MicroRNA-361-5p slows down gliomas development through regulating UBR5 to elevate ATMIN protein expression

MicroRNA (miR)-361-5p has been studied to suppress gliomas development. Based on that, an insight into the regulatory mechanism of miR-361-5p in gliomas was supplemented from ubiquitin protein ligase E3 component N-recognin 5 (UBR5)-mediated ubiquitination of ataxia-telangiectasia mutated interactor (ATMIN). miR-361-5p, ATMIN, and UBR5 levels were clinically analyzed in gliomas tissues, which were further validated in gliomas cell lines. Loss/gain-of-function method was applied to determine the roles of miR-361-5p and UBR5 in gliomas, as to cell viability, migration, invasion, colony formation ability, and apoptosis in vitro and tumorigenesis in vivo. The relationship between miR-361-5p and UBR5 was verified and the interaction between UBR5 and ATMIN was explored. It was detected that reduced miR-361-5p and ATMIN and enhanced UBR5 levels showed in gliomas. Elevating miR-361-5p was repressive in gliomas progression. UBR5 was directly targeted by miR-361-5p. UBR5 can ubiquitinate ATMIN. miR-361-5p suppressed gliomas by regulating UBR5-mediated ubiquitination of ATMIN. Downregulating UBR5 impeded gliomas tumor growth in vivo. Upregulating miR-361-5p targets UBR5 to promote ATMIN protein expression, thus to recline the malignant phenotype of gliomas cells.Subject terms: Cell biology, Diseases     

Synthesis and biological activities of beta-alanyltyrosine derivative of 2',5'-oligoadenylate, and its use in radiobinding assay for 2',5-oligoadenylate

beta-Alanyltyrosine derivative of 2',5'-tetraadenylate 5'-triphosphate, pppA2'p5'A2'-p5'A2'p5'A-beta-Ala-Tyr was prepared by coupling of periodate-oxidized pppA2'p5'-A2'p5'A2'p5'A with beta-alanyltyrosine methyl ester, followed by reduction with sodium cyanoborohydride. Its stability to 2',5'-phosphodiesterase and phosphatase was investigated in mouse L cell extract. The 5'-triphosphate of the compound was cleaved gradually to form the 5'-dephosphorylated derivative, A2'p5'A2'p5'A2'p5'A-beta-Ala-Tyr, followed by slow degradation of the 2',5'-phosphodiester bond. On the other hand, pppA2'p5'A2'p5'A2'p5'A was hydrolyzed very quickly under the same conditions. The tetramer derivative bound tightly to the 2',5'-oligoadenylate-dependent endoribonuclease in rabbit reticulocyte lysate or mouse L cell extract and inhibited protein synthesis of mouse L cells more effectively than the unmodified 2',5'-tetraadenylate 5'-triphosphate. The corresponding trimer derivative had slightly weaker activities than the unmodified trimer for binding to the endoribonuclease and for inhibition of protein synthesis. The compound, pppA2'p5'A2'p5'-A2'p5'A-beta-Ala-Tyr, was iodinated easily at the tyrosine residue with 125I, giving a high-specific-radioactivity derivative which was used as a radio-labeled probe in a radiobinding assay for 2',5'-oligoadenylate.     

Overproduced and purified receptor binding protein pb5 of bacteriophage T5 binds to the T5 receptor protein FhuA

Abstract A promotor-less oad gene of bacteriophage T5, encoding the receptor binding protein pb5, was cloned into pT7-3 under the control of phage T7 promoter Φ10. Induction with IPTG resulted in enhanced production of pb5. Upon fractionation of the producing cells, most of the overproduced pb5 was found in the membrane fraction, which was most likely due to aggregation of the protein. The minor, soluble fraction of pb5 specifically inhibited adsorption of T5 to its FhuA receptor protein. Inhibition was also seen with trace amounts of pb5, and binding of pb5 to FhuA appeared to be almost irreversible. Purification of pb5 from the cytosolic fraction was performed by FPLC using a MonoQ column. pb5, which did not bind to the matrix of the column, was obtained in almost pure form. The purified protein also inhibited T5 adsorption.     

