MicroRNAs are small noncoding RNA molecules that control expression of target genes. Our previous studies show that mir-21 is overexpressed in tumor tissues compared with the matched normal tissues. Moreover, suppression of mir-21 by antisense oligonucleotides inhibits tumor cell growth both in vitro and in vivo. However, it remains largely unclear as to how mir-21 affects tumor growth, because our understanding of mir-21 targets is limited. In this study, we performed two-dimensional differentiation in-gel electrophoresis of tumors treated with anti-mir-21 and identified the tumor suppressor tropomyosin 1 (TPM1) as a potential mir-21 target. In agreement with this, there is a putative mir-21 binding site at the 3-untranslated region (3-UTR) of TPM1 variants V1 and V5. Thus, we cloned the 3-UTR of TPM1 into a luciferase reporter and found that although mir-21 down-regulated the luciferase activity, anti-mir-21 up-regulated it. Moreover, deletion of the mir-21 binding site abolished the effect of mir-21 on the luciferase activity, suggesting that this mir-21 binding site is critical. Western blot with the cloned TPM1-V1 plus the 3-UTR indicated that TPM1 protein level was also regulated by mir-21, whereas real-time quantitative reverse transcription-PCR revealed no difference at the mRNA level, suggesting translational regulation. Finally, overexpression of TPM1 in breast cancer MCF-7 cells suppressed anchorage-independent growth. Thus, down-regulation of TPM1 by mir-21 may explain, at least in part, why suppression of mir-21 can inhibit tumor growth, further supporting the notion that mir-21 functions as an oncogene. MicroRNAs (miRNAs)2 are a class of naturally occurring small noncoding RNAs that regulate gene expression by targeting mRNAs for translational repression or cleavage (1, 2). Like protein-coding mRNAs, miRNAs are transcribed as long primary transcripts in the nucleus. However, unlike protein-coding mRNAs, miRNAs are subsequently cleaved to produce stem-loop-structured precursor molecules of ϳ70 nucleotides in length (pre-miRNAs) by the nuclear RNase III enzyme Drosha (3). The pre-miRNAs are then exported to the cytoplasm, where the RNase III enzyme Dicer further processes them into mature miRNAs (ϳ22 nucleotides). Thus, miRNAs are related to, but distinct from, short inferring RNAs (siRNAs) (4, 5). A key difference between siRNAs and miRNAs is that siRNAs require almost identical sequences to targets to exert their silencing function, whereas miRNAs bind through partial sequence homology to the 3Ј-untranslated region (3Ј-UTR) of target genes. Because of this unique feature, a single miRNA has multiple targets. Thus, miRNAs could regulate a large fraction of protein-coding genes, and as high as 30% of all genes could be miRNA targets (6).As a new layer of gene regulation mechanism, miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation, and apoptosis (7,8). Hence, deregulation of miRNA expression may lead to a variety of disorders. Aberrant expression of miR...

Abstract-It has been suggested in isolated porcine cerebral arteries that stimulation by nicotine of ␣ 7 -nicotinic acetylcholine receptors (␣ 7 -nAChRs) on sympathetic nerves, but not direct stimulation of parasympathetic nitrergic nerves, caused nitrergic neurogenic dilation. Direct evidence supporting this hypothesis has not been presented. The present study, which used in vitro tissue bath and confocal microscopy techniques, was designed to determine whether choline, a selective agonist for ␣ 7 -nAChRs, induced sympathetic-dependent nitrergic dilation of porcine basilar arterial rings. Choline and several nAChR agonists induced exclusive relaxation of basilar arterial rings without endothelium. The relaxation was blocked by tetrodotoxin, nitro-L-arginine, guanethidine, and ␤ 2 -adrenoceptor antagonists. Furthermore, the relaxation was blocked by methyllycaconitine and ␣-bungarotoxin (preferential ␣ 7 -nAChR antagonists) and mecamylamine but was not affected by dihydro-␤-erythroidine (a preferential ␣ 4 -nAChR antagonist). Confocal microscopic study demonstrated that choline and nicotine induced significant calcium influx in cultured porcine superior cervical ganglionic cells but failed to affect calcium influx in cultured sphenopalatine ganglionic cells, providing direct evidence that choline and nicotine did not act directly on the parasympathetic nitrergic neurons. The increased calcium influx in superior cervical ganglionic cells was attenuated by ␣-bungarotoxin and methyllycaconitine but not by dihydro-␤-erythroidine. These results support our hypothesis that activation of ␣ 7 -nAChRs on cerebral perivascular sympathetic nerves causes calcium influx and the release of norepinephrine, which then act on presynaptic Key Words: choline Ⅲ ␣ 7 -nicotinic acetylcholine receptors Ⅲ cerebral neurogenic vasodilation Ⅲ nitric oxide Ⅲ sympathetic-parasympathetic interaction T he ␣-bungarotoxin (␣-BGTX)-sensitive nicotinic acetylcholine receptor (nAChR), which is one of the predominant nAChR subtypes in the brain, consists of the ␣ 7 -subunit, possesses calcium permeability, and exhibits rapid desensitization. 1 Choline, which is a precursor of acetylcholine (ACh) synthesis and a product of ACh hydrolysis by acetylcholinesterase, has been shown to act as a relatively selective agonist for ␣ 7 -nAChRs in the central nervous system. 2,3 By activating presynaptic ␣ 7 -nAChRs, choline has been shown to elicit the release of neurotransmitters, including norepinephrine (NE). 2,4 The presence of nAChRs on sympathetic adrenergic nerve terminals is also well established. 5,6 However, the functional significance of choline and ␣ 7 -nAChRs in the perivascular neurons has not been clarified.We reported recently that nicotine-induced NO-mediated neurogenic vasodilation in porcine basilar arteries and feline middle cerebral arteries was dependent on intact perivascular sympathetic adrenergic innervation. 7-9 Results from pharmacological studies using nicotinic receptor antagonists further suggest that nicotine acts on ␣ 7 -nAChRs ...

