microRNA调控网络与肿瘤

microRNA(miRNA)是一类长度约为22nt的小分子非编码RNA,对基因表达进行转录后调控。基因调控网络由各种回路,如前馈和反馈回路组成。它通过将外界刺激整合成合适的输出信号,控制细胞从增殖到死亡几乎所有的生命活动。已知基因调控网络的失调与肿瘤的发生发展密切相关。新近的实验证据表明,miRNA广泛参与基因调控网络中的反馈和前馈回路。该文主要介绍miRNA在基因表达调控网络中的作用及其与肿瘤发生发展的关系。     

Receptor activity and conformational analysis of 5'-halogenated resiniferatoxin analogs as TRPV1 ligands

A series of 5′-halogenated resiniferatoxin analogs have been investigated in order to examine the effect of halogenation in the A-region on their binding and the functional pattern of agonism/antagonism for rat TRPV1 heterologously expressed in Chinese hamster ovary cells. Halogenation at the 5-position in the A-region of RTX and of 4-amino RTX shifted the agonism of parent compounds toward antagonism. The extent of antagonism was greater as the size of the halogen increased (I > Br > Cl > F) while the binding affinities were similar, as previously observed for our potent agonists. In this series, 5-bromo-4-amino RTX (39) showed very potent antagonism with K_i (ant) = 2.81 nM, which was thus 4.5-fold more potent than 5′-iodo RTX, previously reported as a potent TRPV1 antagonist. Molecular modeling analyses with selected agonists and the corresponding halogenated antagonists revealed a striking conformational difference. The 3-methoxy of the A-region in the agonists remained free to interact with the receptor whereas in the case of the antagonists, the compounds assumed a bent conformation, permitting the 3-methoxy to instead form an internal hydrogen bond with the C4-hydroxyl of the diterpene.     

Pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists

A series of 1-substituted 3-(t-butyl/trifluoromethyl)pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides were investigated for hTRPV1 antagonism. The structure activity relationship indicated that the 3-chlorophenyl group at the 1-position of pyrazole was the optimized hydrophobic group for antagonistic potency and the activity was stereospecific to the S-configuration, providing exceptionally potent antagonists 13S and 16S with K_i(CAP) = 0.1 nM. Particularly significant, 13S exhibited antagonism selective for capsaicin and NADA and not for low pH or elevated temperature. Both compounds also proved to be very potent antagonists for rTRPV1, blocking in vivo the hypothermic action of capsaicin, consistent with their in vitro mechanism. The docking study of compounds 13S and 16S in our hTRPV1 homology model indicated that the binding modes differed somewhat, with that of 13S more closely resembling that of GRT12360.     

Identification of potent, soluble, and orally active TRPV1 antagonists

Optimization of a water soluble, moderately potent lead series of isoxazole-3-carboxamides was conducted, affording a compound with the requisite balance of potency, solubility and physicochemical properties for in vivo use. Compound 8e was demonstrated to be efficacious in a rat model of inflammatory pain, following oral administration.     

2-Aryl substituted pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as highly potent TRPV1 antagonists

A series of 2-aryl pyridine C-region derivatives of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Multiple compounds showed highly potent TRPV1 antagonism toward capsaicin comparable to previous lead 7. Among them, compound 9 demonstrated anti-allodynia in a mouse neuropathic pain model and blocked capsaicin-induced hypothermia in a dose-dependent manner. Docking analysis of 9 with our hTRPV1 homology model provided insight into its specific binding mode.     

The biophysical and molecular basis of TRPV1 proton gating

The capsaicin receptor TRPV1, a member of the transient receptor potential family of non-selective cation channels is a polymodal nociceptor. Noxious thermal stimuli, protons, and the alkaloid irritant capsaicin open the channel. The mechanisms of heat and capsaicin activation have been linked to voltage-dependent gating in TRPV1. However, until now it was unclear whether proton activation or potentiation or both are linked to a similar voltage-dependent mechanism and which molecular determinants underlie the proton gating. Using the whole-cell patch-clamp technique, we show that protons activate and potentiate TRPV1 by shifting the voltage dependence of the activation curves towards more physiological membrane potentials. We further identified a key residue within the pore region of TRPV1, F660, to be critical for voltage-dependent proton activation and potentiation. We conclude that proton activation and potentiation of TRPV1 are both voltage dependent and that amino acid 660 is essential for proton-mediated gating of TRPV1.     

