The role of microRNAs in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary hepatic malignancy with poor prognosis. Despite improvements in its diagnosis and therapy, the prognosis for ICC patients remains poor. An improved understanding of ICC pathogenesis and consequential identification of novel therapeutic targets would improve the prognosis of ICC patients. MicroRNAs (miRNAs) are a class of highly conserved, endogenous, small non‐coding RNA molecules of 18–23 nucleotides in length, which regulate gene expression through complementary base‐pairing with target messenger RNAs and subsequent gene silencing. Several studies have shown deregulated expression of miRNAs in ICC cell lines and tissues, in which these miRNAs play important roles in ICC apoptosis, cell proliferation, invasion, migration and metastasis. In this review, we illustrate the potential role of miRNA in the pathogenesis of ICC and explore the possibilities of using miRNAs as prognostic and diagnostic markers, as well as therapeutic targets in ICC.     

人GLP-1R基因转染BHK细胞及重组细胞的应用

为了构建表达人胰高血糖素样肽-1受体(GLP-1R)基因的BHK细胞株,并利用该重组细胞对GLP-1等相关肽进行活性测定,首先通过酶切、连接方式将人GLP-1R基因克隆至真核表达载体pCDNA3.(1 )中,然后用脂质体转染法将重组质粒转染至BHK-21细胞,转染后的细胞经G418加压筛选、细胞有限稀释等方法获得克隆细胞株。经过该细胞株RT-PCR验证,结果证实目的基因已整合至BHK-21细胞基因组中,并获得成功转录和表达。活性检测实验表明该重组细胞株经过GLP-1的刺激后,其细胞中的cAMP含量得到明显提升。该细胞株的构建为GLP-1及相关肽的活性测定奠定了基础。     

Regulation of adipocyte formation by GLP-1/GLP-1R signaling

Increased nutrient intake leads to excessive adipose tissue accumulation, obesity, and the development of associated metabolic disorders. How the intestine signals to adipose tissue to adapt to increased nutrient intake, however, is still not completely understood. We show here, that the gut peptide GLP-1 or its long-lasting analog liraglutide, function as intestinally derived signals to induce adipocyte formation, both in vitro and in vivo. GLP-1 and liraglutide activate the GLP-1R, thereby promoting pre-adipocyte proliferation and inhibition of apoptosis. This is achieved at least partly through activation of ERK, PKC, and AKT signaling pathways. In contrast, loss of GLP-1R expression causes reduction in adipogenesis, through induction of apoptosis in pre-adipocytes, by inhibition of the above mentioned pathways. Because GLP-1 and liraglutide are used for the treatment of type 2 diabetes, these findings implicate GLP-1 as a regulator of adipogenesis, which could be an alternate pathway leading to improved lipid homeostasis and controlled downstream insulin signaling.     

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation

The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH(2) or GLP-1(7-36)-NH(2) binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca(2+) (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH(2)- and GLP-1(7-36)-NH(2)-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH(2)-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.     

GLP-1 regulates exercise endurance and skeletal muscle remodeling via GLP-1R/AMPK pathway

Exercise-induced physical endurance enhancement and skeletal muscle remodeling can prevent and delay the development of multiple diseases, especially metabolic syndrome. Herein, the study explored the association between glucagon-like peptide-1 (GLP-1) secretion and exercise, and its effect on skeletal muscle remodeling to enhance endurance capacity. We found both acute exercise and short-term endurance training significantly increased the secretion of GLP-1 in mice. Recombinant adeno-associated virus (AAV) encoding Gcg (proglucagon) was used to induce the overexpression of GLP-1 in skeletal muscle of mice. Overexpression of GLP-1 in skeletal muscle enhanced endurance capacity. Meanwhile, glycogen synthesis, glucose uptake, type I fibers proportion, and mitochondrial biogenesis were augmented in GLP-1-AAV skeletal muscle. Furthermore, the in vitro experiment showed that exendin-4 (a GLP-1 receptor agonist) treatment remarkably promoted glucose uptake, type I fibers formation, and mitochondrial respiration. Mechanistically, the knockdown of AMPK could reverse the effects imposed by GLP-1R activation in vitro. Taken together, these results verify that GLP-1 regulates skeletal muscle remodeling to enhance exercise endurance possibly via GLP-1R signaling-mediated phosphorylation of AMPK.     

