miR-145通过靶向吞噬和细胞活力蛋白1抑制乳腺癌细胞侵袭

吞噬和细胞活力蛋白1（engulfment and cell motility protein 1,ELMO1）可以促进多种癌细胞的侵袭和转移,但ELMO1的表达是否受miRNA的调控鲜有研究。本研究旨在探讨miR-145与ELMO1表达的相关性,以及miR-145通过结合ELMO1的mRNA对乳腺癌侵袭的影响。通过TargetScan (http://www.targetscan.org/)靶基因预测软件预测与ELMO1的3′UTR结合的miR-145。荧光素酶结果证实两者互补结合。Transwell侵袭结果显示,miR-145组和siELMO1+miR-145组MDA-231乳腺癌细胞穿膜数较对照组分别降低40%（P＜0.05）和79%（P＜0.05）。siELMO1+miR-145组和siELMO1组细胞穿膜数则无显著差异（P>0.05）。结果提示,miR-145通过与ELMO1的mRNA结合抑制细胞侵袭。qRT-PCR显示,低侵袭的MCF-7乳腺癌细胞miR-145的表达量较高侵袭的MDA-435细胞高80%（P＜0.05）,较MDA-231乳腺癌细胞高75%（P＜0.05）,即miR-145与癌细胞侵袭能力呈负相关。Western印迹结果表明,miR-145组ELMO1表达量低于阴性对照组,miR-145 抑制组ELMO1表达量高于抑制剂NC组（P＜0.05）,证明miR-145抑制ELMO1的表达。qRT-PCR显示,过表达miR-145后ELMO1 mRNA含量与对照组无显著差异（P>0.05）。结果提示,miR-145对ELMO1的调控作用通过抑制其翻译实现。F-肌动蛋白聚合实验表明,miR-145组和阴性对照组于20 s和60 s时F-肌动蛋白聚合结果存在明显区别（P＜0.05）。Western 印迹结果表明,miR-145组活化的Rac1表达量较阴性对照组降低60%（P<0.05）,抑制剂NC组活化的Rac1较miR-145 抑制组降低55%（P<0.05）;miR-145组磷酸化的整合素β1较对照组于15 min时降低42%（P<0.05）,于30 min时降低31%（P<0.05）。由此得出的miR-145过表达显著促进乳腺癌细胞F-肌动蛋白聚合、Rac1活化和整合素β1磷酸化结论。综上所述,miR-145通过靶向ELMO1的 mRNA抑制ELMO1翻译,从而抑制乳腺癌的侵袭。     

长非编码RNA PVT1与消化系统肿瘤

长非编码RNA（long non-coding RNAs, lncRNAs）在肿瘤发生、发展进程中承担重要角色,是近年来的研究热点之一。大量研究表明,浆细胞瘤变异易位基因1（plasmacytoma variant translocation 1, PVT1）可通过多种分子机制参与调控消化系统肿瘤的增殖凋亡、迁移侵袭、细胞自噬、血管生成、多药耐药及肿瘤代谢等过程,从而发挥致癌作用。本文主要就PVT1在消化系统肿瘤中的表达水平变化,及其与临床病理特征和预后的关系,以及PVT1对消化系统肿瘤的致癌作用机制和多药耐药机制等研究进展作一综述。     

苯胺嘧啶衍生物X-9通过下调整合素β1表达抑制肝细胞癌迁移侵袭

整合素在许多肿瘤细胞中高表达,并且参与肿瘤细胞的侵袭转移。在肝细胞癌中,整合素β1被报导高表达,并促进肿瘤细胞的侵袭。目前,对于整合素的表达调控癌细胞机制以及干预其表达进而抑制肿瘤细胞转移的研究较少。本研究探讨利用小分子化合物抑制整合素表达来抑制肿瘤细胞迁移和侵袭的可能。首先,对临床肝癌细胞患者癌组织和癌旁组织中的整合素β1的表达进行检测,发现其在癌组织中的表达显著高于癌旁组织（P<0.05）。对TCGA肿瘤数据库的生物信息学分析结果同样显示,整合素β1的高表达与肝癌的分期（P=0.019）和预后（P=0.013）相关。通过筛选发现,苯胺嘧啶衍生物X09可以抑制肝癌细胞中整合素β1的mRNA和蛋白质的表达（P<0.01）。细胞划痕愈合实验和细胞穿孔实验结果显示,苯胺嘧啶衍生物X-9能够抑制肝癌细胞的迁移和侵袭（P<0.01）。进一步的研究证实,在肝癌细胞中外源表达整合素β1可以逆转X-9对肝癌细胞迁移和侵袭的抑制;而在敲低整合素β1的细胞中,X-9对细胞的迁移和侵袭的抑制被消除。因此,鉴定出苯胺嘧啶衍生物X-9可以通过下调整合素β1表达,进而抑制肝癌细胞的迁移和侵袭。     

Isolation of tetraploid clones with high efficiency from diploid 3Y1 rat fibroblasts treated with sodium butyrate