The Functional Regulation of TRPV1 and Its Role in Pain Sensitization

Transient receptor potential V1 (TRPV1) is specifically expressed in the nociceptive receptors and can detect a variety of noxious stimuli, thus potentiating pain sensitization. While peripheral delivery of capsaicin causes the desensitization of sensory neurons, thus alleviating pain. Therefore capsaicin is used in the clinical treatment of various types of pain; however, these treatments will bring many side effects, such as a strong burning pain in the early stages of treatment which hampers the further use of capsaicin. Thus, the studies of the functional regulation of TRPV1 are mainly focused on two aspects: to develop more potent analogues of capsaicin with less side effects; or to elucidate the mechanisms of TRPV1 in pain sensitivity, especially of that TRPV1 as a target of various protein kinases such as PKD1 and Cdk5 is involved pain hypersensitivity. Thus we would summarize the progress of these two aspects in this mini review. Special issue article in honor of Dr. Ji-Sheng Han.     

Discovery of potent, soluble and orally active TRPV1 antagonists. Structure-activity relationships of a series of isoxazoles

Systematic optimisation of a poorly soluble lead series of isoxazole-3-carboxamides was conducted. Substitution of the 4-position with specific polar functionality afforded the requisite balance of potency, solubility and physicochemical properties. Compound 21a was found to be efficacious in the rat Capsaicin Hargreaves assay following oral administration.     

Lipophilicity of capsaicinoids and capsinoids influences the multiple activation process of rat TRPV1

Analogs of capsaicin, such as capsaicinoids and capsinoids, activate a cation channel, transient receptor potential cation channel vanilloid subfamily 1 (TRPV1), and then increase the intracellular calcium concentration ([Ca2+]i). These compounds would be expected to activate TRPV1 via different mechanism(s), depending on their properties. We synthesized several capsaicinoids and capsinoids that have variable lengths of acyl moiety. The activities of these compounds towards TRPV1 heterologously expressed in HEK293 cells were determined by measuring [Ca2+]i. When an extracellular or intracellular Ca2+ source was removed, some agonists such as capsaicin could increase [Ca2+]i. However, a highly lipophilic capsaicinoid containing C18:0 and capsinoids containing C14:0, C18:0, or C18:1 (the latter was named olvanilate) could not elicit a large increase in [Ca2+]i in the absence of an extracellular or intracellular Ca2+ source. These results suggest that highly lipophilic compounds cause only a slight Ca2+ influx, via TRPV1 in the plasma membrane, and are not able to activate TRPV1 in the endoplasmic reticulum.     

Research on association between variants and haplotypes of TRPV1 and TRPA1 genes with growth traits in three cattle breeds

The objective of this study was to examine the association of transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) genes polymorphisms with growth traits in three Chinese cattle breeds (Jiaxian red cattle, Qinchuan cattle and Luxi cattle). Through experiments we identified three single nucleotide polymorphisms (SNPs) in these three cattle breeds TRPV1 and TRPA1 genes using PCR-SSCP, (forced) PCR-RFLP methods. Three of these polymorphisms are all synonymous mutation which includes (NW_003104493.1: 30327 C?>?T), (NW_003104493.1: 33394 A?>?G) and (NW_003104493.1: 38471?G?>?A) are in exons. The other three polymorphisms are located at 3'UTR. Furthermore, we evaluated the haplotype frequency and the statistical analyses indicated that these SNPs of TRPV1 and TRPA1 genes were associated with bovine body height, body length, waist angle width, hucklebone width, cross ministry height, chest qingwidth (p?<?0.05) and recommendation height, cannon circumference (p?<?0.01) of Qingchuan cattle; body length, waist angle width (p?<?0.05) of Jiaxian red cattle; body weight, Body length, cannon circumference, chest circumference (p?<?0.05) and body height (p?<?0.01) of Luxi cattle. Our result confirms the polymorphisms in the TRPV1 and TRPA1 genes are associated with growth traits that may be used for marker-assisted selection (MAS) in three beef breeds programs.     