大鼠脑内caveolin-1蛋白的表达及其在分辨学习中的作用

Caveolin-1（Cav—1）蛋白作为细胞质膜结构小窝（caveolae）的标志蛋白,在胆固醇运输、膜组装、信号转导和细胞转化过程中扮演重要的角色。为了探讨Cav-1蛋白在中枢神经系统可塑性及学习记忆中的作用,本文以Sprague—Dawley大鼠为实验对象,利用蛋白质免疫印迹杂交方法观察了Car-1蛋白在不同年龄大鼠脑内表达的特征,并研究了Y-迷宫训练前后Cav-1蛋白表达的变化。结果表明：（1）大鼠不同脑区Cav-1蛋白表达的年龄特征不同。海马内的表达属青年鼠最高,其次是老年鼠和幼年鼠;皮层内的表达属幼年鼠最高,其次是老年鼠,青年鼠最低;小脑内的表达无明显年龄差异。（2）Y-迷宫训练引起青年鼠海马和前额叶皮层内Cav-1蛋白的表达显著增加。结果提示,Cav-1蛋白与动物脑发育和学习记忆有密切关系,可能参与中枢可塑性的调节。     

MTA1蛋白在食管鳞癌中的表达和临床意义

目的:探讨肿瘤转移相关基因1(MTA1)蛋白表达与食管鳞癌临床病理参数之间的关系.方法:应用免疫组化SP法和Western blotting方法检测50例食管鳞癌组织中MTA1蛋白的表达.结果:50例食管鳞癌组织中42例MTA1蛋白阳性表达,阳性率为84%.而癌旁组织中10例MTA1蛋白阳性表达,阳性率为20%,差异有显著性(P＜0.05);MTA1高表达与组织分化程度和淋巴结转移有关(P＜0.05),与年龄、性别、肿瘤大小无关(P＞0.05).结论:MTA1在食管鳞癌中高表达,与食管鳞癌的疾病进展密切相关,并有望成为基因治疗的新靶点.     

Molecular modeling of the three-dimensional structure of GLP-1R and its interactions with several agonists

Glucagon-like peptide-1 receptor (GLP-1R) is a promising molecular target for developing drugs treating type 2 diabetes. We have predicted the complete three-dimensional structure of GLP-1R and the binding modes of several GLP-1R agonists, including GLP-1, Boc5, and Cpd1, through a combination of homology modeling, molecular docking, and long-time molecular dynamics simulation on a lipid bilayer. Our model can reasonably interpret the results of a number of mutation experiments regarding GLP-1R as well as the successful modification to GLP-1 by Liraglutide. Our model is also validated by a recently revealed crystal structure of the extracellular domain of GLP-1R. An activation mechanism of GLP-1R agonists is proposed based on the principal component analysis and normal mode analysis on our predicted GLP-1R structure. Before the complete structure of GLP-1R is determined through experimental means, our model may serve as a valuable reference for characterizing the interactions between GLP-1R and its agonists. Figure Comparison of our predicted model of rGLP-1R (left) with the recently revealed crystal structure of hGLP-1R (right)     

