MicroRNAs (miRNAs) are believed to have fundamental roles in tumorigenesis and have great potential for the diagnosis and treatment of cancer. However, the roles of miRNAs in hepatocellular carcinogenesis are still not fully elucidated. We investigated the aberrantly expressed miRNAs involved in hepatoma by comparison of miRNA expression profiles in cancerous hepatocytes with normal primary human hepatocytes, and 37 dysregulated miRNAs were screened out by twofold change with a significant difference (Po0.05). Clustering analysis based on 13 miRNAs with changes over 15-folds showed that the miRNA expression patterns between the cancerous and normal hepatocytes were clearly different. Among the 13 miRNAs, we found that miR-375 was significantly downregulated in hepatocellular carcinoma (HCC) tissues and cell lines. Overexpression of miR-375 in liver cancer cells decreased cell proliferation, clonogenicity, migration/ invasion and also induced G1 arrest and apoptosis. To unveil the molecular mechanism of miR-375-mediated phenotype in hepatoma cells described above, we examined the putative targets using bioinformatics tools and found that astrocyte elevated gene-1 (AEG-1) was a potential target of miR-375. Then we demonstrated that miR-375 bound directly to the 3 0 -untranslated region of AEG-1 and inhibited the expression of AEG-1. TaqMan quantitative reverse transcriptase-PCR and western blot analysis showed that miR-375 expression was inversely correlated with AEG-1 expression in HCC tissues. Knockdown of AEG-1 by RNAi in HCC cells, similar to miR-375 overexpression, suppressed tumor properties. Ectopic expression of AEG-1, conversely, could partially reverse the antitumor effects of miR-375. In a mouse model, therapeutic administration of cholesterol-conjugated 2 0 -O-methyl-modified miR-375 mimics (Chol-miR-375) could significantly suppress the growth of hepatoma xenografts in nude mice. In conclusion, our findings indicate that miR-375 targets AEG-1 in HCC and suppresses liver cancer cell growth in vitro and in vivo, and highlight the therapeutic potential of miR-375 in HCC treatment.

Using extracellular single-unit recordings alone and in combination with neurobiotin juxtacellular labeling and histamine immunohistochemistry, we have identified, for the first time in nonanesthetized, head-restrained mice, histamine neurons in the tuberomammillary nuclei of the posterior hypothalamus. They are all characterized by triphasic broad action potentials. They are active only during wakefulness, and their activity is related to a high level of vigilance. During waking states, they display a slow (Ͻ10 Hz) tonic, repetitive, irregular firing pattern. Their activity varies in the different waking states, being lowest during quiet waking, moderate during active waking, and highest during attentive waking. They cease firing during quiet waking before the onset of EEG synchronization, the EEG sign of sleep (drowsy state), and remain silent during slow-wave sleep and paradoxical (or rapid eye movement) sleep. They exhibit a pronounced delay in firing during transitions from sleep to wakefulness or remain quiescent during the same transitions if the animals are not fully alert. They either respond with a long delay, or do not respond, to an arousing stimulus if the stimulus does not elicit an overt alert state. These data support the view that the activity of histaminergic tuberomammillary neurons plays an important role, not in the induction of wakefulness per se, but in the maintenance of the high level of vigilance necessary for cognitive processes. Conversely, cessation of their activity may play an important role in both the initiation and maintenance of sleep.

Abbreviations AbstractNarcolepsy is characterized by excessive daytime sleepiness(EDS), cataplexy, direct onsets of rapid eye movement(REM) sleep from wakefulness(DREMs) and deficiency of orexins, neuropeptides that promote wakefulness largely via activation of histamine (HA) pathways. The hypothesis that the orexin defect can be circumvented by enhancing HA release was explored in narcoleptic mice and patients using tiprolisant, an inverse H3-receptor agonist. In narcoleptic orexin-/-mice, tiprolisant enhanced HA and noradrenaline neuronal activity, promoted wakefulness and decreased abnormal DREMs, all effects being amplified by co-administration of modafinil, a currently prescribed wake-promoting drug. In a pilot single-blind trial on 22 patients receiving a placebo followed by tiprolisant, both for one week, the Epworth Sleepiness Scale(ESS) score was reduced from a baseline value of 17.6 by 1.0 with the placebo(p>0.05) and 5.9 with tiprolisant(p<0.001). Excessive daytime sleep, unaffected under placebo, was nearly suppressed on the last days of tiprolisant-dosing. H3-receptor inverse agonists could constitute a novel effective treatment of EDS, particularly when associated with modafinil.

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Much experimental and clinical data suggest that the pharmacological profile of modafinil, a newly discovered waking substance, differs from those of amphetamine and methylphenidate, two classical psychostimulants. The brain targets on which modafinil acts to induce wakefulness, however, remain unknown. A double-blind study using the protooncogene c-fos as experimental marker in the cat was, therefore, carried out to identify the potential target neurons of modafinil and compare them with those for amphetamine and methylphenidate. Cats were sacrificed after a single oral administration of amphetamine, methylphenidate, or modafinil at equivalent doses for wake induction (1, 2.5, or 5 mg͞kg, respectively) and brain sections examined for Fos by immunocytochemistry. Administration of either amphetamine or methylphenidate evoked Fos-like immunoreactivity in a large number of neurons in the striatum and whole cortex, especially in the caudate nucleus and mediofrontal cortex, which are known to be dopaminergic targets. In contrast, administration of modafinil resulted in the labeling of few cells in these structures, but did induce marked Fos labeling in neurons of the anterior hypothalamic nucleus and adjacent areas. These results provide evidence for the potential brain targets of modafinil, which differ from those of amphetamine or methylphenidate, and suggest that modafinil induces wakefulness by mechanisms distinct from those of the two stimulants.Modafinil, a newly discovered wake-improving substance, induces prolonged wakefulness in a number of species, apparently without associated behavioral excitation (1-4). Unlike amphetamine, a psychomotor stimulant, its waking effect is not followed by an obvious sleep rebound in the cat (4), either under normal conditions or after sleep deprivation. Moreover, pretreatment with ␣-methylparatyrosine, an inhibitor of catecholamine synthesis prevents the arousal effect of amphetamine but allows that of modafinil to persist (4). Although these results indicate the different pharmacological profiles of modafinil and amphetamine, the underlying mechanisms remain unknown, especially as regards the brain targets by which modafinil induces waking. This is mainly because conventional methods, such as autoradiography, are currently unable to reveal its central sites of action.Recently, visualization of the expression of immediate-early genes (IEGs) has become a promising method in neuroscience. The proto-oncogene, c-fos, is the most frequently activated IEG in the central nervous system following various internal or external stimuli. c-fos encodes a nuclear phosphoprotein, Fos, which is thought to control target gene expression responsible for cell activation. c-fos has, therefore, been considered as the third messenger, reacting to short-term stimuli and triggering longterm cell reaction cascades (5-7). Although a basic level of expression of IEGs occurs in the brain, c-fos is strongly expressed in various specific cerebral areas following different stimuli, such as electric...

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