Vanilloid Receptor-1 (TRPV1) Expression and Function in the Vasculature of the Rat

Transient receptor potential (TRP) cation channels are emerging in vascular biology. In particular, the expression of the capsaicin receptor (TRPV1) was reported in vascular smooth muscle cells. This study characterized the arteriolar TRPV1 function and expression in the rat. TRPV1 mRNA was expressed in various vascular beds. Six commercially available antibodies were tested for TRPV1 specificity. Two of them were specific (immunostaining was abolished by blocking peptides) for neuronal TRPV1 and one recognized vascular TRPV1. TRPV1 was expressed in blood vessels in the skeletal muscle, mesenteric and skin tissues, as well as in the aorta and carotid arteries. TRPV1 expression was found to be regulated at the level of individual blood vessels, where some vessels expressed, while others did not express TRPV1 in the same tissue sections. Capsaicin (a TRPV1 agonist) evoked constrictions in skeletal muscle arteries and in the carotid artery, but had no effect on the femoral and mesenteric arteries or the aorta. In blood vessels, TRPV1 expression was detected in most of the large arteries, but there were striking differences at level of the small arteries. TRPV1 activity was suppressed in some isolated arteries. This tightly regulated expression and function suggests a physiological role for vascular TRPV1.     

TRPV1 Agonist Monoacylglycerol Increases UCP1 Content in Brown Adipose Tissue and Suppresses Accumulation of Visceral Fat in Mice Fed a High-Fat and High-Sucrose Diet

The administration of such a transient receptor potential vanilloid 1 (TRPV1) agonist as capsaicin, which is a pungent ingredient of red pepper, promotes energy metabolism and suppresses visceral fat accumulation. We have recently identified monoacylglycerols (MGs) having an unsaturated long-chain fatty acid as the novel TRPV1 agonist in foods. We investigated in this present study the effects of dietary MGs on uncoupling protein 1 (UCP1) expression in interscapular brown adipose tissue (IBAT) and on fat accumulation in mice fed with a high-fat, high-sucrose diet. The MG30 diet that substituted 30% of all lipids for MGs (a mixture of 1-oleoylglycerol, 1-linoleoylglycerol and 1-linolenoylglycerol) significantly increased the UCP1 content of IBAT and decreased the weight of epididymal white adipose tissue, and the serum glucose, total cholesterol and free fatty acid levels. The diet containing only 1-oleoylglycerol as MG also increased UCP1 expression in IBAT. MGs that activated TRPV1 also therefore induced the expression of UCP 1 and prevented visceral fat accumulation as well as capsaicin.     

Regulation of the epithelial calcium channel TRPV6 by the serum and glucocorticoid-inducible kinase isoforms SGK1 and SGK3

Epithelial calcium (re)absorption is mediated by TRPV5 and TRPV6 channels. TRPV5 is modulated by the SGK1 kinase, a process requiring the PDZ-domain containing scaffold protein NHERF2. The present study explored whether TRPV6 is similarly regulated by SGKs and the scaffold proteins NHERF1/2. In Xenopus oocytes, SGKs activate TRPV6 by increasing its plasma membrane abundance. Deletion of the putative PDZ binding motif on TRPV6 did not abolish channel activation by SGKs. Furthermore, coexpression of neither NHERF1 nor NHERF2 affected TRPV6 or potentiated the SGKs stimulating effect. The present observations disclose a novel TRPV6 regulatory mechanism which presumably participates in calcium homeostasis.     