下丘脑室旁核注射GLP-1 对糖尿病大鼠中枢GLP-1 受体表达

目的:探讨下丘脑室旁核(hypothalamic paraventricular nucleus,PVN)注射GLP-1(胰高血糖素样肽-1)对糖尿病大鼠胃排空的影响及机制。方法:30只Wistar大鼠随机分为正常对照组(NC组)、糖尿病组(DM组)和GLP-1干预组(GLP-1组),每组各10只。DM组和GLP-1组腹腔注射链脲佐菌素,三组大鼠均PVN区埋置套管,恢复7d,GLP-1组微量注射0.5μg/0.5μl的GLP-1,NC组和DM组大鼠PVN区微量注射等体积生理盐水。甲基纤维素-酚红灌胃法检测胃排空;半定量RT-PCR检测大鼠下丘脑GLP-1RmRNA的表达。结果:DM组胃排空率较NC组明显升高(P<0.05),GLP-1组胃排空明显低于DM组(P<0.05),GLP-1组和NC组差异无统计学意义(P>0.05)。GLP-1组下丘脑GLP-1RmRNA的表达明显高于DM组和NC组(P<0.05),并与胃排空率成负相关(P<0.05)。DM组和NC组差异无统计学意义(P>0.05)。结论:PVN区注射GLP-1可以抑制糖尿病大鼠早期胃排空加速,作用机制可能和促进下丘脑GLP-1受体表达有关。     

WWOX蛋白在胰腺癌组织中的表达及其临床意义

目的探讨WWOX蛋白在胰腺癌组织中的表达及其临床意义。方法应用免疫组织化学孓P法检测人胰腺癌组织芯片150芯（包括70例胰腺癌组织和5例胰腺炎）中WWOX蛋白的表达,采用HPIAS-1000图文报告管理系统对WWOX蛋白的表达进行定量分析,并用SPSS11．5软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK（q）检验。结果胰腺癌组织中WWOX蛋白呈低表达,胰腺炎组织中WWOX蛋白呈高表达。结论提示WWOX基因表达下调可能在胰腺癌的发生、发展中起了重要作用。     

Loureirin B activates GLP-1R and promotes insulin secretion in Ins-1 cells

Loureirin B (LB) is a natural product derived from Sanguis draconis, which has hypoglycaemic effects. In order to research the possible target of LB in the treatment of diabetes, molecular docking was used to simulate the interaction between LB and potential targets, and among them, glucagon-like peptide-1 receptor (GLP-1R) had the optimal results. Further, spectroscopy and surface plasmon resonance (SPR) experiments were applied to detect the interaction between LB and GLP-1R. Ultimately, after GLP-1R siRNA interfering the expression of GLP-1R in Ins-1 cell, the promoting insulin secretion of LB was weaken, which directly proved that GLP-1R plays an important role. These results show that LB promotes insulin secretion of Ins-1 cells through GLP-1R. Hence, the strategy of LB as a prodrug will provide a potential approach for non-peptide GLP-1R agonist.     

Identification of intrahepatic cholangiocarcinoma related genes by comparison with normal liver tissues using expressed sequence tags

Intrahepatic cholangiocarcinoma (ICC), a malignant tumor derived from the bile duct epithelium, is one of the leading causes of death from cancer, worldwide. However, the mechanisms related to it remain largely unknown. In this study, an analysis of the gene expression profiles for ICC was done using the frequency of the ESTs obtained from nine cDNA libraries that constructed from 4 ICC cell lines and 4 normal liver tissues. One hundred and thirty-seven genes were identified as being either up- or down-regulated in human ICC cells. Thirty genes were randomly selected to confirm their differential expression in 4 human ICC cell lines and 5 ICC tissues compared to normal liver tissues by semi-quantitative RT-PCR. Among these genes, ANXA1, ANXA2, AMBP, and SERPINC1 were further verified by immunohistochemical analyses. In conclusion, these identified genes represent potential biomarkers for ICC and represent potential targets for elucidating the molecular mechanisms that are associated with ICC.     

Expression and purification of exendin-4 dimer in Escherichia coli and its interaction with GLP-1 receptor in vitro

Exendin-4 is a 39 amino acid peptide isolated from the Gila monster salivary gland. It is 53% homologous to GLP-1 and exhibits similar glucoregulatory activities. In this study, exendin-4 dimer (D-Ex4) was constructed, cloned into plasmid pET32a(+) and expressed in E. coli BL21(DE3). The fusion protein with His-tag at the N-terminus was purified with a Ni-NTA-agarose column. After proteolytic cleavage, D-Ex4 peptide with high purity was obtained by HPLC. The results obtained by chemical cross-linking showed that D-Ex4 maintained affinity to GLP-1 receptor.     