Inhibition of Coprinus cinereus by 5-fluoroindole

5-Fluoroindole (5FI) was more inhibitory than 5-fluorotryptophan (5FT) to Coprinus cinereus. A mutant blocked in the conversion of indole to tryptophan, but not one blocked earlier in the tryptophan pathway, was resistant to 5FI. This is consistent with the hypothesis that 5FI was converted in vivo to 5FT which inhibited growth.     

Biosynthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid from 5-hydroperoxyeicosatetraenoic acid in the murine macrophage

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a metabolite of arachidonic acid shown to possess important biological activities within different cell types. In the neutrophil, a specific NADP(+)-dependent dehydrogenase utilizes 5-lipoxygenase-derived 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) as the required substrate. In the present study, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HpETE), rather than 5-HETE, was found to be the biosynthetic precursor of 5-oxo-ETE in the murine macrophage. The macrophage was not able to convert 5-HETE into 5-oxo-ETE even when preincubated with phorbol ester or with other lipid hydroperoxides. The factor responsible for the conversion of 5-HpETE into 5-oxo-ETE was found predominantly in the cytosolic fraction of the macrophage, with an approximate molecular weight of 50,000-60,000, as assessed by size exclusion chromatography. Formation of 5-oxo-ETE was rapid and the catalytic protein was found to have an apparent K(m) of 5.3 microM for the eicosanoid. Furthermore, the protein could efficiently utilize 5(R,S)-HpETE as substrate and was heat and protease labile. This novel pathway of 5-oxo-ETE biosynthesis in the murine macrophage was consistent with reduction of a 5-hydroperoxy group to an intermediate alkoxy radical that could be subsequently oxidized to the 5-oxo product. Such a mechanism would enable racemic 5-HpETE, derived from free radical oxidation of arachidonic acid, to be efficiently converted into this potent chemotactic eicosanoid.     

High lung PDE5: a strong basis for treating pulmonary hypertension with PDE5 inhibitors

[3H]Vardenafil (Levitra) or [3H]tadalafil (Cialis) binding was used to quantify PDE5 in rat lung and heart tissue. Each radioligand bound to purified recombinant phosphodiesterase-5 (PDE5) or to PDE5 in crude extracts with strong affinity, high specificity, slow dissociation, and good stoichiometry. PDE5, the only 3H inhibitor-binding protein detected in extracts, was 15 times higher in lung than in heart extracts, and the level measured by PDE5 catalytic activity agreed with that determined by 3H inhibitor binding. High level of PDE5 in lung approximated that in penile corpus cavernosum, the tissue targeted by PDE5 inhibitors. PDE5 was the predominant cGMP-PDE in lung, and on a molar basis was five times higher than cGMP-dependent protein kinase (PKG), which phosphorylates PDE5 in vivo. The PDE5 level was one-half that of PKG in heart. Thus, abundance of PDE5 in lung vascular smooth muscle provides a strong molecular basis for PDE5 inhibitor treatment of pulmonary hypertension.     

Sensitive radioimmuno assay for 2',5'-oligoadenylates using a novel 125I-labeled derivative of 2',5'-triadenylate 5'-triphosphate

A novel 125I-labeled derivative of 2',5'-triadenylate 5'-triphosphate, pppA2'p5'A2'p5'A, with high specific radioactivity was synthesized by coupling of periodate-oxidized pA2'p5'A2'p5'A with beta-alanyltyrosine methyl ester followed by 5'-triphosphorylation and iodination with 125I. Antisera toward 2',5'-oligoadenylate 5'-triphosphate were produced in rabbits by immunization with the conjugate of pppA2'p5'A2'p5'A2'p5'A with bovine serum albumin, and an antiserum with high specificity and high sensitivity for 2',5'-oligoadenylates was selected and tested extensively. Radioimmuno assaying of 2',5'-oligoadenylates was carried out by a competitive double antibody method in which the amount of the antibody bound to the 125I-labeled probe was measured after precipitation with goat anti-rabbit IgG. The concentration of pppA2'p5'A2'p5'A required for 50% inhibition of the binding between the antiserum and the probe was 0.6 nM. The cross reactivity of the antiserum with the 3',5'-triadenylate was more than 10,000 times weaker compared to in the case of 2',5'-oligoadenylates. Very low or no cross reaction was observed with ATP, AMP, and adenosine. The radioimmuno assay using the 125I-labeled compound and the antiserum allows the direct analysis of 2',5'-oligoadenylates in the range of 4 fmol to 1 pmol (0.04-10 nM in a 100 microliter sample). This assay was applied to the measurement of the activity of 2',5'-oligoadenylate synthetase in cells stimulated by interferon. The properties of the 125I-labeled derivative of pppA2'p5'A2'p5'A are described.     

