Synergistic Effect of Metformin and Lansoprazole Against Gastric Cancer through Growth Inhibition.

Cancer has been linked to metabolic disorders and diverse gene mutations. Metformin, which is widely used to treat type 2 diabetes, inhibits the growth of cancer cells in animal models. Here we investigated the effects of metformin on human gastric cancer cell lines. We also investigated the synergistic anticancer effect of metformin and proton pump inhibitors. Lansoprazole, a proton pump inhibitor, is effective for treating gastroesophageal reflux disease. Our results revealed that metformin and lansoprazole can significantly inhibit cancer cell growth in a dose-dependent manner by suppressing cell cycle progression and inducing apoptosis. Low concentrations of metformin and lansoprazole have a synergistic effect on AGS cell growth inhibition. In summary, our findings suggest a new and safe treatment protocol for treating stomach cancers.

Mycena genomes resolve the evolution of fungal bioluminescence.

Mushroom-forming fungi in the order Agaricales represent an independent origin of bioluminescence in the tree of life; yet the diversity, evolutionary history, and timing of the origin of fungal luciferases remain elusive. We sequenced the genomes and transcriptomes of five bonnet mushroom species (Mycena spp.), a diverse lineage comprising the majority of bioluminescent fungi. Two species with haploid genome assemblies ∼150 Mb are among the largest in Agaricales, and we found that a variety of repeats between Mycena species were differentially mediated by DNA methylation. We show that bioluminescence evolved in the last common ancestor of mycenoid and the marasmioid clade of Agaricales and was maintained through at least 160 million years of evolution. Analyses of synteny across genomes of bioluminescent species resolved how the luciferase cluster was derived by duplication and translocation, frequently rearranged and lost in most Mycena species, but conserved in the Armillaria lineage. Luciferase cluster members were coexpressed across developmental stages, with the highest expression in fruiting body caps and stipes, suggesting fruiting-related adaptive functions. Our results contribute to understanding a de novo origin of bioluminescence and the corresponding gene cluster in a diverse group of enigmatic fungal species.

Urine miR-21-5p as a potential non-invasive biomarker for gastric cancer.

Many reports have implicated that microRNAs involve in cancer development and progression, such as miR-155 in breast cancers and miR-196 in gastric cancers. Furthermore, microRNAs are more stable than typical protein-coding gene mRNAs in varieties of clinical samples including body fluids. This suggests that they are potentially valuable biomarkers for cancer monitoring. In this study, we have used urine samples of gastric cancer patients to demonstrate the feasibility of urine microRNAs for gastric cancer detection. Urine samples of gastric cancer patients were extracted for total RNA, which were examined for the expression of miR-21-5p using quantitative stem-loop PCR. Our results demonstrated that miR-21-5p could be detected in small amounts of urine samples with good stability, and the expression levels of miR-21-5p were reduced following surgical removal of gastric cancer tissues. These results implicate that urine miR-21-5p could be utilized as a novel non-invasive biomarker of gastric cancer detection and monitoring.

Phylogeography, Historical Demography, and Genetic Structure of the Rose Bitterling, Rhodeus ocellatus (Kner, 1866) (Cypriniformes: Acheilognathidae), in East Asia.

Yao-Feng Tsao, Wen-Wen Lin, Chia-Hao Chang, Takayoshi Ueda, Nian-Hong Jang-Liaw, Ya-Hui Zhao, and Hsiao-Wei Kao (2016) Rose bitterling, Rhodeus ocellatus, is a small cyprinid fish distributed in East Asia. To infer its phylogeography and genetic structure, specimens from Taiwan, China, and Japan were collected, and complete mitochondrial cytochrome b (cyt b) DNA sequences were amplified and sequenced. Phylogenetic analyses identified seven mitochondrial lineages (A-G). Among them, three lineages (A, B, and C) distributed in mainland China. Lineages D, E, and F distributed in Japan, Korea, and Taiwan, respectively. Lineage G distributed in both China and Japan. The results of the Bayesian Binary MCMC analysis (BBM) suggested that the most recent common ancestor of R. ocellatus was from Lower Yangtze region. Divergence times among lineages inferred by molecular clock ranged from 7.55 to 1.44 million years ago. We propose that topography and climate changes by uplift of the Tibetan Plateau in the Late Miocene-Pliocene and the glacial-interglacial cycles in the Pleistocene might account for population expansion and genetic differentiation. Divergence times among lineages A, B, and C in Yangtze River basin ranged from 7.55 to 2.27 million years ago that might result from changes of flow directions of rivers from westward to eastward driven by the uplift of the Tibetan Plateau. The glacial-interglacial cycles in the Pleistocene might further cause population expansion to the northward of lineage G at about 0.19 million years ago. Lineage D in Japan was dispersed from the mainland China before the opening of the Sea of Japan, and lineage F in Taiwan was dispersed from the mainland China through the land bridge in the Pleistocene. Because of the genetic differentiation is statistically significant among populations, protection of genetic diversity and distinctness of R. ocellatus should be considered in the future conservation management.

