proteins reduced the extent of cell-cell fusion. Interestingly, siRNA-mediated knockdown of the matrix (M) protein not only enhanced cell-cell fusion but also increased the levels of both mRNAs and genomic RNA by a factor of 2 to 2.5 so that the genome-to-mRNA ratio was constant. These findings indicate that M acts as a negative regulator of viral polymerase activity, affecting mRNA transcription and genome replication to the same extent.

Animals↗
PubMed2000

Inhibition of Trypanosoma brucei gene expression by RNA interference using an integratable vector with opposing T7 promoters.

RNA interference is a powerful method for inhibition of gene expression in Trypanosoma brucei (Ngo, H., Tschudi, C., Gull, K., and Ullu, E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14687-14692). Here we describe a vector (pZJM) for in vivo tetracycline-inducible synthesis of double-stranded RNA (dsRNA) in stably transformed cells. The dsRNA is synthesized from opposing T7 promoters. We tested the vector with genes involved in processes such as kinetoplast DNA replication, mitochondrial mRNA synthesis, glycosyl phosphatidylinositol biosynthesis, glycosome biogenesis, and polyamine biosynthesis. In most cases the induction of dsRNA caused specific and dramatic loss of the appropriate mRNA, and in many cases there was growth inhibition or cell death. One striking phenotype was the loss of kinetoplast DNA after interference with expression of a topoisomerase II. The gene being analyzed by this procedure need not even be fully sequenced. In fact, many of the genes we tested were derived from partial sequences in the T. brucei genome data base that were identified by homology with known proteins. It takes as little as 3 weeks from identification of a gene sequence in the data base to the appearance of a phenotype.

Animals↗
PubMed2004

Efficient RNA interference depends on global context of the target sequence: quantitative analysis of silencing efficiency using Eulerian graph representation of siRNA.

Several aspects of gene silencing by small interfering RNA duplexes (siRNA) influence the efficiency of the silencing. They can be divided into two categories, one covering the cell-specific factors and the other covering molecular factors of the RNA interference (RNAi). A prerequisite for sequence-based siRNA design is that hybridization thermodynamics is the dominant factor. Our assumption is that cell-specific parameters (cell line, degradation, cross-hybridization, target conformation, etc.) can be pooled into an average cellular factor. Our hypothesis is that the molecular basis of the positional dependence of siRNA-induced gene silencing is the uniqueness of context of a corresponding target sequence segment relative to all other such segments along the attacked RNA. We encode this context into descriptors derived from Eulerian graph representation of siRNAs and show that the descriptor based upon the contextual similarity and predicted thermodynamic stability correlates with the experimentally observed silencing efficiency of human lamin A/C gene. We further show that information encoded in this regression function is generalizable and can be used as a predictor of siRNA efficiency in unrelated genes (CD54 and PTEN). In summary, our method represents an evolution of siRNA design from the currently used algorithms which are only qualitative in nature.

Base Sequence↗
PubMed2004

RNA interference reveals that ligand-independent met activity is required for tumor cell signaling and survival.

Hepatocyte growth factor/scatter factor-Met signaling has been implicated in tumor growth, invasion, and metastasis. Suppression of this signaling pathway by targeting the Met protein tyrosine kinase may be an ideal strategy for suppressing malignant tumor growth. Using RNA interference technology and adenovirus vectors carrying small-interfering RNA constructs (Ad Met small-interfering RNA) directed against mouse, canine, and human Met, we can knock down c-met mRNA. We show a dramatic dependence on Met in both ligand-dependent and ligand-independent mouse, canine, and human tumor cell lines. Mouse mammary tumor (DA3) cells and Met-transformed NIH3T3 (M114) cells, as well as both human and canine prostate cancer (PC-3 and TR6LM, human sarcoma (SK-LMS-1), glioblastoma (DBTRG), and gastric cancer (MKN45) cells, all display a dramatic reduction of Met expression after infection with Ad Met small-interfering RNA. In these cells, we observe suppression of tumor cell growth and viability in vitro as well as inhibition of hepatocyte growth factor/scatter factor-mediated scattering and invasion in vitro, whether Met activation was ligand dependent or not. Importantly, Ad Met small-interfering RNA led to apoptotic cell death in many of the tumor cell lines, especially DA3 and MKN45, but did not adversely affect MDCK canine kidney cells. Met small-interfering RNA also abrogated downstream Met signaling to molecules such as Akt and p44/42 mitogen-activated protein kinase. We further show that intratumoral infection with c-met small-interfering RNA adenovirus results in a substantial reduction in tumor growth. Thus, Met small-interfering RNA adenoviruses are reliable tools for studying Met function and raise the possibility of their application for cancer therapy.

