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BackgroundAccurate identification of protein complexes in protein-protein interaction (PPI) networks is crucial for understanding the principles of cellular organization. Most computational methods ignore the fact that proteins in a protein complex have a functional similarity and are co-localized and co-expressed at the same place and time, respectively. Meanwhile, the parameters of the current methods are specified by users, so these methods cannot effectively deal with different input PPI networks.ResultTo address these issues, this study proposes a new method called MP-AHSA to detect protein complexes with Multiple Properties (MP), and an Adaptation Harmony Search Algorithm is developed to optimize the parameters of the MP algorithm. First, a weighted PPI network is constructed using functional annotations, and multiple biological properties and the Markov cluster algorithm (MCL) are used to mine protein complex cores. Then, a fitness function is defined, and a protein complex forming strategy is designed to detect attachment proteins and form protein complexes. Next, a protein complex filtering strategy is formulated to filter out the protein complexes. Finally, an adaptation harmony search algorithm is developed to determine the MP algorithm’s parameters automatically.ConclusionsExperimental results show that the proposed MP-AHSA method outperforms 14 state-of-the-art methods for identifying protein complexes. Also, the functional enrichment analyses reveal that the protein complexes identified by the MP-AHSA algorithm have significant biological relevance.

Complement component 4 (C4) has critical immunological functions in vertebrates. In the current study, a C4 homolog (gcC4) was identified in grass carp (Ctenopharyngodon idella). The full-length 5458 bp gcC4 cDNA contained a 5148 bp open reading frame (ORF) encoding a protein of 1715 amino acids with a signal peptide and eight conservative domains. The gcC4 protein has a high level of identity with other fish C4 counterparts and is phylogenetically clustered with cyprinid fish C4. The gcC4 transcript shows wide tissue distribution and is inducible by Aeromonas hydrophila in vivo and in vitro. Furthermore, its expression also fluctuates upon lipopolysaccharide or flagellin stimulation in vitro. During infection, the gcC4 protein level decreases or increases to varying degrees, and the intrahepatic C4 expression location changes. With gcC4 overexpression, interleukin 1 beta, tumor necrosis factor alpha, and interferon transcripts are all upregulated by A. hydrophila infection. Meanwhile, overexpression of gcC4 reduces bacterial invasion or proliferation. Moreover, gcC4 may activate the NF-κB signaling pathway. These findings demonstrate the vital role of gcC4 in the innate immunity of grass carp.

Background:: Genome-Wide Association Study (GWAS) plays a very important role in identifying the causes of a disease. Because most of the existing methods for genetic-interaction detection in GWAS are designed for a single-correlation model, their performances vary considerably for different disease models. These methods usually have high computation cost and low accuracy. Method:: We present a new multi-objective heuristic optimization methodology named HSMMGKG for detecting genetic interactions. In HS-MMGKG, we use harmony search with five objective functions to improve the efficiency and accuracy. A new strategy based on p-value and MDR is adopted to generate more reasonable results. The Boolean representation in BOOST is modified to calculate the five functions rapidly. These strategies take less time complexity and have higher accuracy while detecting the potential models. Results:: We compared HS-MMGKG with CSE, MACOED and FHSA-SED using 26 simulated datasets. The experimental results demonstrate that our method outperforms others in accuracy and computation time. Our method has identified many two-locus SNP combinations that are associated with seven diseases in WTCCC dataset. Some of the SNPs have direct evidence in CTD database. The results may be helpful to further explain the pathogenesis. Conclusion:: It is anticipated that our proposed algorithm could be used in GWAS which is helpful in understanding disease mechanism, diagnosis and prognosis.

Ascl2 has been shown to be involved in tumorigenesis in colorectal cancer (CRC), although its epigenetic regulatory mechanism is largely unknown. Here, we found that methylation of the Ascl2 promoter (bp -1670 ∼ -1139) was significantly increased compared to the other regions of the Ascl2 locus in CRC cells and was associated with elevated Ascl2 mRNA expression. Furthermore, we found that promoter methylation was predictive of CRC patient survival after analyzing DNA methylation data, RNA-Seq data, and clinical data of 410 CRC patient samples from the MethHC database, the MEXPRESS database, and the Cbioportal website. Using the established TET methylcytosine dioxygenase 2 (TET2) knockdown and ectopic TET2 catalytic domain-expression cell models, we performed glucosylated hydroxymethyl-sensitive quatitative PCR (qPCR), real-time PCR, and Western blot assays to further confirm that hypermethylation of the Ascl2 promoter, and elevated Ascl2 expression in CRC cells was partly due to the decreased expression of TET2. Furthermore, BCLAF1 was identified as a TET2 interactor in CRC cells by LC-MS/MS, coimmunoprecipitation, immunofluorescence colocalization, and proximity ligation assays. Subsequently, we found the TET2-BCLAF1 complex bound to multiple elements around CCGG sites at the Ascl2 promoter and further restrained its hypermethylation by inducing its hydroxymethylation using chromatin immunoprecipitation-qPCR and glucosylated hydroxymethyl-qPCR assays. Finally, we demonstrate that TET2-modulated Ascl2-targeted stem gene expression in CRC cells was independent of Wnt signaling. Taken together, our data suggest an additional option for inhibiting Ascl2 expression in CRC cells through TET2-BCLAF1-mediated promoter methylation, Ascl2-dependent self-renewal of CRC progenitor cells, and TET2-BCLAF1-related CRC progression.