Evolution of infectious bronchitis virus in Taiwan: positively selected sites in the nucleocapsid protein and their effects on RNA-binding activity.

RNA recombination has been shown to underlie the sporadic emergence of new variants of coronavirus, including the infectious bronchitis virus (IBV), a highly contagious avian pathogen. We have demonstrated that RNA recombination can give rise to a new viral population, supported by the finding that most isolated Taiwanese (TW) IBVs, similar to Chinese (CH) IBVs, exhibit a genetic rearrangement with the American (US) IBV at the 5' end of the nucleocapsid (N) gene. Here, we further show that positive selection has occurred at two sites within the putative crossover region of the N-terminal domain (NTD) of the TW IBV N protein. Based on the crystal structure of the NTD, the stereographic positions of both predicted selected sites do not fall close to the RNA-binding groove. Surprisingly, converting either of the two residues to the amino acid present in most CH IBVs resulted in significantly reduced affinity of the N protein for the synthetic RNA repeats of the viral transcriptional regulatory sequence. These results suggest that modulating the amino acid residue at either selected site may alter the conformation of the N protein and affect the viral RNA-N interaction. This study illustrates that the N protein of the current TW IBV variant has been shaped by both RNA recombination and positive selection and that the latter may promote viral survival and fitness, potentially by increasing the RNA-binding capacity of the N protein.

Aberrant expression of miR-196a in gastric cancers and correlation with recurrence.

MicroRNAs (miRNAs) are short noncoding RNAs (~22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. Here, we examined the relationship between miR-196a and gastric cancer.By the analysis of 72 gastric cancer samples, we found that the expression level of miR-196a microRNA significantly increased in primary gastric cancer tissues versus adjacent normal tissues. In addition, extracellular miR-196a detected in conditioned medium was strongly correlated with its cellular expression status and increased circulating miR-196a in patient serum was associated with gastric cancer disease status and relapse. Furthermore, ectopic expression of miR-196a microRNA promoted the epithelial-mesenchymal transition and migration/invasion capabilities of transfected cells, suggesting its oncogenic potential in gastric cancer progression. Altogether, our data demonstrate that miR-196a exerts an oncogenic role in gastric cancer and miR-196a may be a novel biomarker for detecting gastric cancer and for monitoring disease recurrence.

Cytoskeleton network and cellular migration modulated by nuclear-localized receptor tyrosine kinase ROR1.

UNLABELLED: Biological functions of receptor tyrosine kinase-like orphan receptor 1 (ROR1) remain to be elucidated due to the lack of identified genuine ligands. Previously, transiently expressed ROR1 was unexpectedly found to exhibit nuclear localization, the functions of which are unknown. MATERIALS AND METHODS: We constructed nuclear-homing peptidyl-prolyl cis-trans isomerases (FKBP) domain fusion ROR1-expressing cells and used a synthetic dimerizer to specifically activate FKBP-fused ROR1 proteins for subsequent functional characterization. RESULTS: Activation of nuclear-homing ROR1 by treating cells with AP20187 dimerizer led to significant increase in actin stress fibers and increased cellular migration. Following gene expression microarray analysis, we demonstrated that activated ROR1 affects several genes involved in the regulation of the actin cytoskeleton (radixin (RDX), ezrin (EZR), son of sevenless homolog 2 (SOS2) and caldesmon 1 (CALD1)). CONCLUSION: Our data indicate that nuclear-localized ROR1 may play an important role in cell migration and cytoskeleton remodeling. This might explain the critical roles of ROR1 in neuron development.

Epigenetic regulation of miR-34b and miR-129 expression in gastric cancer.

