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RNA interference (RNAi) has been established as an important research tool that carries great potential for gene therapy. However, targeted induction of RNAi in vivo has met with significant challenges. In this study, a novel pSLS plasmid capable of expressing short hairpin RNAs (shRNAs) was transformed into attenuated Salmonella enterica serovar typhimurium strain 7207 (SL). In vitro infection studies with the transformed S. enterica containing pSLS (SL-pSLS-CAT) demonstrated that expression of shRNA targeting the CTNNB1 gene induced potent and specific silencing of CTNNB1 expression in cultured SW480 cells. CTNNB1 knockdown in SW480 cells was associated with markedly reduced proliferation and cell death compared with that of control infected cells. In addition, SL-pSLS-CAT-mediated CTNNB1 knockdown markedly reduced tumor growth in SW480 xenograft mice. These tumors exhibited reduced levels of CTNNB1, as well as c-Myc and cyclin D1. Finally, SL-pSLS-CAT treatment also resulted in reduced expression levels of these genes in polyps, mucosal tissues and in small intestines of APC(Min) mice. Taken together, these data suggest that attenuated shRNA-expressing Salmonella may be a powerful new tool for in vitro gene silencing, functional genomics, and the development of RNAi-based anticancer or human immunodeficiency virus therapeutics.

The molecular biology of circadian rhythms has been extensively studied in mice, and the widespread expression of canonical circadian clock genes in peripheral organs is well established in this species. In contrast, much less information about the peripheral expression of haPer1, haPer2, and haBmal1 is available in Syrian hamsters despite the fact that this species is widely used for studies of circadian organization and photoperiodic responses. Furthermore, examination of oscillating expression of these genes in mouse testis has generated discrepant results, and little is known about gonadal expression of haPer1 and haBmal1 or their environmental control. To address these questions, the authors examined the pattern of haPer1 and haBmal1 in heart, kidney, liver, muscle, spleen, and testis of hamsters exposed to DD. In most organs, Northern blots suggested the existence of single transcripts of each of these messenger RNAs (mRNAs). haPer1 peaked in late subjective day and haBmal1 during the late subjective night. Closer inspection of SCN and muscle haPer1, however, revealed the existence of two major transcripts of similar size, as well as minor transcripts that varied in the 3′-untranslated region. In hamster testis, two haPer1 transcripts were found, both of which are truncated relative to the corresponding mouse transcript and both of which contain a sequence homologous to intron 18 of mPer1. Neither testis transcript contains a nuclear localization signal, and haPer1 transcripts lacked the putative C-terminal CRY1-binding domain. Furthermore, the testis deviated from the general pattern in that haPer1 and haBmal1 both peaked in the subjective night. In situ hybridization revealed that haPer1, but not haBmal1, showed a heterogeneous distribution among seminiferous tubules. Hamster testis also expresses 2 haPer2 transcripts, but no circadian variation is evident. In a second experiment, long-term exposure to DD sufficient to induce gonadal regression was found to eliminate circadian oscillations of both testicular haPer1 transcripts. In contrast, gonadal regression was accompanied by a more robust rhythm of haBmal1.

Background— Response gene to complement 32 (RGC-32) is induced by activation of complement and regulates cell proliferation. To determine the mechanism of RGC-32 in angiogenesis, we examined the role of RGC-32 in hypoxia-related endothelial cell function. Methods and Results— Hypoxia/ischemia is able to stimulate both angiogenesis and apoptosis. Hypoxia-inducible factor-1/vascular endothelial growth factor is a key transcriptional regulatory pathway for angiogenesis during hypoxia. We demonstrated that the increased RGC-32 expression by hypoxia was via hypoxia-inducible factor-1/vascular endothelial growth factor induction in cultured endothelial cells. However, overexpression of RGC-32 reduced the proliferation and migration and destabilized vascular structure formation in vitro and inhibited angiogenesis in Matrigel assays in vivo. Silencing RGC-32 had an opposing, stimulatory effect. RGC-32 also stimulated apoptosis as shown by the increased apoptotic cells and caspase-3 cleavage. Mechanistic studies revealed that the effect of RGC-32 on the antiangiogenic response was via attenuating fibroblast growth factor 2 expression and further inhibiting expression of cyclin E without affecting vascular endothelial growth factor and fibroblast growth factor 2 signaling in endothelial cells. In the mouse hind-limb ischemia model, RGC-32 inhibited capillary density with a significant attenuation in blood flow. Additionally, treatment with RGC-32 in the xenograft tumor model resulted in reduced growth of blood vessels that is consistent with reduced colon tumor size. Conclusions— We provide the first direct evidence for RGC-32 as a hypoxia-inducible gene and antiangiogenic factor in endothelial cells. These data suggest that RGC-32 plays an important homeostatic role in that it contributes to differentiating the pathways for vascular endothelial growth factor and fibroblast growth factor 2 in angiogenesis and provides a new target for ischemic disorder and tumor therapies.