The Wnt signaling pathway controls stem cell identity in the intestinal epithelium and cancer stem cells (CSCs). The transcription factor Ascl2 (Wnt target gene) is fate decider of intestinal cryptic stem cells and colon cancer stem cells. It is unclear how Wnt signaling is translated into Ascl2 expression and keeping the self-renewal of CRC progenitor cells. We showed that the exogenous Ascl2 in colorectal cancer (CRC) cells activated the endogenous Ascl2 expression via a direct autoactivatory loop, including Ascl2 binding to its own promoter and further transcriptional activation. Higher Ascl2 expression in human CRC cancerous tissues led to greater enrichment in Ascl2 immunoprecipitated DNA within the Ascl2 promoter in the CRC cancerous sample than the peri-cancerous mucosa. Ascl2 binding to its own promoter and inducing further transcriptional activation of the Ascl2 gene was predominant in the CD133(+)CD44(+) CRC population. R-spondin1/Wnt activated Ascl2 expression dose-dependently in the CD133(+)CD44(+) CRC population, but not in the CD133(−)CD44(−) CRC population, which was caused by differences in Ascl2 autoregulation under R-spondin1/Wnt activation. R-spondin1/Wnt treatment in the CD133(+)CD44(+) or CRC CD133(−)CD44(−) populations exerted a different pattern of stemness maintenance, which was defined by alterations of the mRNA levels of stemness-associated genes, the protein expression levels (Bmi1, C-myc, Oct-4 and Nanog) and tumorsphere formation. The results indicated that Ascl2 autoregulation formed a transcriptional switch that was enhanced by Wnt signaling in the CD133(+)CD44(+) CRC population, thus conferring their self-renewal.

The attachment of cell-surface carbohydrates to proteins mediated by the amino acids serine or threonine (O-glycan) is involved in tumor metastasis; the roles of O-glycans vary depending on their structure, but the detailed mechanisms by which O-glycans trigger signaling to control tumor metastasis are largely unknown. In this study, we found that the reduced expression of core 3 synthase correlated with metastasis to lymph nodes and distant organs, resulting in poor prognosis for colorectal cancer (CRC) patients. Mechanically, we revealed that mucin-type core 3 O-glycan was synthesized at the membrane-tethered MUC1 N terminus because of core 3 synthase expression in colon cancer cells. This further inhibited the translocation of MUC1-C to the nucleus, initiated p53 gene transcription that was dependent on the inhibition of MUC1-C nucleus translocation, activated p53-mediated miR-200c expression and resulted in mesenchymal-epithelial transition (MET). Inhibition of MUC1 via small interfering RNA (siRNA) in re-expressed core 3 synthase colon cancer cells further inhibited MUC1-C nucleus translocation, increased p53 and miR-200c expression, and enhanced MET. However, inhibition of p53 via siRNA or miR-200c via miR-200c inhibitor in re-expressed core 3 synthase colon cancer cells promoted the epithelial-mesenchymal transition (EMT) in a reversible manner. Core 3 synthase mRNA levels and the p53 mRNA levels or miR-200c levels in the colon cancerous samples were positively correlated. Our findings suggest a novel mechanism linking mucin-type core 3 O-glycan to the EMT-MET plasticity of CRC cells via MUC1/p53/miR-200c-dependent signaling cascade and shed light on therapeutic strategies to treat this malignancy.

BackgroundProtein complex identification from protein-protein interaction (PPI) networks is crucial for understanding cellular organization principles and functional mechanisms. In recent decades, numerous computational methods have been proposed to identify protein complexes. However, most of the current state-of-the-art studies still have some challenges to resolve, including their high false-positives rates, incapability of identifying overlapping complexes, lack of consideration for the inherent organization within protein complexes, and absence of some biological attachment proteins.ResultsIn this paper, to overcome these limitations, we present a protein complex identification method based on an edge weight method and core-attachment structure (EWCA) which consists of a complex core and some sparse attachment proteins. First, we propose a new weighting method to assess the reliability of interactions. Second, we identify protein complex cores by using the structural similarity between a seed and its direct neighbors. Third, we introduce a new method to detect attachment proteins that is able to distinguish and identify peripheral proteins and overlapping proteins. Finally, we bind attachment proteins to their corresponding complex cores to form protein complexes and discard redundant protein complexes. The experimental results indicate that EWCA outperforms existing state-of-the-art methods in terms of both accuracy andp-value. Furthermore, EWCA could identify many more protein complexes with statistical significance. Additionally, EWCA could have better balance accuracy and efficiency than some state-of-the-art methods with high accuracy.ConclusionsIn summary, EWCA has better performance for protein complex identification by a comprehensive comparison with twelve algorithms in terms of different evaluation metrics. The datasets and software are freely available for academic research athttps://github.com/RongquanWang/EWCA.

