MicroRNAs (miRNAs) are endogenously expressed 20 -24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.The first miRNA 1 was identified in Caenorhabditis elegans as a gene important for timing of larval development (5). miRNAs have since been implicated in many processes in invertebrates, including cell proliferation and apoptosis (6, 7), fat metabolism (6), and neuronal patterning (8). As many miRNAs are conserved across species (9 -11), they are likely to be involved in developmental processes in all animals. Only a few mammalian miRNAs have been assigned any function, and at least two of these are involved in developmental processes: miR-181 promotes B cell development in mice (12) and miR196a regulates several Hox genes (13), which code for a family of transcription factors involved in various developmental programs in animals (14).We hypothesized that miRNAs may play a role in maturation of human adipocytes. Understanding the molecular events involved in adipocyte differentiation is of interest for development of therapeutics for metabolic diseases such as obesity and diabetes. In vitro cell culture systems, such as human primary subcutaneous pre-adipocytes, have been crucial in uncovering signaling pathways important for adipocyte differentiation (15). These cells can be cultured with differentiation-promoting hormonal stimuli, causing them to develop into cells that morphologically and functionally resemble mature adipocytes. In this study we have inhibited a panel of miRNAs in pre-adipocytes using antisense oligonucleotides and evaluated the effect on adipocyte differentiation. Combined with expression analysis of miRNAs in differentiating adipocytes by microarray, one miRNA, miR-143, was identified which normally promotes adipocyte differentiation. These results indicate that miRNAs do play a role in adipocyte differentiation and are potential therapeutic targets for obesity and metabolic diseases. EXPERIMENTAL PROCEDURESOligonucleotide Synthesis-Oligonucleotides were prepared using conventional phosphoramidite chemistry and...
Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.
ASO treatment alone has no effect, it can sensitize myeloma cell lines to dexamethasone (Dex), whereas Bcl-x L ASO in combination with Dex still had no effect. As MM remains an incurable disease despite intensive chemotherapy, these results suggest that Mcl-1 antisense strategy rather than Bcl-2 antisense strategy could be of considerable importance in the treatment of MM. (Blood. 2002;100:194-199)
RNA interference can be considered as an antisense mechanism of action that utilizes a double-stranded RNase to promote hydrolysis of the target RNA. We have performed a comparative study of optimized antisense oligonucleotides designed to work by an RNA interference mechanism to oligonucleotides designed to work by an RNase H-dependent mechanism in human cells. The potency, maximal effectiveness, duration of action, and sequence specificity of optimized RNase H-dependent oligonucleotides and small interfering RNA (siRNA) oligonucleotide duplexes were evaluated and found to be comparable. Effects of base mismatches on activity were determined to be position-dependent for both siRNA oligonucleotides and RNase H-dependent oligonucleotides. In addition, we determined that the activity of both siRNA oligonucleotides and RNase H-dependent oligonucleotides is affected by the secondary structure of the target mRNA. To determine whether positions on target RNA identified as being susceptible for RNase H-mediated degradation would be coincident with siRNA target sites, we evaluated the effectiveness of siRNAs designed to bind the same position on the target mRNA as RNase H-dependent oligonucleotides. Examination of 80 siRNA oligonucleotide duplexes designed to bind to RNA from four distinct human genes revealed that, in general, activity correlated with the activity to RNase H-dependent oligonucleotides designed to the same site, although some exceptions were noted. The one major difference between the two strategies is that RNase H-dependent oligonucleotides were determined to be active when directed against targets in the pre-mRNA, whereas siRNAs were not. These results demonstrate that siRNA oligonucleotide-and RNase Hdependent antisense strategies are both valid strategies for evaluating function of genes in cell-based assays.
MicroRNAs (miRNAs) are believed to play important roles in developmental and other cellular processes by hybridizing to complementary target mRNA transcripts. This results in either cleavage of the hybridized transcript or negative regulation of translation. Little is known about the regulation or pattern of miRNA expression. The predicted presence of numerous miRNA sequences in higher eukaryotes makes it highly likely that the expression levels of individual miRNA molecules themselves should play an important role in regulating multiple cellular processes. Therefore, determining the pattern of global miRNA expression levels in mammals and other higher eukaryotes is essential to help understand both the mechanism of miRNA transcriptional regulation as well as to help identify miRNA regulated gene expression. Here, we describe a novel method to detect global processed miRNA expression levels in higher eukaryotes, including human, mouse and rats, by using a high-density oligonucleotide array. Array results have been validated by subsequent confirmation of mir expression using northern-blot analysis. Major differences in mir expression have been detected in samples from diverse sources, suggesting highly regulated mir expression, and specific gene regulatory functions for individual miRNA transcripts. For example, five different miRNAs were found to be preferentially expressed in human kidney compared with other human tissues. Comparative analysis of surrounding genomic sequences of the kidney-specific miRNA clusters revealed the occurrence of specific transcription factor binding sites located in conserved phylogenetic foot prints, suggesting that these may be involved in regulating mir expression in kidney.
Heterologous expression of the transient receptor potential-1 gene product (Trp1) encodes for a Ca2+ entry pathway, though it is unclear whether endogenous Trp1 contributes to a selective store-operated Ca2+ entry current. We examined the role of Trp1 in regulating both store-operated Ca2+ entry and a store-operated Ca2+ entry current, I(SOC), in A549 and endothelial cells. Twenty different 'chimeric' 2'-O-(2-methoxy)ethylphosphothioate antisense oligonucleotides were transfected separately using cationic lipids and screened for their ability to inhibit Trp1 mRNA. Two hypersensitive regions were identified, one at the 5' end of the coding region and the second in the 3' untranslated region beginning six nucleotides downstream of the stop codon. Antisense oligonucleotides stably decreased Trp1 at concentrations ranging from 10 to 300 nM, for up to 72 h. Thapsigargin increased global cytosolic Ca2+ and activated a I(SOC), which was small (-35 pA @ -80 mV), reversed near +40 mV, inhibited by 50 microM La3+, and exhibited anomalous mole fraction dependence. Inhibition of Trp1 reduced the global cytosolic Ca(2+) response to thapsigargin by 25% and similarly reduced I(SOC) by 50%. These data collectively support a role for endogenously expressed Trp1 in regulating a Ca2+-selective current activated upon Ca2+ store depletion.
The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3,4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G 1 in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27 KIP1 . Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.
Resistance to apoptosis, which plays an important role in tumors that are refractory to chemotherapy, is regulated by the ratio of antiapoptotic to proapoptotic proteins. By manipulating levels of these proteins, cells can become sensitized to undergo apoptosis in response to chemotherapeutic agents. Alternative splicing of the bcl-x gene gives rise to two proteins with antagonistic functions: Bcl-xL, a well-characterized antiapoptotic protein, and Bcl-xS, a proapoptotic protein. We show here that altering the ratio of Bcl-xL to Bcl-xS in the cell using an antisense oligonucleotide permitted cells to be sensitized to undergo apoptosis in response to ultraviolet B radiation and chemotherapeutic drug treatment. These results demonstrate the ability of a chemically modified oligonucleotide to alter splice site selection in an endogenous gene and illustrate a powerful tool to regulate cell survival.
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