Short interfering RNA (siRNA) is widely used for studying gene function and holds great promise as a tool for validating drug targets and treating disease. A critical assumption in these applications is that the effect of siRNA on cells is specific, i.e., limited to the specific knockdown of the target gene. In this article, we characterize the specificity of siRNA by applying gene expression profiling. Several siRNAs were designed against different regions of the same target gene for three different targets. Their effects on cells were compared by using DNA microarrays to generate gene expression signatures. When the siRNA design and transfection conditions were optimized, the signatures for different siRNAs against the same target were shown to correlate very closely, whereas the signatures for different genes revealed no correlation. These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA-mediated gene silencing as a reliable approach for large-scale screening of gene function and drug target validation.DNA microarray ͉ RNA interference ͉ gene knockdown T he use of RNA interference for inhibiting gene expression represents a powerful tool for exploring gene function (1-7), identifying and validating new drug targets, and treating disease (8, 9). The process of RNA interference is mediated by doublestranded RNA, which is cleaved by the enzyme DICER into 21-to 23-nt duplexes containing a 2-nt overhang at the 3Ј end of each strand. These duplexes are incorporated into a protein complex called the RNA-induced silencing complex (RISC). Directed by the antisense strand of the duplex, RISC recognizes and cleaves the target mRNA (for recent reviews, see refs. 1 and 10-13). Although long double-stranded RNAs (Ͼ30 nt) invoke an interferon response, short interfering RNAs (siRNAs) that resemble the products produced by DICER have been reported to specifically inhibit gene expression in many different mammalian cell lines (1-7). It has been shown that even single nucleotide mismatches between the antisense strand of the siRNA and target mRNA can abolish RNA interference (14). In addition, mapping of mRNA cleavage sites has revealed no cleavage sites outside of the region of complementarity (10). However, the specificity of siRNA at the cellular level remains to be comprehensively studied.For siRNAs to be a useful tool in gene knockdown experiments, it is critical that siRNA-mediated transcriptional silencing be specific. It is not enough to simply show that a control siRNA with a scrambled nucleotide sequence fails to knock down the protein of interest or produce the same cellular phenotype. Ideally, the siRNA must not cause any effects other than those related to the knock down of the target gene. There are several types of nonspecific effects that siRNA could potentially display. In addition to the possibility for cross-hybridization of the antisense strand of the siRNA to different mRNAs, siRNAs could bind in a sequencedependent manner to various cellular proteins. I...

Observations in hemodialysis patients suggest a survival advantage associated with activated vitamin D therapy. Left ventricular (LV) structural and functional abnormalities are strongly linked with hemodialysis mortality. Here, we investigated whether paricalcitol (PC, 19-nor-1,25(OH)2D2), an activated vitamin D compound, attenuates the development of LV abnormalities in the Dahl salt-sensitive (DSS) rat and whether humans demonstrate comparable findings. Compared with DSS rats fed a high-salt (HS) diet (6% NaCl for 6 weeks), HS؉PC was associated with lower heart and lung weights, reduced LV mass, posterior wall thickness and end diastolic pressures, and increased fractional shortening. Blood pressures did not significantly differ between the HS groups. Plasma brain natriuretic peptide levels, and cardiac mRNA expression of brain natriuretic peptide, atrial natriuretic factor, and renin were significantly reduced in the HS؉PC animals. Microarray analyses revealed 45 specific HS genes modified by PC. In a retrospective pilot study of hemodialysis patients, PC-treated subjects demonstrated improved diastolic function and a reduction in LV septal and posterior wall thickness by echocardiography compared with untreated patients. In summary, PC attenuates the development of LV alterations in DSS rats, and these effects should be examined in human clinical trials.cardiac hypertrophy ͉ heart failure ͉ paricalcitol ͉ renal failure T he rate of cardiovascular-related mortality in hemodialysis patients is 10-20 times higher than that observed in the general population (1). Left ventricular hypertrophy (LVH) and diastolic dysfunction are present in Ͼ50% of patients at dialysis initiation, and these abnormalities are strongly linked with dialysis-related mortality (2). Currently, there are no well accepted means to modify cardiac structural and functional alterations in renal-failure patients.We recently demonstrated in observational studies that therapy with activated vitamin D to chronic hemodialysis patients is associated with reduction in cardiovascular-related mortality (3, 4). Conversion of nutritional vitamin D (25(OH)D 3 ) to the hormonally active form of vitamin D (1,25(OH) 2 D 3 ) occurs primarily in the kidney; thus, patients with kidney failure commonly present with altered vitamin D status (5). There is growing evidence that vitamin D either directly or indirectly affects cardiac structure and function. The vitamin D receptor knockout mouse model demonstrates increased cardiac renin expression and marked cardiomyocyte hypertrophy (6), and 1,25(OH) 2 D 3 attenuates cardiomyocyte proliferation (7) and hypertrophy (8) in vitro. Here, we demonstrate that treatment with an activated vitamin D compound attenuates the development of cardiac hypertrophy and dysfunction in a recognized animal model of such abnormalities and that comparable findings are evident in humans.

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob͞ob and db͞db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA 1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50␣, were increased and PI3-kinase p85␣ expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its analogues have been shown to inhibit proliferation of human cancer cells mediated by vitamin D receptor (VDR). The over-expression of 25-hydroxyvitamin D-24-hydroxylase (CYP24A1), an enzyme involved in the metabolism of 1,25(OH)2D3 and its analogues, is associated with poor prognosis of some human cancers. In this study, we employed real-time reverse transcription PCR to examine the expression of VDR and CYP24A1 mRNA in a cohort of human breast, lung, colon and ovary tumor samples. We found that CYP24A1 mRNA was significantly up-regulated in colon, ovary and lung tumors, but down-regulated in breast tumor relative to the analogous normal tissues. As a comparison, VDR mRNA was modestly down-regulated in colon, breast and lung tumors, but highly up-regulated in ovarian tumors. Treatment of two breast cancer cell lines, SW-620 and MCF-7, and one colon cancer cell line, HT-29, by 1,25(OH)2D3 for 48 h profoundly stimulated CYP24A1 mRNA expression (EC50=0.6, 0.8 and 29.5 nM in SW-620, HT-29 and MCF-7, respectively), but did not significantly affect VDR mRNA expression. Growth as assessed by DNA synthesis was modestly arrested by 1,25(OH)2D3 after 72 h of incubation, but was not altered after a 5-day incubation period. These data suggest that the VDR signaling pathway may be compromised via the modulation of CYP24A1 and VDR in human tumors.

The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiae ELO2 gene, a consensus cDNA sequence was identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5' and 3' ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897 bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo-gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n-3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response.