Background & Aims Multidrug resistance-associated protein 2 (MRP2) excretes conjugated organic anions including bilirubin and bile acids. Malfunction of MRP2 leads to jaundice in patients. Studies in rodents indicate that Radixin plays a critical role in determining Mrp2 canalicular membrane expression. However, it is not known how human hepatic MRP2 expression is regulated in cholestasis. Methods We assessed liver MRP2 expression in patients with obstructive cholestasis caused by gallstone blockage of bile ducts, and investigated the regulatory mechanism in HepG2 cells. Results Western blot detected that liver MRP2 protein expression in obstructive cholestatic patients (n=30) was significantly reduced to 25% of the non-cholestatic controls (n=23). Immunoprecipitation identified Ezrin but not Radixin associating with MRP2 in human livers, and the increased amount of phospho-Ezrin Thr567 was positively correlated with the amount of co-precipitated MRP2 in cholestatic livers, whereas Ezrin and Radixin total protein levels were unchanged in cholestasis. Further detailed studies indicate that Ezrin Thr567 phosphorylation plays an important role in MRP2 internalization in HepG2 cells. Since increased expression of PKCα, δ and e were detected in these cholestatic livers, we further confirmed that these PKCs stimulated Ezrin phosphorylation and reduced MRP2 membrane expression in HepG2 cells. Finally, we identified GP78 as the key ubiquitin ligase E3 involved in MRP2 proteasome degradation. Conclusions Activation of liver PKCs during cholestasis leads to Ezrin Thr567 phosphorylation resulting in MRP2 internalization and degradation where ubiquitin ligase E3 GP78 is involved. This process provides a mechanistic explanation for jaundice seen in patients with obstructive cholestasis.

Background In recent decades, detecting protein complexes (PCs) from protein-protein interaction networks (PPINs) has been an active area of research. There are a large number of excellent graph clustering methods that work very well for identifying PCs. However, most of existing methods usually overlook the inherent core-attachment organization of PCs. Therefore, these methods have three major limitations we should concern. Firstly, many methods have ignored the importance of selecting seed, especially without considering the impact of overlapping nodes as seed nodes. Thus, there may be false predictions. Secondly, PCs are generally supposed to be dense subgraphs. However, the subgraphs with high local modularity structure usually correspond to PCs. Thirdly, a number of available methods lack handling noise mechanism, and miss some peripheral proteins. In summary, all these challenging issues are very important for predicting more biological overlapping PCs. Results In this paper, to overcome these weaknesses, we propose a clustering method by core-attachment and local modularity structure, named CALM, to detect overlapping PCs from weighted PPINs with noises. Firstly, we identify overlapping nodes and seed nodes. Secondly, for a node, we calculate the support function between a node and a cluster. In CALM, a cluster which initially consists of only a seed node, is extended by adding its direct neighboring nodes recursively according to the support function, until this cluster forms a locally optimal modularity subgraph. Thirdly, we repeat this process for the remaining seed nodes. Finally, merging and removing procedures are carried out to obtain final predicted clusters. The experimental results show that CALM outperforms other classical methods, and achieves ideal overall performance. Furthermore, CALM can match more complexes with a higher accuracy and provide a better one-to-one mapping with reference complexes in all test datasets. Additionally, CALM is robust against the high rate of noise PPIN. Conclusions By considering core-attachment and local modularity structure, CALM could detect PCs much more effectively than some representative methods. In short, CALM could potentially identify previous undiscovered overlapping PCs with various density and high modularity. Electronic supplementary material The online version of this article (10.1186/s12859-018-2309-9) contains supplementary material, which is available to authorized users.

// Yangyang Shang 1,* , Qiong Pan 1,* , Lei Chen 1,* , Jun Ye 1 , Xiaoli Zhong 1 , Xiaohuan Li 1 , Linkuan Meng 1 , Jin Guo 1 , Yin Tian 1 , Yonghong He 1 , Wensheng Chen 1 , Zhihong Peng 1 and Rongquan Wang 1 1 Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing, P. R. China * These authors have contributed equally to this study Correspondence to: Zhihong Peng , email: // Rongquan Wang, email: // Keywords : Achaete scute-like 2, CDX2, transcriptional regulation, colorectal carcinoma, differentiation Received : March 26, 2015 Accepted : August 13, 2015 Published : August 17, 2015 Abstract The role of Achaete scute-like 2 (Ascl2) in colorectal cancer (CRC) cell differentiation is unknown. LS174T, HT-29 and Caco-2 cells have high Ascl2 expression, while Lovo and SW480 cells have low Ascl2 expression. LS174T and HT-29 cells with Ascl2 knockdown were transfected with caudal type homeobox 2 (CDX2) promoter constructs and used for luciferase assays and chromatin immunoprecipitation (ChIP) assays. Ascl2 knockdown promoted differentiation of CRC cells into a goblet cell phenotype, as determined by increased expression of MUC2, TFF3, and CDX2. Ascl2 knockdown activated CDX2 expression through a transcriptional mechanism via direct binding of Ascl2 to the proximal E-box of the CDX2 promoter. Ascl2 over-expression in Lovo and SW480 cells inhibited a goblet cell phenotype, as determined by reduced CDX2 and MUC2 expression. Inverse correlations between Ascl2 and CDX2, and Ascl2 and MUC2 mRNA levels, as well as Ascl2 and CDX2 protein levels were observed in CRC cancerous samples. This study demonstrates CDX2 repression by Ascl2 and highlights a role for Ascl2 in CRC cell differentiation. These findings suggest that the Ascl2/CDX2 axis may serve as a potential therapeutic target in colorectal cancer.