MicroRNAs (miRNAs) are small noncoding RNAs that play fundamental roles in diverse biological and pathological processes by targeting the expression of specific genes. Here, we identified 38 methylation-associated miRNAs, the expression of which could be epigenetically restored by cotreatment with 5-aza-2'-deoxycytidine and trichostatin A. Among these 38 miRNAs, we further analyzed miR-34b, miR-127-3p, miR-129-3p and miR-409 because CpG islands are predicted adjacent to them. The methylation-silenced expression of these miRNAs could be reactivated in gastric cancer cells by treatment with demethylating drugs in a time-dependent manner. Analysis of the methylation status of these miRNAs showed that the upstream CpG-rich regions of mir-34b and mir-129-2 are frequently methylated in gastric cancer tissues compared to adjacent normal tissues, and their methylation status correlated inversely with their expression patterns. The expression of miR-34b and miR-129-3p was downregulated by DNA hypermethylation in primary gastric cancers, and the low expression was associated with poor clinicopathological features. In summary, our study shows that tumor-specific methylation silences miR-34b and miR-129 in gastric cancer cells.

Aberrant hypermethylation of miR-9 genes in gastric cancer.

Carcinogenesis of the stomach involves multiple steps including genetic mutation or epigenetic alteration of tumor suppressor genes or oncogenes. Recently, tumor suppressive miRNAs have been shown to be deregulated by aberrant hypermethylation during gastric cancer progression. In this study, we demonstrate that three independent genetic loci encoding for miR-9 (miR-9-1, miR-9-2 and miR-9-3) are simultaneously modified by DNA methylation in gastric cancer cells. Methylation-mediated silencing of these three miR-9 genes can be reactivated in gastric cancer cells through 5-Aza-dC treatment. Subsequent analysis of the expression levels of miR-9 showed that it was significantly down-regulated in gastric cancers compared with adjacent normal tissues (P value < 0.005). A similar tendency toward a tumor-specific DNA methylation pattern was shown for miR-9-1, miR-9-2 and miR-9-3 in 72 primary human gastric cancer specimens. Ectopic expression of miR-9 inhibited cell proliferation, migration and invasion, suggesting its tumor suppressive potential in gastric cancer progression.

Epigenetic regulation of miR-196b expression in gastric cancer.

MicroRNAs (miRNAs) are short noncoding RNAs that play important roles in cellular processes and disease pathogenesis via the control of specific targeted gene expression. The miR-196s miRNA is encoded at three paralogous loci in three HOX clusters and acts as an oncogenic miRNA in cancer progression. Recent studies have demonstrated that the expression of miR-196b increases cell proliferation and survival in leukemic cells. Here, we used a sequential methylation analysis to reveal that the methylation status correlated well with miR-196b expression in different cell lines. Treatment with the demethylating drug 5-Aza-dC reactivated miR-196b transcription in methylation-silenced cells. Using in vitro methylation approach, we further provide evidences that promoter hypermethylation represses miR-196b transcriptional activation tightly in human cancer cell lines. We also demonstrate that the expression of miR-196b is significantly elevated in gastric cancer and that hypomethylation status of miR-196b CpG islands frequently is observed in primary gastric tumors. Our results provide important information on miR-196s regulation and demonstrate that abnormal DNA hypomethylation induces overexpression of miR-196b in gastric cancer.

Evolution of infectious bronchitis virus in Taiwan: characterisation of RNA recombination in the nucleocapsid gene.

Avian infectious bronchitis virus (IBV) belongs to the Coronaviridae family and causes significant economic loss in Taiwan (TW), even in flocks that have been extensively immunised with Massachusetts (Mass)-serotype vaccines. Phylogenetic analysis of all non-structural and most structural genes shows that TW IBV is genetically distinct from the US strain and more similar to Chinese (CH) IBV. In contrast, the nucleocapsid (N) gene of TW IBV presents phylogenetic incongruence. RNA recombination at the 5' end of the N gene between TW and US IBV is shown to be responsible for this discordance. Surprisingly, the recombinant N gene is found in all of tested TW IBV isolates, suggesting that a recombination event gave origin to a founder lineage. Our data indicate that RNA recombination in the recombinant 5' end of the N gene may have caused the emergence of the current IBV population in Taiwan.

Epigenetic control of the expression of a primate-specific microRNA cluster in human cancer cells.