Summary Sodium butyrate causes proliferation arrest with a G2 (4C) DNA content and induces formation of tetraploid cells upon removal of the inhibitor, in rat 3Y1 diploid fibroblasts. We isolated tetraploid clones from the butyrate-treated 3Y1 cells with high efficiency; among 21 clones randomly isolated, 5 were pure diploid, 7 were mainly tetraploid with a small contaminating diploid population, and 7 were pure tetraploid. Among the pure tetraploid clones, two showed doubled chromosome numbers with slightly broader distributions than that seen in parental 3Y1 cells. Butyrate further induced polyploid formation in the tetraploid cells thus produced, but octaploid cells that resulted could not be maintained for prolongeed, cultivation. We found no difference between the tetraploid and the (parental and parallel isolated) diploid clones in terms of colony-forming ability, proliferation rate, and sensitivity to density-dependent inhibition of proliferation. These results suggest that doubling of chromosome number by itself does not cause a change in proliferation property. The tetraploid clones had lower average saturation densities possibly due to enlargement of cell size represented by higher cellular protein content.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Effects of glucagon on the redox states of cytochromes in mitochondria in situ in perfused rat liver

The effects of glucagon on the respiratory function of mitochondria in situ were investigated in isolated perfused rat liver. Glucagon at the concentrations higher than 20 pM and cyclic AMP (75 microM) stimulated hepatic respiration, and shifted the redox state of pyridine nucleotide (NADH/NAD) in mitochondria in situ to a more reduced state as judged by organ fluorometry and beta-hydroxybutyrate/acetoacetate ratio. The organ spectrophotometric study revealed that glucagon and cyclic AMP induced the reduction of redox states of cytochromes a(a3), b and c+c1. Atractyloside (4 micrograms/ml) abolished the effects of glucagon on these parameters and gluconeogenesis from lactate. These observations suggest that glucagon increases the availability of substrates for mitochondrial respiration, and this alteration in mitochondrial function is crucial in enhancing gluconeogenesis.     

Electrolysis-mediated irreversible inactivation of lipoxygenase directed toward electroaffinity labelling

Irreversible inhibition of soybean lipoxygenase-1 (SL-1) was accomplished via a controlled potential oxidative electrolysis of 1,5-dihydroxynaphthalene (1,5-DHN) at +0.8 V vs SCE. The inactivation of SL-1 with this known inhibitor was greatly enhanced under these electrolytic conditions to which the enzyme itself was stable. Electrolyses were run at 0 degree C in a 0.05 M phosphate buffer, pH 7.0, using graphite cloth electrodes. The rate of inactivation was observed to be limited by and dependent on the anodic oxidation of 1,5-DHN. The non-oxidizable (at this potential) inhibitor indomethacin was shown to protect the enzyme from irreversible inactivation, however, an external nucleophile (2-mercaptoethanol) had little effect. These initial studies support the capability of such electrochemical methods for the site-specific covalent modification (affinity labelling) of lipoxygenase, and perhaps other enzymes.     

Highly potent and specific inhibitors of human renin

We designed aldehyde derivatives of small peptides representing the C-terminal portion of angiotensin I sequence as an inhibitor of human renin. Among compounds that we synthesized, benzyloxycarbonyl (Z)-Phe-His-Leucinal (compound V), Z-Pro-Phe-His-Leucinal (Compound IV) and Z-[3-(1'-naphthyl)Ala]-His-Leucinal (compound VII) markedly inhibited human renin (IC50, 7.5 X 10(-7), 3.2 X 10(-7) and 8.0 X 10(-8) mol/l, respectively). Compound VII was shown to be noncompetitive (Ki = 2.4 X 10(-7) mol/l). It did not inhibit either cathepsin D or pepsin. Compound V had slight or no inhibitory effect at the concentration of 10(-5) mol/l on six animal renins except for monkey and rabbit renins. Results obtained show that these aldehyde compounds are highly selective and species specific inhibitors for human and monkey renins.     

Receptor-mediated regulation of calcium mobilization and cyclic GMP synthesis in neuroblastoma cells

In neuroblastoma N1E 115 cells, carbachol, histamine and PGE1 elevated cyclic GMP content and, induced the efflux of preloaded 45Ca2+, the release of membrane-bound Ca2+ measured by fluorescent CTC, and the increase in [Ca2+]i as measured by Quin 2 fluorescence. The time course of the responses, the absolute requirement of extracellular Ca2+, the inhibition by receptor blockers, and the concentration dependency on histamine were all similar between these responses. The observation indicates that the mobilization of Ca2+, especially the increase of [Ca2+]i, may be intimately linked to the synthesis of cyclic GMP in the cells.     

In vitro alkylation of calf thymus DNA by acrylonitrile. Isolation of cyanoethyl-adducts of guanine and thymine and carboxyethyl-adducts of adenine and cytosine

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 5.0 and/or pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), N6-methyl-2'-deoxyadenosine (N6-Me-dAdo) and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. Adducts were not detected after 4 h. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, 1-CE-N6-Me-dAdo and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. Evidence is presented which strongly suggests that N6-CE-dAdo was formed by Dimroth rearrangement of 1-CE-dAdo during the reaction between AN and dAdo. The carboxyethyl adducts resulted from initial cyanoethylation (by Michael addition) at a ring nitrogen adjacent to an exocyclic nitrogen atom followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is an autocatalyzed reaction resulting from the formation of a cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated were 1-CE-Ade (26%), N6-CE-Ade (8%), 3-CE-Cyt (1%), 7-CNE-Gua (26%), 7,9-bis-CNE-Gua (4%), imidazole ring-opened 7,9-bis-CNE-Gua (19%) and 3-CNE-Thy (16%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts. Three of the adducts (1-CE-Ade, N6-CE-Ade and 3-CE-Cyt) were identical to adducts previously reported by us to be formed following in vitro reaction of the carcinogen beta-propiolactone (BPL) and calf thymus DNA. The results demonstrate that AN can directly alkylate DNA in vitro at a physiological pH and temperature.