Ascl2, a basic helix-loop-helix transcription factor, is a downstream target of WNT signaling that controls the fate of intestinal cryptic stem cells and colon cancer progenitor cells. However, its involvement in colon cancer and downstream molecular events is largely undefined; in particular, the mechanism by which Ascl2 regulates the plasticity of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) programs in colon cancer cells remains unknown. In this study, we systematically demonstrate that Ascl2 loss of function in colon cancer cells promotes MET by derepressing the expression of microRNA (miR)-200s (i.e. miR-200b, miR-200a, miR-429, miR-200c, and miR-141) and further activating their expression through a transcriptional mechanism that involves direct binding to the most proximal E-box (E-box2) in the miR-200b-a-429 promoter. Activation of miR-200s due to Ascl2 deficiency led to the inhibition of ZEB1/2 expression and the alteration of epithelial and mesenchymal features. Transfection of miR-200b, miR-200a, and miR-429 inhibitors into Ascl2-deficient colon cancer cells promoted the epithelial-mesenchymal transition in a reversible manner. Transfection of miR-200a or miR-429 inhibitors into Ascl2-deficient colon cancer cells increased cellular proliferation and migration. Ascl2 mRNA levels and the miR-200a, miR-200b, miR-200c, miR-141, or miR-429 levels in the colon cancerous samples were inversely correlated. These results provide the first evidence of a link between Ascl2 and miR-200s in the regulation of EMT-MET plasticity in colon cancer.

NFIL3 (nuclear factor interleukin 3-regulated) is an important bZIP transcription factor in the immune response and immune cells' development. Here, we identified the NFIL3 gene from grass carp (Ctenopharyngodon idella; gcNFIL3). The deduced amino acid sequence of gcNFIL3 is 468 residues with a typical bZIP domain. Phylogenetics demonstrated that gcNFIL3 clustered closely with NFIL3 of zebrafish. Real-time PCR revealed gcNFIL3 is constitutively expressed in all tissues examined. Its expression was significantly upregulated in head kidney and trunk kidney after stimulation by bacteria. Immunofluorescence microscopy revealed that gcNFIL3 is mainly expressed in the nucleus. Overexpression of gcNFIL3 reduces Aeromonas hydrophila invasion and proliferation. In CIK cells, gcNFIL3 could induce the activation of NF-kappa B and upregulates the expression of IL10 and IFN. These results indicated that gcNFIL3 has immunoregulatory properties and might play a role in the immune response of fish.

Nanog is an important embryonic stem cell (ESC) gene that does not function as a classical oncogene, but needs to cooperate with other molecules to potentiate tumorigenic activity. The question addressed by the present study was whether a miRNA link exists between Nanog and epithelial-mesenchymal transition (EMT)-mesenchymal-epithelial transition (MET) plasticity. Here, we found that Nanog mRNA expression level was inversely correlated with miR-200c and miR-200b expression levels in colon cancer cell lines and human colorectal cancer tissues. Forced Nanog expression in low-Nanog colon cancer cells inhibited miR-200c and miR-200b expression, and interfered Nanog expression in high-Nanog colon cancer cells promoted miR-200c and miR-200b expression. Furthermore, we confirmed that Nanog directly repressed transcription of the miR-200c and miR-200b genes, and miR-200c and miR-200b mediated Nanog-induced EMT occurrence. Luciferase and ChIP assays determined that Nanog bound directly to the potential Nanog binding sites in the miR-200c and miR-200b promoters and repressed their transcription. In conclusion, our findings suggest that Nanog modulates EMT-MET plasticity by regulating miR-200 clusters via a direct transcriptional mechanism, and the Nanog-miR-200 axis may be a good therapeutic target for CRC control.

We characterized and identified the cDNA sequence of Toll-like receptor 20.2 in Ctenopharyngodon idella (gctlr20.2); it consisted of 3197 bp, with an open reading frame of 2835 bp that encoded a 944 amino acid polypeptide. Relatively, high expression levels of gctlr20.2 were observed in the spleen, head kidney, liver and brain tissues, with lower expression levels in the trunk kidney, intestine and heart tissues. In vivo and in vitro, after being challenged with Aeromonas hydrophila or grass carp reovirus (GCRV), gctlr20.2 expression was induced in C. idella kidney cells stimulated with lipopolysaccharide, flagellin or polyinosinic-polycytidylic acid. Overexpression of gctlr20.2 increased the expression of il1β, il8 and tnf-α, but not ifn, and also increased the activity of the nf-κB signal pathway. Silencing, via siRNA-tlr20.2, inhibited gctlr20.2 transcription by 65.7% and down-regulated the expression of inflammatory cytokine genes, but not tnf-α. This study increases understanding of the immune system in C. idella.

Chronic gastritis associated with Helicobacter pylori is a leading cause of gastric intestinal metaplasia (IM), which arises from abnormal cell differentiation of the epithelium in the gastric mucosa. However, the mechanisms involved in H. pylori-mediated IM remain elusive. The aim of our study was to explore the effects and the underlying mechanisms of H. pylori on the abnormal expression of Hath1 and Sox2 and to reveal its relationship to the development of gastric IM. We found that Hath1 and Sox2 were overexpressed in gastric IM tissue. Hath1 expression was up-regulated, whereas Sox2 expression, which was independent of the CagA virulence factor, was down-regulated in gastric epithelial cells and coincided with increased IL-6 and IL-8 levels in the culture media. Stimulation with H. pylori-related cytokine IL-8, but not IL-6 or IL-1β, was induced by Hath1 expression in the gastric epithelial cells. Although IL-8 and IL-6 levels correlated with STAT3 (signal transducer and activator of transcription) phosphorylation before and after H. pylori eradication in the gastric mucosa, only the blocking of IL-8-induced STAT3 activation using AG490 or STAT3-targeting RNA interference altered Hath1 expression. Additionally, we found that H. pylori down-regulated Hes1, which is a direct downstream target gene of Notch signaling and a repressor of Hath1 expression. These findings suggest that H. pylori induced inflammation up-regulate Hath1 expression via interleukin-8/STAT3 (IL-8) phosphorylation while suppressing Hes1, which provides a novel molecular connection between a H. pylori infection and intestinal metaplasia.