Differential expression of an endogenous mannose-binding protein R1 during muscle development and regeneration delineating its role in myoblast fusion

The role of the endogenous brain carbohydrate-binding proteinR1 in muscle cell development and regeneration was analysedboth in vivo and in vitro. In vivo, R1 was developmentally regulated,with an embryonic 65 000 subunit and a neonatal 67 000 subunit,being replaced progressively by a 135 000 adult form. LectinR1 was intracellularly localized at birth and in the prenatalperiod. During development and at the time of myoblast fusion,the antigen was progressively found at the surface, where itremained at low levels in the adult. In vitro, in pure myoblastcultures, only the embryonic form was present. The ultrastructuralstudies indicated that the lectin could participate in the membranefusion process during myoblast fusion. The specific role inmyoblast fusion, derived from the ultrastructural localizationof R1, was evidenced by a strong inhibitory effect of anti-R1 Fab fragments (10–100 µg/ml), relative to controlFab fragments. In vivo, the embryonic subunit pattern and subcellulardistribution of R1 reappeared in muscle cells after lesion ofthe adult muscle. This suggested that, as observed in vitro,R1 participated in vivo in the phenomenon of myoblast fusion.Similar modifications in subunit expression were observed inmuscles after denemation (the embryonic form of lectin R1 reappearingafter lesion), suggesting that R1 could be involved in the processof neuromuscular junction formation. Thus, it is proposed thatthe carbohydrate-binding protein R1 is an important recognitionmolecule for the formation of myotubes. Its potential involvementin a recognition process between axons and muscle cells duringneuromuscular junction formation is discussed. culture development fusion N-glycan glycoprotein lectin mannose myoblast     

Expression of the adenovirus E1B 175R protein and its association with membranes of Escherichia coli.

The E1B 175-amino-acid (175R) protein of adenovirus 2 is required for cellular transformation of primary cells and establishing cell morphology in lytically infected cells. To investigate the biochemical function of this protein, we constructed a bacterial expression vector (pKHB1-T) to produce the 175R protein in sufficient amounts for purification and biochemical analysis. On the basis of DNA sequencing, gel electrophoresis, and immunoblot analysis, the pKHB1-T-encoded 175R protein appears to be identical to that expressed transiently in mammalian or adenovirus-transformed cells. The bacterially produced viral protein was also found to be quite stable and without any modifications. Partial purification of the pKHB1-T-encoded protein revealed that the majority of its associates with the inner membrane of the bacterial cell. This, together with the possibility of the 175R protein containing an N-terminal amphipathic alpha-helix as a potential translocation signal, suggests that there may be a common mechanism of protein transport operating in both eucaryotic and procaryotic systems.     

Thymidine phosphorylase promotes angiogenesis and tumour growth in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer, and thymidine phosphorylase (TP) is a regulator of angiogenesis. To investigate the biological activities of TP in ICC, we established human cholangiocarcinoma RBE cell lines overexpressing TP or silencing TP. Overexpression of TP enhanced viability, suppressed apoptosis and increased tube formation in human umbilical vein endothelial cells, while downregulation of TP reversed these effects. Moreover, an orthotopic xenograft mouse model of ICC was built to further explore TP's function in ICC in vivo. Histological analysis using H&E, TUNEL and Ki67 staining showed that TP promoted tumour growth and inhibited cell apoptosis. Immunostaining for CD31 revealed an elevation in microvessel density in the presence of TP. Besides, upregulation of TP increased the expression of vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8 and tumour necrosis factor alpha. In contrast, TP knockdown inhibited tumour growth, suppressed microvessel formation and decreased the expression of angiogenesis-related proteins. Therefore, we suggest that TP promotes angiogenesis and tumour growth in ICC, which can be a potent therapeutic target for ICC treatment.