RNA interference (RNAi) is a potent and specific mechanism for eliminating the mRNA of specific genes. This gene silencing mechanism occurs naturally and is highly conserved from plants to human cells, holding promise for functional genomics and for revolutionizing medicine due to its unlimited potential to treat genetic, epigenetic, and infectious disease. However, efforts to unleash the enormous potential of RNAi have met with significant challenges. Delivery is problematic because short interfering RNAs (siRNA) are negatively charged polymers that inefficiently enter cells and undergo rapid enzymatic degradation in vivo. In addition, the synthesis of siRNAs is expensive for long-term research and therapeutic applications. Recently, we have shown that nonpathogenic bacteria can be engineered to activate RNAi in mammalian cells (TransKingdom RNA interference; tkRNAi). This new approach offers several advantages and has significant implications. First, this method allows the establishment of a long-term stable gene silencing system in the laboratory against genes of interests in vitro and in vivo, and enables high-throughput functional genomics screening in mammalian systems. RNAi libraries can be constructed, stored, reproduced, amplified, and used with the help of E. coli as currently done with gene cloning. Second, this technology provides a clinically compatible way to achieve RNAi for therapeutic applications due to the proven clinical safety ofnonpathogenic bacteria as a gene carrier, tkRNAi also eliminates the siRNA manufacture issue, and may circumvent or mitigate host interferon-like responses since siRNA is produced intracellularly.

Neurotransplantation of the suprachiasmatic nucleus (SCN) was used to assess communication between the central circadian pacemaker and peripheral oscillators in Syrian hamsters. Free-running rhythms of haPer1, haPer2, and Bmal1 expression were documented in liver, kidney, spleen, heart, skeletal muscle, and adrenal medulla after 3 d or 11 weeks of exposure to constant darkness. Ablation of the SCN of heterozygote tau mutants eliminated not only rhythms of locomotor activity but also rhythmic expression of these genes in all peripheral organs studied. The Per:Bmal ratio suggests that this effect was attributable not to asynchronous rhythmicity between SCN-lesioned individuals but to arrhythmicity within individuals. Grafts of wild-type SCN to heterozygous, SCN-lesioned tau mutant hamsters not only restored locomotor rhythms with the period of the donor but also led to recovery of rhythmic expression of haPer1, haPer2, and haBmal1 in liver and kidney. The phase of these rhythms most closely resembled that of intact wild-type hamsters. Rhythmic gene expression was also restored in skeletal muscle, but the phase was altered. Behaviorally effective SCN transplants failed to reinstate rhythms of clock gene expression in heart, spleen, or adrenal medulla. These findings confirm that peripheral organs differ in their response to SCN-dependent cues. Furthermore, the results indicate that conventional models of internal entrainment may need to be revised to explain control of the periphery by the pacemaker.

Although dependent on the integrity of a central pacemaker in the suprachiasmatic nucleus of the hypothalamus (SCN), endogenous daily (circadian) rhythms are expressed in a wide variety of peripheral organs. The pathways by which the pacemaker controls the periphery are unclear. Here, we used parabiosis between intact and SCN-lesioned mice to show that nonneural (behavioral or bloodborne) signals are adequate to maintain circadian rhythms of clock gene expression in liver and kidney, but not in heart, spleen, or skeletal muscle. These results indicate that the SCN regulates expression of circadian oscillations in different peripheral organs by diverse pathways.