Aim To investigate the antitumor activities and safety of Ad-KDRscFv, Ad-sTRAIL (114–281) and Ad-KDRscFv:sTRAIL in vivo and in vitro. Methods Recombinant replication-defective adenovirus vectors encoding either the extracellular domain (114–281 aa) of TRAIL, the KDRscFv (single chain antibody (scFv) against human vascular endothelial growth factor (VEGF) receptor KDR) or the fusion gene of KDRscFv:sTRAIL were constructed and transfected into HEK 293 cells for virus packaging. The recombinant virus particles were then infected human tumor cell lines of liver cancer (HepG2), gastric cancer (SGC-7901), colorectal cancer (SW480) and normal human liver cell line (LO2) to investigate the antitumor activities. Nude mice of the subcutaneous tumor models were established with HepG2 cells and were randomly divided into different groups to investigate the therapeutic effect and safety of these adenovirus particles on hepatocellular carcinoma. The expression of foreign proteins and the effect on microvascular number were also evaluated. Results All three adenovirus particles could induce apoptosis of cancer cells lines HepG2, SGC-7901 and SW480, but had no obvious lethal effect on LO2 cells. Ad-KDRscFv:sTRAIL showed the strongest tumoricidal effect. After intratumoral injection with these adenovirus particles on nude mice model, all the three adenoviruses could inhibit the tumor growth and angiogenesis, and the expression of foreign proteins (sTRAIL, KDRscFv and KDRscFv:sTRAIL fusion protein) was restricted to liver and tumor tissues. In coincidence with the result in vitro, Ad-KDRscFv:sTRAIL also had the strongest antitumor activity in vivo. No obvious pathological changes were detected in vivo. Conclusions Replication-defective recombinant adenovirus of Ad-KDRscFv, Ad-sTRAIL and Ad-KDRscFv:sTRAIL all had tumoricidal activities and Ad-KDRscFv:sTRAIL showed the strongest effect. All three adenoviruses had no obvious toxicity to normal cells and tissues in vitro and in vivo.

Multidrug resistance-associated protein 3 (MRP3, ABC subfamily C [ABCC]3) plays an important role in protecting hepatocytes and other tissues by excreting an array of toxic organic anion conjugates, including bile salts. MRP3/ABCC3 expression is increased in the liver of some cholestatic patients, but the molecular mechanism of this up-regulation remains elusive. In this report, we assessed liver MRP3/ABCC3 expression in patients (n = 22) with obstructive cholestasis caused by gallstone blockage of bile ducts and noncholestatic patient controls (n = 22). MRP3/ABCC3 messenger RNA (mRNA) and protein expression were significantly increased by 3.4- and 4.6-fold, respectively, in these cholestatic patients where elevated plasma tumor necrosis factor alpha (TNFα) (4.7-fold; P < 0.01) and hepatic specificity protein 1 transcription factor (SP1) and liver receptor homolog 1 expression (3.1- and 2.1-fold at mRNA level, 3.5- and 2.5-fold at protein level, respectively) were also observed. The induction of hepatic MRP3/ABCC3 mRNA expression is significantly positively correlated with the level of plasma TNFα in these patients. In HepG2 cells, TNFα treatment induced SP1 and MRP3/ABCC3 expression in a dose- and time-dependent manner, where increased phosphorylation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) was also detected. These inductions were significantly reduced in the presence of the JNK inhibitor, SP600125. TNFα treatment enhanced HepG2 cell nuclear extract-binding activity to the MRP3/ABCC3 promoter, but was abolished by SP600125, as demonstrated by electrophoretic mobility shift assay (EMSA). An increase in nuclear protein-binding activity to the MRP3/ABCC3 promoter, consisting primarily of SP1, was also observed in liver samples from cholestatic patients, as assessed by supershift EMSA assays. Conclusions: Our findings indicate that up-regulation of hepatic MRP3/ABCC3 expression in human obstructive cholestasis is likely triggered by TNFα, mediated by activation of JNK/SAPK and SP1. (HEPATOLOGY 2012)

The human cervical cancer oncogene HCCR-2 is overexpressed in various malignant tumors and cell lines, and might function as a negative regulator of the p53 tumor suppressor. Here, we used RNA interference strategies to evaluate the role of HCCR-2 in liver cancer, and to explore its potential therapeutic effect. Changes of HepG2 cells stably transfected by an HCCR-2 RNA interference vector were detected by real-time PCR, MTT staining, plate colony formation, flow cytometry, and cell migration experiments. Apoptosis-related protein Bcl-2 and Bax levels were measured by Western blot. Our results showed that of the three siRNA-expressing vectors, siRNA-H3 had a suppressive effect on the expression of HCCR-2 mRNA, interfering with proliferation and migration of HCCR-2. Moreover, the apoptotic rate also increased, and cells transfected by siRNA-H3 were blocked in the G0/G1 stage. Plate colony formation experiments demonstrated that the single cell clone formation capacity of HepG2-H3 cells was clearly lower than that of HepG2 and HepG2-N cells. Western blot results indicated that the expression of Bcl-2 was inhibited, and the expression of Bax was increased. In summary, RNAi targeting HCCR-2 could be an effective means for suppressing malignant features of hepatocellular carcinoma cells.