Adenoviridae↗
PubMed2006

Screening for genes essential for mouse embryonic stem cell self-renewal using a subtractive RNA interference library.

The pluripotency of mouse embryonic stem (ES) cells is maintained by self-renewal. To screen for genes essential for this process, we constructed an RNA interference (RNAi) library by inserting subtracted ES cell cDNA fragments into plasmid containing two opposing cytomegalovirus promoters. ES cells were transfected with individual RNAi plasmids and levels of the pluripotency marker Oct-4 were monitored 48 hours later by real time RT-PCR. Of the first 89 RNAi plasmids characterized, 12 downregulated Oct-4 expression to less than 50% of the normal level and 7 of them upregulated Oct-4 expression to more than 150% of the normal level. To investigate their long-term effect on self-renewal, ES cells were transfected by these 19 RNAi plasmids individually and G418-resistant colonies were subjected to alkaline phosphatase (AP) staining after 7 days selection. Except for 4 plasmids that caused cell death, the ratio of AP positive colonies was repressed to less than 60% of the control group by the other 15 plasmids and even below 20% by 10 plasmids. The cDNA fragments in these 10 plasmids correspond to eight genes, including Zfp42/Rex-1, which was chosen for further functional analysis. RNAi knockdown of Zfp42 induced ES cells differentiate to endoderm and mesoderm lineages, and overexpression of Zfp42 also caused ES cells to lose the capacity of self-renewal. Our results indicate that RNAi screen is a feasible and efficient approach to identify genes involved in ES cells self-renewal. Further functional characterization of these genes will promote our understanding of the complex regulatory networks in ES cells.

Animals↗
PubMed2004

Phospholamban ablation by RNA interference increases Ca2+ uptake into rat cardiac myocyte sarcoplasmic reticulum.

Phospholamban (PLB) inhibits SR Ca(2+)-ATPase 2 (SERCA2) Ca(2+) uptake and is a potential therapeutic target in the context of heart failure. RNA interference (RNAi) is a technique that produces sequence-specific, post-transcriptional gene silencing through the use of double-stranded RNA directed against the homologous target gene. The goal of the current study was to investigate the efficacy of the RNAi method for ablation of PLB gene expression and restoration of Ca(2+) uptake function in cultured neonatal rat cardiac myocytes in which SERCA2 protein levels were decreased. Myocytes were transfected with 21-nucleotide duplexes of small interfering RNA (siRNA) targeting PLB (30 nmol/l) or with scramble sequence using a haemagglutinating virus of Japan (HVJ) envelope vector. Administration of PLB siRNA resulted in the reduction of PLB mRNA level to approximately 6% of that observed after administration of scramble siRNA group at 12 h after transfection. Further, PLB protein levels in the PLB siRNA groups were 12% of that in cells treated with scramble siRNA on day 2, and the mRNA and protein levels for SERCA2 and calsequestrin were not affected. In addition, Ca(2+) uptake affinity was increased in total homogenates from the PLB siRNA group (a 29% decrease in EC(50) value when compared with scramble siRNA group). Finally, PLB siRNA restored Ca(2+) uptake affinity following hydrogen peroxide-induced decreases in SERCA2 and PLB mRNA expression. These results demonstrate that PLB-targeted RNAi inhibited endogenous PLB expression in neonatal rat myocytes and restored Ca(2+) uptake affinity in cardiac myocytes in which SERCA2 protein levels were decreased. This technique may represent a novel therapeutic strategy for heart failure.

Animals↗
PubMed2003

Use of RNA interference to validate Brk as a novel therapeutic target in breast cancer: Brk promotes breast carcinoma cell proliferation.