Many of the known microRNAs (miRNAs) are encoded by polycistronic transcripts and are thought to be co-expressed. In this study, we discovered that the expression of a large miRNA cluster (C19MC) on human chromosome 19 is upregulated by the demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), in AGS gastric cancer cells. We found that C19MC was rarely expressed in most cells, but its expression was restored through DNA demethylation. We confirmed that this miRNA cluster was mainly expressed in the placenta, as previously reported. Furthermore, its expression pattern was highly correlated with the methylation state of a distal CpG-rich region located about 17.6 kb upstream of the miRNA cluster. Using combined bisulfite restriction analysis (COBRA) and bisulfite-sequencing techniques, we determined that this CpG-rich region is hypermethylated in the AGS and HeLa cells, but hypomethylated in the placenta tissue. In conclusion, we demonstrated that the expression pattern of the C19MC was activated in human cancer cells through demethylation of a CpG-rich region.

Genetic diversity and differentiation of gray mullet (Mugil cephalus) in the coastal waters of Taiwan.

From mid-December to late January, schools of mature gray mullet (Mugil cephalus) migrate southward along the coastal waters of China to Taiwan for spawning. It has been proposed that there is no genetic differentiation of gray mullet in the coastal waters of Taiwan. To test this hypothesis, complete cytochrome b (cyt b) DNA sequences of 98 Individuals of gray mullet, two Individuals of Liza macrolepis, and three individuals of L. affinis were amplified by polymerase chain reaction and sequenced. Phylogenetic trees reconstructed with the Bayesian, maximum likelihood, maximum parsimony, and neighbor-Joining methods all support the existence of three monophyletic groups (denoted as groups 1, 2, and 3), with net evolutionary divergences (p-distances) between groups ranging from 5.1% to 6.6%. To estimate the relative abundance of each group, a PCR-RFLP method was developed to examine 600 juveniles collected from October 2006 to March 2007. Groups 1, 2, and 3 comprised 85%, 3%, and 12% of the samples, respectively. Juveniles of groups 1 and 3 could be found as early as November, but Juveniles of group 2 were not found until February. Based on the dates of specimen collection and phylogenetic analyses, we propose that groups 1 and 2 are migratory populations from China and Japan, respectively, whereas group 3 is a resident population in Taiwan.

Differential expression of zebrafish gpia and gpib during development.

Glucose 6-phosphate isomerase (GPI), alternatively named phosphoglucose isomerase (PGI), autocrine motility factor (AMF) or neuroleukin (NLK), is a sugar metabolic enzyme catalyzing the interconversion between glucose-6-phosphate and fructose-6-phosphate. When secreted out of the cell, it can induce cellular activities of neighboring cells. As a prerequisite to study the function of gpi during development, we analyzed the sequences and expression patterns of zebrafish gpia and gpib. Phylogenetic analyses indicate that gpia and gpib are two paralogs resulting from the teleost fish-specific whole genome duplication. In adult zebrafish, gpia is widely expressed in many tissues, whereas gpib is only expressed in heart, muscle, and at lower abundance in eye. During embryonic development, gpia mRNA is maternally deposited in cleavage and blastula embryos, but gpib mRNA is not. Zygotic expression of gpia mRNA initiates in the primitive gut of segmentation embryos, and its expression increases in eye, cerebellum, hindbrain, heart, pharyngeal arches, pectoral fin buds and gut of hatching embryos at 60hpf (hours post fertilization). At 72hpf, gpia mRNA is strongly expressed in the retina of eye, tectum, hindbrain, and gut derivatives such as liver, intestine and circular smooth muscle layer of the swim bladder. By contrast, gpib is expressed in the yolk syncytial nuclei of late blastula, gastrula and segmentation embryos. Somitic gpib expression initiates in late pharyngula embryos, and it is most prominent in the muscle of hatching embryos around 48-60hpf. Transcript of gpib is also expressed in the pectoral fins and portions of the pharyngeal arches in the hatching period. The differential expression of gpia and gpib in zebrafish suggests partition or divergence of gpi functions between the two duplicates.

Specific induction of the high-molecular-weight microtubule-associated protein 2 (hmw-MAP2) by betel quid extract in cultured oral keratinocytes: clinical implications in betel quid-associated oral squamous cell carcinoma (OSCC).