rMuc3 is a typical transmembrane mucin and contains a 174 amino acid domain called an SEA module in its C-terminal domain which is cleaved in eukaryotic cells. However, the mechanism by which the rMuc3 SEA module is proteolyzed and its biological significance has to be elucidated. In this study, we showed that the rMuc3 C-terminal domain was cleaved at LSKGSIVV motif within SEA module in prokaryotic cells, the time-dependence of the cleavage was found in the purified rMuc3 C-terminal domain carrying a mutated LSKASIVV motif expressed in bacteria. Thus, the cleavage of rMuc3 SEA module depended on autoproteolysis. The autoproteolysis of the SEA module of rMuc3 C-terminal domain played a critical role in the migration and invasion of the LoVo human colon cancer cells with rMuc3 C-terminal domain in vitro. The rMuc3 C-terminal domain induced a significant activation of HER/ErbB2 phosphorylated form (py1248) in LoVo cells. Inhibition of the phosphorylation by gefitinib (ZD1839) did attenuate migration and invasion of LoVo cells with rMuc3 C-terminal domain. Thus, rMuc3 C-terminal domain undergoes autoproteolysis at its SEA module, which maintains its availability for the potentiation of the signaling process that is modulated by HER/ErbB2 phosphorylation to promote the migration and invasion of LoVo cells.

A prominent feature of the rodent Muc3 SEA module is the precursor cleavage event that segregates the O-glycosylated N-terminal fragment and transmembrane domain into the noncovalently attached heterodimer. There are seven potential N-glycosylation sites that occur in a cluster in the SEA module of Muc3. However, it is unknown if these sites are modified or what the function of these N-glycans may be in the SEA module. Our data show that the proteolytic cleavage of the rodent Muc3 SEA module was partially prevented by treatment with tunicamycin, an inhibitor of N-glycosylation. Each single mutant of the seven N-glycosylation sites (N1A, N2A, N3A, N4A, N5A, N6A, and N7A) and multiple mutants, including double (N34A) and triple (N345A) mutants, and mutants with four (N3457A), five (N34567A), six (N134567A and N234567A), seven (N1234567A) mutations, confirmed that all seven of these potential sites are N-glycosylated simultaneously. The proteolytic cleavage of the SEA module was not affected when it lacked only one, two, or three N-glycans, but was partially inhibited when lacking four, five, and six N-glycans. In all, 2%, 48%, 85%, and 73% of the products from N3457A, N34567A, N134567A, and N234567A transfectants, respectively, remained uncleaved. The proteolytic cleavage was completely prevented in the N1234567A transfectant, which eliminated all seven N-glycans in the SEA module. The interaction of the heterodimer was independent of the N-glycans within the rodent Muc3 SEA module. Thus, the N-glycosylation pattern constituted a control point for the modulation of the proteolytic cleavage of the SEA module.

Multidrug resistance-associated protein 2 (MRP2) plays an important role in bile acid metabolism by transporting toxic organic anion conjugates, including conjugated bilirubin, glutathione, sulfate, and multifarious drugs. MRP2 expression is reduced in cholestatic patients and rodents. However, the molecular mechanism of MRP2 down-regulation remains elusive. In this report, we treated human hepatoma HepG2 cells with interleukin-18 (IL-18) and measured the expression of MRP2, nuclear factor kappa B (NF-κB), farnesoid X receptor (FXR), and the transcription factor Yin Yang 1 (YY1) by quantitative real-time quantitative polymerase chain reaction (PCR) and western blotting. We found that expression of MRP2 was repressed by IL-18 at both the mRNA and protein levels in a dose- and time-dependent manner. Furthermore, the activated NF-κB pathway increased YY1 and reduced FXR. These changes were all attenuated in HepG2 cells with knockdown of the NF-κB subunit, p65. The reduced expression of FXR and MRP2 in HepG2 cells that had been caused by IL-18 treatment was also attenuated by YY1 knockdown. We further observed significantly elevated IL-18, NF-κB, and YY1 expression and decreased FXR and MRP2 expression in bile duct-ligated Sprague Dawley rat livers. Chromatin immunoprecipitation assays also showed that FXR bound to the promoter region in MRP2 was less abundant in liver extracts from bile duct-ligated rats than sham-operated rats. Our findings indicate that IL-18 down-regulates MRP2 expression through the nuclear receptor FXR in HepG2 cells, and may be mediated by NF-κB and YY1.

Within the C-terminal domain of many secretory mucins is a ‘cystine knot’ (CK), which is needed for dimer formation in the endoplasmic reticulum. Previous studies indicate that in addition to an unpaired cysteine, the three intramolecular cystine bonds of the knot are important for stability of the dimers formed by rat intestinal mucin Muc2. The present study was undertaken to determine whether the two N-glycans N9 and N10, located near the first and second cysteines of the knot, also play a role in dimer formation. The C-terminal domain of rat Muc2 (RMC), a truncated RMC mutant containing the CK, and mutants lacking N9 and N10 sites, were expressed in COS-1 cells and the products monitored by radioactive [35S]Met/Cys metabolic pulse–chase and immunoprecipitation. Mutation of N9, but not N10, caused increased synthesis of dimers over a 2-h chase period. The N9 mutant remained associated with calreticulin for a prolonged period. About 34–38% of the total labelled products of RMC and its mutants was secreted into the media by 2 h, but the proportion in dimer form was dramatically reduced for the N9 mutant, suggesting lower dimer stability relative to RMC or its N10 mutant. We conclude that under normal conditions the presence of the N9 glycan functions to maintain a folding rate for mucin monomers that is sufficiently slow to allow structural maturation and stability of Muc2 dimers. To our knowledge this report is the first demonstration that a specific N-glycan plays a definitive role in mucin dimer formation.