Molecular Determinants of Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Binding to Transient Receptor Potential V1 (TRPV1) Channels

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P₂ is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P₂ binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P₂ interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P₂ behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P₂ binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P₂ in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P₂ binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.     

The effect of T1 and T2 cytokines on the cytotoxic T cell response to mannan-MUC1

MUC1 is a mucin over-expressed in breast cancer and a proposed target for immunotherapy. By immunising mice with MUC1 conjugated to mannan (M-FP), CD8⁺ MHC-class-I restricted cytotoxic T lymphocytes (CTL), of high CTL precursor (CTLp) frequency (1/8000) and with significant tumour protection, can be induced. The effect of various cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-7, interferon γ (IFNγ), and granulocyte/macrophage-colony-stimulating factor (GM-CSF)] on the MUC1 CTL immune response was investigated (a) by measuring the frequencies of CTLp in mice immunised with vaccinia virus constructs containing recombinant cytokines and M-FP, or (b) by immunising cytokine- or cytokine-receptor-knockout (−/−) mice with M-FP. Vaccinia virus (VV) constructs containing recombinant cytokines were used either individually or in combination in vivo with M-FP immunisation. M-FP immunisations combined with VV-IL-2, VV-IL-7 and VV-GM-CSF, and combinations of VV-IFNγ + VV-IL-2, VV-IFNγ + VV-IL-4 or VV-GM-CSF + VV-IL-7 increased CTLp frequencies up to threefold (1/17 666: M-FP + VV-GM-CSF + VV-IL-7) compared to M-FP (1/77 500) alone. By contrast, M-FP combined with VV-IL-4 decreased the CTLp frequency threefold whereas VV-IL-6 and VV-IFNγ had no effect. Studies in cytokine- and cytokine-receptor-gene-knockout (−/−) mice demonstrated that mice that are IL-2 −/− and IL-7 receptor −/− produce the same CTLp response to M-FP as do control mice, whereas responses in the IL-6 −/−, IL-10 −/− and IFNγ−/− mice were marginally improved and responses to M-FP in IL-4 −/− and tumour necrosis factor receptor 2 −/− mice were weaker. In spite of the increase in CTLp frequency, this was not reflected in an in vivo tumour model. Tumour challenges using MUC1⁺ P815 cells, demonstrated that the addition of cytokines had little additive effect on the already effective tumour-regression capabilities of M-FP alone. Received: 24 September 1998 / Accepted: 21 September 1999     

基于TRPV1和P2X3交互作用的大鼠外周痛感觉调控机制

目的 观察大鼠外周TRPV1和P2X3的相互关系,以期部分阐明外周痛感觉调控机制。方法雄性SD大鼠随机分为空白对照组、TRPV1激动剂组、P2X3激动剂组、TRPV1激动剂+P2X3激动剂组、TPRV1激动剂+P2X3抑制剂组、P2X3激动剂+TRPV1抑制剂组。通过足底皮下注射TRPV1或P2X3激动剂和(或)抑制剂,分别观察20min内各组大鼠缩足次数、抬腿/舔足持续时间;采用免疫荧光法观察L4DRG水平TRPV1和P2X3阳性面积表达及共表达情况;采用免疫共沉淀法观察L4DRG水平TRPV1和P2X3的相互关系。结果 P2X3激动剂不能提升TRPV1激动剂诱发的痛行为学,P2X3抑制剂能减轻TRPV1激动剂诱发的痛行为学;TRPV1激动剂能增加P2X3激动剂诱发的痛行为学,TRPV1抑制剂不会减轻P2X3激动剂诱发的痛行为。P2X3激动剂能增加L4DRG水平TRPV1阳性面积表达,TRPV1激动剂能增加L4DRG水平P2X3阳性面积表达;TRPV1和P2X3在DRG水平有共表达且存在共沉淀现象。结论 外周神经元水平,TRPV1和P2X3之间存在一定的相互作用。两者可以相互促进对方的表达。当其中一方受到抑制时,另一方的功能也会相应的降低。