Brk (PTK6) is a nonreceptor protein tyrosine kinase, which is expressed in over 60% of breast carcinoma tissue samples and breast tumour cell lines, but not normal mammary tissue or benign lesions. Since experimental Brk expression in nontransformed mammary epithelial cells enhances their mitogenic response to epidermal growth factor, it was important to determine the role Brk plays in the proliferation of breast carcinoma cells and validate it as a therapeutic target. We have used RNA interference to efficiently and specifically downregulate Brk protein levels in breast carcinoma cells, and determined that this results in a significant suppression of their proliferation. Additionally, through the expression of a kinase-inactive mutant, we have determined that Brk can mediate promotion of proliferation via a kinase-independent mechanism, potentially functioning as an 'adapter'. These data identify Brk as a novel target for antiproliferative therapy in the majority of breast cancers, and illustrate the power of RNA interference for rapidly validating candidate therapeutic targets.

Breast Neoplasms↗
PubMed2007

Gene silencing by RNA interference in the koji mold Aspergillus oryzae.

We found the orthologous genes required for RNA interference (RNAi) in the Aspergillus oryzae genome database, and constructed a set of tools for gene silencing using RNAi in A. oryzae. This system utilizes compatible restriction enzyme sites so that only a single target gene fragment is required to create the hairpin RNA cassette. For ease of handling, we also separated the construction of the hairpin RNA cassette for the target gene from its subsequent introduction into the expression vector. Using the brlA gene as a target for RNAi, we detected decreased mRNA levels and a delayed conidiation phenotype in the transformants. Furthermore, even though A. oryzae possesses three copies of the alpha-amylase gene, a single copy of an alpha-amylase RNAi construct was sufficient to downregulate the mRNA levels and decrease the enzymatic activity to 10% of control levels. Gene silencing by RNAi should provide a powerful genetic tool for post-genomic studies of the industrially important fungus A. oryzae.

Aspergillus oryzae↗
PubMed2002

RNA interference.

A conserved biological response to double-stranded RNA, known variously as RNA interference (RNAi) or post-transcriptional gene silencing, mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. RNAi has been cultivated as a means to manipulate gene expression experimentally and to probe gene function on a whole-genome scale.

Animals↗
PubMed2006

Mutation of siRNA results in thermodynamically unstable duplex which influences knockdown of dmrt1 by RNA interference.

MicroRNAs (miRNAs) constitute a growing class of non-coding RNAs that are thought to regulate gene expression by translational repression and mRNA degradation. We report here that short interfering RNA (siRNA) mutation significantly changed kinetics of the folding and unfolding of secondary structures and decreased Tm value of the duplex melting. The mutant duplex was more unstable thermodynamically than the normal structure. Furthermore negative effects of the mutation on RNA interference (RNAi) was observed in both mouse dmrt1 transfected COS-7 and Sertoli cells in which endogenous dmrt1 was expressed. However, interference efficiency of mutational and normal duplex in Sertoli cells was not significant in comparison with those in dmrt1 transfected COS-7 cells, suggesting complex regulatory mechanisms in RNAi in endogenous dmrt1 expression. Abundance of intronic miRNA structures observed in the dmrt1 gene may also contribute to the precise regulation cascade in the dmrt1 expression. These findings help in further understanding RNA silence in vertebrate development and drug design for target gene.

Animals↗
PubMed2001

RNA interference of gene expression (RNAi) in cultured Drosophila cells.

RNA interference (RNAi) can be used to silence genes in a number of taxa, including plants, nematodes, protozoans, flies, and mammals represented by mouse embryos and cultured mammalian cells. To investigate signal transduction pathways, we used RNAi on Drosophila-cultured cells, which affords the opportunity to study protein function in a simple, well-defined cell culture system. Furthermore, the results obtained from experiments performed on cultured cells can be confirmed and extended in the whole organism, which, in the case of Drosophila, is also RNAi responsive. RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce posttranscriptional gene silencing in a highly specific manner. This silencing is efficacious and long-lived, as it is passed to subsequent generations in insect cell culture. To date, all Drosophila cell lines tested (S2, KC, BG2-C6, and Shi) respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested (more than 15) have been effective at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss-of-function studies applicable to a wide variety of questions.

Animals↗