In a previous study we showed, by transient expression studies in COS-1 cells, that the C-terminal domain of rat intestinal membrane mucin Muc3 was cleaved between glycine and serine within a GSIVV (one-letter) amino acid sequence during its residence in the endoplasmic reticulum. The extracellular domain fragment remained linked to the membrane-associated fragment by non-covalent interactions. The present study demonstrates that cleavage depends not only on the presence of the G/SIVV site (where G/S is the glycine↓serine cleavage site), but also on more distant C-terminal sequences in the SEA (sea-urchin sperm protein, enterokinase and agrin) module. Inhibition of N-glycosylation by tunicamycin treatment of transfected cells did not prevent re-association of fragments, although cleavage was partially impaired, as some of the non-glycosylated, non-cleaved products were seen to accumulate in cells. Membrane targeting of the Muc3 domain and its cleavage products occurred in transfected cells and was not impaired in mutants in which the cleavage site was mutated. Targeting was also not impaired for products devoid of N-linked oligosaccharides. Our studies thus indicate that (a) cleavage within the SEA module of rat Muc3 requires participation of peptide sequences located C-terminal of and distant from the cleavage site, (b) re-association of the fragments requires the SEA module, but is independent of N-linked oligosaccharides, and (c) membrane targeting of the mucin is independent of the SEA-module-cleavage reaction.

Aim To investigate the effects of the nitrous oxide (NO)-donor sodium nitroprusside (SNP) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric cancer cells. Methods The MTT assay and flow cytometry were used to detect cellular proliferation and markers of apoptosis, respectively. Expression levels of caspases-8, and 9 were determined by Western blot. Changes in Nitric Oxide Synthase (NOS) activity, NO production, and caspase activation were also evaluated. Results We found that TRAIL induced apoptosis and cell cycle arrest in human gastric cancer cell lines, and that this effect was mediated by NO production, and activation of both the extrinsic and intrinsic signaling pathways of apoptosis. In addition, we found that the NO-donor SNP sensitizes gastric cancer cells to TRAIL-mediated apoptosis. Treatment of cells with both TRAIL and SNP resulted in increased activation of caspase-8 and caspase-9 and NO release. Inhibition of caspase-8 blocked cell TRAIL-induced apoptosis, while a selective caspase-9 inhibitor was unable to prevent apoptosis induced by either TRAIL or TRAIL plus SNP. Inhibition of NOS could block the activation of caspase-9, but had no obvious effect on cell apoptosis. Conclusions SNP-sensitized gastric cancer cells to TRAIL-induced cytotoxicity by stimulating the release of NO, in turn facilitating the mitochondria-mediated signal transduction pathway. The engagement of the mitochondria signaling pathways along with the TRAIL death receptor signaling pathway synergistically increase levels of apoptosis in these cells.

We investigated whether a synthetic tetrameric branched peptide based on the conserved TFLK motif from mammary-associated serum amyloid A3 (M-SAA3) is more efficient than the monomeric peptide at up-regulating MUC3 expression and examined the possible mechanism(s) and biological significance of this process. We used standard solid-phase methods to synthesize a tetrameric branched peptide (sequence GWLTFLKAAG) containing a trilysine core, termed the TFLK-containing 10-mer BP. The aberrant expression of transcription factors was analyzed using a transcription factor protein/DNA array. MUC3 and relevant transcription factors were detected using real-time PCR and/or Western blots. The luciferase assay, EMSA, and ChIP assays were used to analyze the activity of the human MUC3 promoter. The bacterial adherence assay was used to evaluate the in vitro inhibition of enteropathogenic Escherichia coli or enterohemorrhage E. coli serotype O157:H7 (EHEC O157:H7) adherence to HT-29-Gal cells after treatment with the TFLK-containing 10-mer BP. In HT-29-Gal cells, the TFLK-containing 10-mer BP induced higher levels of MUC3 expression than the M-SAA3-derived N-terminal 10-mer monomeric peptide, and MUC3 expression was activated through transcriptional mechanisms, including the induction of multiple transcription factors and further binding with their cis-elements between nucleotides −242 and −62 within MUC3 promoter. Interestingly, the TFLK-containing 10-mer BP dramatically inhibited enteropathogenic E. coli and EHEC O157:H7 adherence to the HT-29-Gal cells compared with the controls. This finding suggests a potential therapeutic use for this peptide to prevent gastrointestinal infection.

Resistance to anoikis, the subtype of apoptosis induced by lack of matrix adhesion, contributes to malignant transformation and development of metastasis. MicroRNAs play key regulatory roles in tumorigenesis and metastasis. In this study, we described that miR-26a, which is usually downregulated in tumor cells, is involved in the acquisition of anoikis-resistance of human esophageal adenocarcinoma (EA) cells. Results of qRT-PCR in clinical samples showed that downregulated miR-26a expression is related to tumorigenesis and metastasis of EA. In vitro experiments determined that miR-26a directly participates in the regulation of cell cycle and anoikis of human EA OE33 cells. Further, we identified that Rb1 is the direct functional target of miR-26a, and revealed that the reduction of miR-26a expression leads to increased Rb1 protein level and thus inhibits the function of E2F1, by which it influences the phenotypes of cell cycle and anoikis. The findings we reported here presented the evidence that miR-26a may be involved in regulation of anoikis-resistance of EA cells. Targeting miR-26a may provide a novel strategy to inhibit metastasis.