Chronic exposure to inorganic lead (Pb 2ϩ ) has been shown to facilitate peripheral vasoconstriction causing hypertension. Effect of lead on cerebral vascular function has not been reported. We have suggested in isolated porcine cerebral arteries that ␣ 7 -nicotinic acetylcholine receptors (␣ 7 -nAChRs) on perivascular sympathetic nerves mediate calcium influx in these neurons, resulting in release of norepinephrine. The released norepinephrine then acts on presynaptic ␤ 2 -adrenoceptors located on the neighboring nitrergic nerve terminals, causing nitric oxide (NO) release and vasodilation. Because Pb 2ϩ has been shown to inhibit ␣ 7 -nAChR-mediated responses in the central nervous system, effects of Pb 2ϩ on ␣ 7 -nAChRmediated nitrergic neurogenic dilation in isolated porcine basilar arteries and calcium influx in cultured superior cervical ganglion (SCG) cells of the pig were examined using in vitro tissue bath and confocal microscopic techniques. The results indicated that Pb 2ϩ (but not Cd 2ϩ , Zn 2ϩ , or Al 3ϩ ) in a concentration-dependent manner blocked relaxation of endothelium-denuded basilar arterial rings induced by nicotine (100 M) and choline (1 mM) without affecting relaxation induced by sodium nitroprusside or isoproterenol. Furthermore, significant calcium influx in cultured SCG cells induced by choline and nicotine was attenuated specifically by Pb 2ϩ with IC 50 values comparable with those from tissue bath study. These results provide evidence supporting that lead is a likely antagonist for ␣ 7 -nAChRs that are found on postganglionic sympathetic adrenergic nerve terminals of SCG origin. Furthermore, these results indicate that lead can attenuate dilation of cerebral arteries by blocking sympathetic nerve-mediated release of NO from the perivascular nitrergic nerves.

Cholinesterase inhibitors (ChEIs) have been used to treat Alzheimer's disease (AD). The efficacy of these drugs, however, is less than satisfactory. The possibility that ChEIs may have effects unrelated to ChE activity, such as negatively modulate neuronal nicotinic acetylcholine receptors (nAChRs) was evaluated. Since a7-nAChRs on cerebral perivascular sympathetic neurons mediate cerebral parasympathetic-nitrergic vasodilation, effects of physostigmine, neostigmine, and galantamine on a7-nAChR-mediated dilation in isolated porcine basilar arterial rings denuded of endothelium was examined using in vitro tissue bath technique. The results indicated that these ChEIs blocked vasodilation induced by choline (0.3 mmol/L), nicotine (0.1 mmol/L), and transmural nerve stimulation (TNS). The ChEI inhibition of dilation induced by TNS but not by choline or nicotine was prevented by atropine (0.1 lmol/L) pretreatment. Furthermore, using confocal microscopy, significant calcium influx induced by choline and nicotine in cultured porcine superior cervical ganglion (SCG) cells was attenuated by ChEIs. In a7-nAChR-expressed Xenopus oocytes, nicotine-induced inward currents were attenuated by a-bungarotoxin and ChEIs. Moreover, ChEI inhibition of nicotine-and choline-induced dilation was prevented by pretreatment with mevastatin and lovastatin (10 lmol/L), which did not affect ChEI inhibition of TNS-induced relaxation. These findings suggest that ChEIs inhibit the a7-nAChRs located on postganglionic sympathetic nerve terminals of SCG origin, causing a decreased release of nitric oxide in the neighboring nitrergic nerves and cerebral vasodilation. Inhibition of a7-nAChRs leading to a potential cerebral hypoperfusion may contribute to the limitation of ChEIs and question the validity of using a ChEI alone in treating AD. The efficacy of ChEIs may be improved by concurrent use of statins.

The regulation of heme oxygenase (HO) activity and its dependence on iron was studied in bovine aortic endothelial cells (BAEC) subjected to hypoxia-reoxygenation (H/R). HO activity was induced by hypoxia (10 h) and continued to increase during the reoxygenation phase. HO-1 protein levels were strongly induced by hypoxia from undetectable levels and remained elevated at least 8 h postreoxygenation. Addition of the Fe(3+) chelator desferrioxamine mesylate (DFO) or the Fe(2+) chelator o-phenanthroline during hypoxia alone or during the entire H/R period inhibited the induction of HO activity and HO-1 protein levels. However, DFO had no effect and o-phenanthroline had a partial inhibitory effect on HO activity and protein levels when added only during reoxygenation. Loading of BAEC with Fe(3+) enhanced the activation of the HO-1 gene by H/R, whereas loading with L-aminolevulinic acid, which stimulates heme synthesis, had little effect. These results suggest that chelatable iron participates in regulating HO expression during hypoxia.