Comparison of 5-HT4 and 5-HT7 receptor expression and function in the circular muscle of the human colon

Serotonin receptors are potential targets for treating functional bowel disorders. This study investigated the functional roles and expression of the 5-HT4 and the 5-HT7 receptor, which coexist in human colon circular smooth muscle. 5-HT3 receptor expression was also investigated. Part of the relaxant response to 5-HT was due to activation of 5-HT4 receptors as the apparent pKB value of the selective 5-HT4 antagonist, GR 113808, was 9.36. 5-HT4 mRNA levels were low in five tissues and undetectable in four others, but all responded to 5-HT with an EC50 value of 102.54+/-19.32 nM. The contribution of 5-HT7 receptors to the response was not readily demonstrated using the selective 5-HT7 antagonist, SB-269970, as its apparent pKB value of 7.19 (5-HT4 block with 1 microM GR 113808) was lower than the value obtained using the 5-HT7 guinea pig ileum assay (8.62). Nevertheless, the 5-HT7 receptor was expressed more consistently than the 5-HT4, but at similar levels. The 5-HT(3Ashort) and 5-HT(3B) subunits were co-expressed at similar levels, but the 5-HT(3Along) subunit was detected in only five of the nine samples tested. The findings show that 5-HT4-induced relaxation occurs at low to undetectable levels of tissue mRNA, as measured by qPCR. Although 5-HT7 receptor mRNA is detected at low, but consistent levels, the functional activity of this receptor is not readily identified given the currently available drugs.     

Metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one and 5 alpha-androstan-3 alpha,17 beta-diol in the rat.

Significant metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one was recorded in several tissues and organs from rats and humans. This bioconversion was further investigated in rat testis homogenates. 5 alpha-Androstane-3 beta,17 beta-diol was readily metabolized to 17 beta-hydroxy-5 alpha-androstan-3-one with NAD and/or NADP added as cofactors. When a NADPH generating system was included in the incubation, 5 alpha-androstane-3 beta,17 beta-diol was metabolized to 5 alpha-androstan-3 alpha,17 beta-diol. Only small amounts of 17 beta-hydroxy-5 alpha-androstan-3-one accumulated under the latter condition.     

Expression of CD5 regulates responsiveness to IL-1

The role of the CD5 surface molecule in T cell responsiveness to IL-1 was examined. A CD5-mutant Jurkat cell line was generated from a CD5+ parent cell line. This CD5- mutant subclone was infected with a defective retrovirus containing the CD5 cDNA and/or the neo gene encoding G418 resistance. The CD5+ wild type Jurkat produced IL-2 in response to anti-CD3 mAb, OKT3, cross-linked to a solid surface. IL-2 production was enhanced by co-culture with IL-1 or anti-CD5 Mab. Neither the CD5- mutant nor the CD5- G418-resistant infectant responded to anti-CD5 mAb or to IL-1. Responsiveness to IL-1 was restored by cell surface expression of CD5 in the CD5+ infectant. Both the CD5+ wild type Jurkat and the CD5+ infectant responded equivalently to purified IL-1, IL-1 alpha and rIL-1 beta. Optimal concentrations of IL-1 and anti-CD5 mAb had an additive effect on the enhancement of IL-2 production stimulated with cross-linked anti-CD3 mAb suggesting that IL-1 and CD5 act through distinct, complementary pathways to augment T cell activation. The correlation of CD5 expression and specific binding of rIL-1 beta was examined in these cell lines. Both the specific binding (at 4 degrees C) and subsequent internalization (at 37 degrees C) of 125I labeled rIL-1 beta was equivalent in the CD5+ infectant and the CD5+ wild type Jurkat cell, whereas specific binding of 125I-labeled rIL-1 beta was markedly decreased in the CD5-G418-resistant infectant. These observations strongly suggest that cell surface expression of CD5 regulates binding of and responsiveness to IL-1.     

Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro.

Two major stable oxidation products of 2'-deoxycytidine are 2'-deoxy-5-hydroxycytidine (5-OHdC) and 2'-deoxy-5-hydroxyuridine (5-OHdU). In order to study the in vitro incorporation of 5-OHdC and 5-OHdU into DNA by DNA polymerase, and to check the base pairing specificity of these modified bases, 5-OHdCTP and 5-OHdUTP were synthesized. Incorporation studies showed that 5-OHdCTP can replace dCTP, and to a much lesser extent dTTP, as a substrate for Escherichia coli DNA polymerase I Klenow fragment (exonuclease free). However, 5-OHdUTP can only be incorporated into DNA in place of dTTP. To study the specificity of nucleotide incorporation opposite 5-hydroxypyrimidines in template DNA, 18- and 45-member oligodeoxyribonucleotides, containing an internal 5-OHdC or 5-OHdU in two different sequence contexts, were used. Translesion synthesis past 5-OHdC and 5-OHdU in both oligonucleotides occurred, but pauses both opposite, and one nucleotide prior to, the modified base in the template were observed. The specificity of nucleotide incorporation opposite 5-OHdC and 5-OHdU in the template was sequence context dependent. In one sequence context, dG was the predominant nucleotide incorporated opposite 5-OHdC with dA incorporation also observed; in this sequence context, dA was the principal nucleotide incorporated opposite 5-OHdU. However in a second sequence context, dC was the predominant base incorporated opposite 5-OHdC. In that same sequence context, dC was also the predominant nucleotide incorporated opposite 5-OHdU. These data suggest that the 5-hydroxypyrimidines have the potential to be premutagenic lesions leading to C-->T transitions and C-->G transversions.     

Developmental changes in localization of the main ganglioside during sea urchin embryogenesis

Ganglioside M5 (NeuGcalpha2-6Glcbeta1-1'Cer), the main ganglioside in sea urchin and sand dollar eggs, exists mainly in the endoplasmic reticulum and yolk granules in unfertilized eggs. To study the localization of ganglioside M5 after fertilization, early embryos were stained with an anti-ganglioside M5 monoclonal antibody. Using immunofluorescent and immunoelectron microscopy, intense label was observed in the outer surface and cytoplasm of embryos. These results indicate that ganglioside M5 was secreted during embryogenesis and localized in the extracellular matrix (ECM). When living embryos were incubated in sea water containing 7-nitrobenz-2-oxa-1,3-diazole labeled-ganglioside M5 (NBD-M5), the ECM and plasma membrane were strongly stained. Since the localization of NBD-M5 in the ECM was similar to that of extracellular M5, NBD-M5 was likely to be useful to examine the fate of extracellular ganglioside M5. Interestingly, NBD-M5 was incorporated in subcortical vesicles during embryogenesis, suggesting that the extracellular ganglioside M5 is transported into the cytoplasm. When fertilized eggs were incubated with NBD-M5 and tetramethylrhodamine dextran (a marker dye for endocytotic vesicles), colocalization of the dyes was observed in the vesicles. Thus, it was concluded that NBD-M5 in the ECM and/or plasma membrane was internalized in the cells by endocytosis, suggesting that extracellular M5 is transported from the ECM to endocytotic vesicles.     