CpG oligodeoxynucleotides (CpG ODN) have the potential to enhance the antigen-presenting cells function of human naïve B cells. In this study, we aim to define the effect of CpG ODNs on the binding capacity of human naïve B cells for different Hepatitis B virus (HBV) epitopes. Three HLA-A2 restricted epitopes were selected to incubate with CpG ODN-primed human naïve B cells. Binding capacity for each epitope and expression of CD80, CD86, class I major histocompatibility complex (MHC), and class II MHC of naïve B cells was tested, respectively, by flow cytometry. CpG ODNs, especially ODN 2216, enhanced the binding capacity of human naïve B cells for HBV epitopes (p < 0.01), and induced markedly higher expression of CD80, CD86, class I MHC, and class II MHC. The binding capacity of CpG-treated naive B cells for each epitope was significantly different. In all the 3 subjects, CpG ODN 2216-primed naïve B cells showed the highest binding ability for Env172–180 compared with the other epitopes with a high expression of co-stimulatory and MHC molecules. CpG ODN showed the potential to selectively enhance the binding capacity of human naïve B cells for HBV epitopes. These results suggest new strategies for development of vaccine design.

Recent investigations discovered that CIAPIN1 might be another drug resistance-associated molecule in cancer cells. However, the underlying mechanisms of CIAPIN1-related multidrug resistance (MDR) remain elusive. In the present study, we investigated the role and possible mechanisms of CIAPIN1 in MDR of human colon carcinoma LoVo/Adr cells which express the wild-type p53 gene. By using small interference RNA and gene transfection techniques, we found that knockdown of CIAPIN1 expression re-sensitized LoVo/Adr cells to anti-cancer drugs and up-regulation of CIAPIN1 in sensitive LoVo cells resulted in a distinct MDR phenotype. We further revealed that CIAPIN1 conferred the MDR phenotype in LoVo/Adr cells through up-regulating expression of MDR-1 (P-gp) and Bcl-xL. Finally, by analyzing the effect of inactivation of wild-type p53 on CIAPIN1-induced up-regulation of P-gp and Bcl-xL, we determined that CIAPIN1 could exhibit its MDR-related function independently of the p53 signaling pathway. Overall, the results presented here further suggest that over-expression of CIAPIN1 is an important mechanism of drug resistance in human cancers, even if not the sole one.

Although human MUC3 and rodent Muc3 are both membrane-associated intestinal mucins, the present study has explored the possibility that rodent Muc3 might exist in soluble as well as membrane forms. No evidence was obtained for the existence of soluble splice variants; however, experiments with heterologous cells transfected with cDNA encoding the 381-residue C-terminal domain of rodent Muc3 showed that a definitive proteolytic cleavage occurs during processing in the endoplasmic reticulum. The products consisted of a V5-tagged 30 kDa extracellular glycopeptide and a Myc-tagged 49 kDa membrane-associated glycopeptide. Throughout their cellular transport to the plasma membrane, the two fragments remained associated by non-covalent SDS-sensitive interactions. Site-specific mutagenesis pinpointed the need for glycine and serine residues in the cleavage sequence Leu-Ser-Lys-Gly-Ser-Ile-Val-Val, which is localized between the two epidermal-growth-factor-like motifs of the mucin. A similar cleavage sequence (Phe-Arg-Pro-Gly downward arrow Ser-Val-Val-Val, where downward arrow signifies the cleavage site) has been reported in human MUC1 and analogous sites are present in human MUC3, MUC12 and MUC17. Thus early proteolytic cleavage may be a conserved characteristic of many membrane-associated mucins, possibly as a prelude to later release of their large extracellular domains at cell surfaces.

Background Achaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controls the fate of intestinal stem cells. However, the role of Ascl2 in colon cancer progenitor cells remains unknown. The cell line HT-29 (47.5–95% of CD133+ population) and LS174T (0.45% of CD133+ population) were chosen for functional evaluation of Ascl2 in colon cancer progenitor cells after gene knockdown by RNA interference. Methodology/Principal Findings Immunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas. Downregulation of Ascl2 using RNA interference in cultured colonic adenocarcinoma HT-29 and LS174T cells reduced cellular proliferation, colony-forming ability, invasion and migration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2 protein level in CD133+ HT-29 cells was significantly higher than in CD133− HT-29 cells. Ascl2 blockade via shRNA interference in HT-29 cells (shRNA-Ascl2/HT-29 cells) resulted in 26.2% of cells staining CD133+ compared with 54.7% in control shRNA-Ctr/HT-29 cells. The levels of ‘stemness’ associated genes, such as CD133, Sox2, Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft (CD133 was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/HT-29 cells had inhibited abilities to form tumorspheres compared with control. The microRNA (miRNAs) microarrays, identified 26 up-regulated miRNAs and 58 down-regulated miRNAs in shRNA-Ascl2/HT-29 cells. Expression levels of let-7b, miRNA-124, miRNA-125b, miRNA-17, miRNA-20a and miRNA-302b, involved in the regulation of ‘stemness’, were quantified with qPCR, which confirmed their identities. Restoration of miRNA-302b, via its mimic, led to the restoration of shRNA-Ascl2/HT-29 ‘stemness’ characteristics, including tumorsphere formation and ‘stemness’ associated genes levels, and the recovery of cellular behaviors, including colony-forming ability, invasion and migration in vitro. Conclusions/Significance Ascl2 may be a potential target for the inhibition of colon cancer progenitor cells, and functions through a miR-302b-related mechanism.