Betel quid (BQ) chewing, a popular habit in numerous Asian countries including India and Taiwan, has a strong correlation with an increased risk of oral squamous cell carcinoma (OSCC). While substantial efforts have been made to test the cytotoxic, genotoxic and mutagenic effects of BQ extract and its components, the disease mechanisms underlying BQ-induced oral carcinogenesis remain obscure. Here, we show that a neuronal protein, microtubule-associated protein 2 (MAP2), was induced by BQ extract in cultured normal human oral keratinocytes (NHOKs). Subsequent analyses demonstrated that such induction was more eminent and consistent in the high-molecular-weight isoform of MAP2 (hmw-MAP2) than that in its low-molecular-weight counterpart (lmw-MAP2). Furthermore, we analyzed expression of hmw-MAP2 protein in 88 oral specimens consisting of clinicopathologically pre-malignant (leukoplakia) and malignant (OSCC) lesions, along with their adjacent normal mucosa. Immunohistochemistry revealed that, with the exposure to BQ, the hmw-MAP2 was over-expressed in 41.2% (7/17) of OSCC, 11.2% (1/9) of leukoplakia and none (0/19) of normal mucosa. In contrast, expression of the hmw-MAP2 was barely detected in BQ-free OSCC. These results suggest a significant correlation between expression of the hmw-MAP2 and BQ-associated progression of oral carcinogenesis (P=0.0046). Interestingly, the hmw-MAP2 was found to preferentially express in histopathologically less differentiated OSCC (P=0.014); the percentages of positive staining in poorly, moderately and well differentiated OSCC were 62.5, 21.4 and 7.1%, respectively. However, BQ chewing appeared to have marginal correlation with such propensity. Finally, we show that the majority of hmw-MAP2-positive poorly differentiated lesions were also histopathologically invasive. Taken together, these findings suggest the possibility that the hmw-MAP2 may be a diagnostic marker for BQ-chewing lesions and a potential therapeutic target. To our knowledge, this study has provided the first clinical implication that closely links a cytoskeletal protein to BQ-associated oral cancer.

Phosphoglucose isomerases of hagfish, zebrafish, gray mullet, toad, and snake, with reference to the evolution of the genes in vertebrates.

Phosphoglucose isomerase (PGI) is a protein with multiple functions. To infer its structure changes and evolution in vertebrates, we cloned cDNAs encoding PGI genes from hagfish (Paramyxine yangi), gray mullet (Mugil cephalus), zebrafish (Danio rerio), toad (Bufo melanosticus), and snake (Boiga kraepelini). Only one PGI gene was cloned in each of hagfish, toad, and snake, but two PGI genes were found in zebrafish and gray mullet, respectively. The PGI of hagfish encodes 554 amino acids, in contrast to the PGIs of bonyfishes, toad, and snake which encode 553 amino acids and the PGIs of mammals which encode 558 amino acids. Among 558 aligned amino acid sites, there are 314 sites (56.27%) totally conserved. To see if diversifying selection acts on PGI amino acids of vertebrates, we calculated the pairwise ratio of nonsynonymous versus synonymous substitution per site (Ka/Ks) and the ratio of radical amino acid changes versus conservative amino acid changes per sites (dR/dC) between PGI sequences. The average pairwise ratio between nonsynonymous substitutions per nucleotide (Ka) and synonymous substitutions per nucleotide (Ks) among vertebrate PGI sequences equals 0.047 +/- 0.019. The average pairwise ratio between radical amino acid changes and conservative amino acid changes (dR/dC) among the vertebrate PGIs equal 0.938 +/- 0.158 for charge changes, 0.558 +/- 0.085 for polarity changes, and 0.465 +/- 0.0714 when both polarity and volume are considered. There is no amino acid within the vertebrate PGIs under diversifying selection as analyzed by the method of Yang et al. (2000b). The results suggest that the present vertebrate PGIs are at evolutionary stasis and are being subjected to intense purifying selection. The purifying selection is to maintain polarity and volume of the protein but not the charge groups of amino acids. Phylogenetic analysis reveals that vertebrate PGIs can be classified into three major groups: the mammalian, amphibian-reptilian, and teleostean PGIs. The gene tree suggests that the gene duplication event of PGI in bonyfishes occurred before diversification of Acanthopterygii but after the split of bonyfishes and tetrapods. The evolution of multiple functions of PGI is discussed.