Serotonin Metabolism by Monoamine Oxidase in Rat Primary Astrocyte Cultures

The oxidative deamination of serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA) by rat primary astrocyte cultures was investigated in intact cells using HPLC. All detectable 5-HIAA accumulated in the extracellular medium, and its rate of production was proportional to the 5-HT concentration over the tested range of 5 x 10(-7) to 10(-4) M. At 5 x 10(-7) M 5-HT, intracellular 5-HT was detectable only in astrocytes treated with monoamine oxidase (MAO) inhibitors. These findings are consistent with the idea that 5-HT taken up into astrocytes is not stored for re-release, but is rapidly metabolized to 5-HIAA, which is then extruded from the cell. At 5 x 10(-7) M 5-HT, 5-HIAA formation in intact cells was blocked 63% by the selective high-affinity 5-HT uptake inhibitor fluoxetine. 5-HT oxidation to 5-HIAA is carried out principally by MAO-A, because clorgyline was more effective at inhibiting the production of 5-HIAA than was pargyline. Radioenzymatic determinations of MAO activity in cell homogenates supported these findings, because under these conditions clorgyline was 1,000-fold more effective than pargyline at inhibiting MAO activity toward 14C-labelled 5-HT. However, the relatively selective MAO-B substrate beta-phenylethylamine (PEA) was also oxidized, showing that these cultures also contained MAO-B activity; the Km values for MAO-A oxidation of 5-HT and MAO-B oxidation of PEA were 135 and 45 microM, and Vmax values were 88 and 91 nmol/mg of total cell protein/h, respectively. Higher concentrations of PEA (greater than 20 microM) were oxidized by both MAO-A and MAO-B isozymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Only one 3'-hydroxyl group of ppp5' A2'p5'A2'p5' A (2-5A) is required for activation of the 2-5A-dependent endonuclease

To investigate the relative importance of each of the ribose 3'-hydroxyl groups of 2-5A (ppp5' A2'p5'A2'-p5' A) in determining binding to and activation of the 2-5A-dependent endonuclease (RNase L), the 3'-hydroxyl functionality of each adenosine moiety of 2-5A trimer triphosphate was sequentially replaced by hydrogen. The analog in which the 5'-terminal adenosine was replaced by 3'-deoxyadenosine (viz. ppp5'(3'dA)-2'p5' A2'p5' A) was bound to RNase L as well as 2-5A itself and was only 3 times less potent than 2-5A as an activator of RNase L. On the other hand, when the second adenosine unit was replaced by 3'-deoxyadenosine (viz. ppp5' A2'p5'(3'dA)2'p5' A), binding to RNase L was decreased by a factor of eight relative to 2-5A trimer and, even more dramatically, there was a 500-1000-fold drop in ability to activate the 2-5A-dependent endonuclease. Finally, when the 3'-hydroxyl substituent was converted to hydrogen in the 2'-terminal residue of 2-5A, a significant increase in both binding and activation ability occurred. We conclude that only the 3'-hydroxyl group of the second (from the terminus) nucleotide residue of 2-5A is needed for effective activation of RNase L.     

The identification of (3R,4S)-5-fluoro-5-deoxy-D-ribulose-1-phosphate as an intermediate in fluorometabolite biosynthesis in Streptomyces cattleya

(3R,4S)-5-Fluoro-5-deoxy-D-ribulose-1-phosphate (5-FDRulP) has been identified as the third fluorinated intermediate on the biosynthetic pathway to fluoroacetate and 4-fluorothreonine in Streptomyces cattleya. 5-FDRulP is generated after formation of 5'-fluoro-5'-deoxyadenosine (5'-FDA) and then phosphorolysis of 5'-FDA to 5-fluoro-5-deoxy-D-ribose-1-phosphate (5-FDRP) by the action of a purine nucleoside phosphorylase. An isomerase mediates the conversion of 5-FDRP to 5-FDRulP. The identity of the (3R,4S) diastereoisomer of 5-FDRulP was established by comparative (19)F{(1)H} NMR studies whereby 5-FDRulP that accumulated in a cell free extract of S. cattleya, was treated with a phytase to generate the non-phosphorylated sugar, 5-fluoro-5-deoxy-D-ribulose (5-FDRul). This S. cattleya product was compared to the product of an in-vitro biotransformation where separately 5-fluoro-5-deoxy-D-ribose and 5-fluoro-5-deoxy-D-xylose were converted to 5-fluoro-5-deoxy-D-ribulose and 5-fluoro-5-deoxy-D-xylulose respectively by the action of glucose isomerase. It was demonstrated that 5-fluoro-5-deoxy-D-ribose gave the identical diastereoisomer to that observed from 5-FDRulP.     

Metabolism and binding in vitro of 5 alpha-androstane-3 beta, 17 beta-diol and of 5 alpha-androstane-3 alpha, 17 beta-diol in cell fractions of rat ventral prostate and liver.

Rat ventral prostate and liver were investigated for the binding in vitro to particulate fractions and for the metabolism of 5 alpha-androstane-3 beta, 17 beta-diol. Comparative investigations were carried out on the metabolism of 5 alpha-androstane-3 alpha, 17 beta-diol. Preparations of the liver were investigated in order to establish the organ specificity of the method. In the prostate, the bulk of the metabolites of 5 alpha-androstane-3 beta, 17 beta-diol was present as steroids of high polarity. Of the less polar metabolites, 17 beta-hydroxy-5 alpha-androstan-3-one, 3 beta-hydroxy-5 alpha-androstan, 17-one and 5 alpha-androstane-3 alpha, 17 beta-diol were detectable. The binding of a 5 alpha-androstane-3 beta, 17 beta-diol to mitochondria and microsomes was unspecific. In the liver, among the less polar metabolites, 3 beta-hydroxy-5 alpha-androstan-17-one was the main metabolite, and the binding was unspecific. The main metabolite in the prostate homogenate of 5 alpha-androstane-3 alpha, 17 beta-diol was 17 beta-hydroxy-5 alpha-androstan-3-one. The portion of highly polar steroids was very low. The portion of unmetabolized hormone was distributed almost equally among the different cell preparations except the nuclei, in which 17 beta-hydroxy-5 alpha-androstan-3-one was higher and 5 alpha-androstane-3 alpha, 17 beta-diol was lower than in the remaining cell fractions.     

Enzyme assay for 5alpha-reductase type 2 activity in the presence of 5alpha-reductase type 1 activity in rat testis

The relative abundance and physiological role of 5alpha-reductase (5alphaR) isoforms in rat testis, in particular 5alpha-reductase Type 2 (5alphaR2) are poorly understood. Investigation of 5alphaR2 activity using enzyme kinetic studies was hampered by the high concentrations of 5alpha-reductase Type 1 (5alphaR1) in rat testis. Therefore, an assay was developed which exploited the differences in pH optima of the two isoforms. The 5alphaR assays measured the conversion of 3[H]-testosterone to 5alpha-reduced metabolites (dihydrotestosterone+3alpha-Androstanediol) at pH 5.0 and 7.0. To compensate for the overlap of 5alphaR1 activity at pH 5.0, the amount of 5alphaR1 activity at pH 5.0 was determined by measuring recombinant rat 5alphaR1 expressed in COS-7 cells at pH 5.0 and 7.0. The amount of activity at pH 5.0 that was attributed to 5alphaR1 was determined to be 12.4+/-1.4% (mean+/-S.D., n=14). The 5alphaR2 assay was validated by determining recombinant rat 5alphaR2 activity in the presence of recombinant rat 5alphaR1 activity in COS cells. A 99.3+/-14.7% recovery of 5alphaR2 activity was obtained when comparing 5alphaR2 activity recovered versus activity added. 5alphaR1 and 5alphaR2 activities were then assayed in rat testis extracts from 30, 75 and 147 days. Both isoforms markedly declined (50-100-fold) over this age range, with 5alphaR1 as the predominant isoform. In conclusion, an enzymatic assay that detects 5alphaR2 activity in the presence of high concentrations of 5alphaR1 was developed and is applicable in the measurement of 5alphaR2 activity in rat testis.     

G蛋白偶联胆汁酸受体1促进胃癌细胞的增殖、迁移和侵袭的实验研究

目的:探讨G蛋白偶联胆汁酸受体1(G-protein coupled bile acid receptor 1,GPBAR1/TGR5)对胃癌细胞增殖、迁移和侵袭的影响。方法:免疫组织化学染色方法(Immunohistochemistry,IHC)检测胃癌及癌旁组织芯片中TGR5表达情况;qRT-PCR及Western blot检测胃癌细胞系中TGR5表达水平;小干扰RNA处理AGS、MKN-45胃癌细胞后构建TGR5敲减细胞系,慢病毒载体转染胃癌SGC-7901细胞构建TGR5过表达细胞系;CCK-8实验、平板克隆形成实验、裸鼠皮下移植瘤实验检测TGR5对细胞增殖的影响;流式细胞仪检测TGR5对细胞周期及凋亡的影响;Tanswell实验检测TGR5对胃癌细胞迁移及侵袭的影响;Western blot检测上皮间充质转化(Epithelial-mesenchymal transition,EMT)相关分子β-连环蛋白(β-catenin)、锌脂蛋白转录因子(Snail)、E盒结合锌指蛋白(Zinc finger E-box binding homeobox 1,ZEB)1在AGS、MKN-45及SGC-7901胃癌细胞中的表达。结果:TGR5在胃癌及癌旁组织中均有表达,胃癌组织TGR5高表达率(41.0%)显著高于癌旁组织(9.5%),伴肠化生癌旁组织TGR5高表达率(50%)显著高于不伴肠化生的癌旁组织(0%),胃癌组织TGR5表达与肿瘤大小相关。TGR5在正常人胃上皮永生化细胞株GES-1及各胃癌细胞系中均有表达。TGR5表达敲低的AGS和MKN-45细胞增殖能力减弱、凋亡率显著升高、侵袭和迁移能力显著降低。过表达TGR5的SGC-7901细胞增殖能力增强、克隆形成能力提高、凋亡率明显减低、侵袭和迁移能力显著升高。此外,TGR5过表达显著上调了间质细胞标志物β-catenin、Snail、ZEB1的表达水平。结论:TGR5能够增强胃癌细胞增殖及迁移能力,并抑制细胞凋亡。TGR5可能通过EMT途径介导胃癌细胞转移。     

Protein kinase A-regulated membrane trafficking of a green fluorescent protein-aquaporin 5 chimera in MDCK cells

The green fluorescent protein (GFP) of the jellyfish, Aeqorea victoria, was used as an autofluorescent tag to track the trafficking of aquaporin 5 (AQP5), an exocrine gland-type water channel. Two groups of chimeric proteins were constructed; one in which GFP was fused to the amino-terminus of AQP5 (GFP-AQP5) and the other, in which it was fused to the carboxyl terminus of it (AQP5-GFP). In each group, 2 chimeras were produced, a wild-type AQP5 with its normal sequence and a mutant AQP5 having a mutated amino acid at 259, i.e., GFP-AQP5-T259A and AQP5-GFP-T259A. They were used to transfect Madin-Darby canine kidney (MDCK) cells. The GFP-AQP5 chimera was localized in the intracellular vesicles, which trafficked to the plasma membrane in response to N(6), 2'-O-dibutyryladenosine 3', 5'-cyclic monophosphate (dbcAMP). Membrane trafficking was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquimolinesulfonamide (H-89) but not by palmitoyl-dl-carnitine chloride (PCC). In contrast, the AQP5-GFP chimera expressed in MDCK cells was localized constitutively on the plasma membrane. The cellular localization of the latter chimera was not affected by stimulation with dbcAMP in the presence or absence of H-89 or PCC. Replacement of Thr-259 with Ala-259 did not affect the dbcAMP-induced translocation of the chimeric protein, suggesting that phosphorylation of Thr-259 was not necessary for AQP5 trafficking under the present experimental conditions. Thus, the GFP-AQP5 chimera will be a useful tool to study AQP5 trafficking in vitro, whereas the constitutive membrane localization of the AQP5-GFP chimera suggests the importance of the carboxyl terminus of the AQP5 protein for its sorting, whether it is translocated to intracellular vesicles or to the plasma membrane.