Identification of Novel Core Genes Involved in Malignant Transformation of Inflamed Colon Tissue Using a Computational Biology Approach and Verification in Murine Models.

Inflammatory bowel disease (IBD) is a complex and multifactorial systemic disorder of the gastrointestinal tract and is strongly associated with the development of colorectal cancer. Despite extensive studies of IBD pathogenesis, the molecular mechanism of colitis-driven tumorigenesis is not yet fully understood. In the current animal-based study, we report a comprehensive bioinformatics analysis of multiple transcriptomics datasets from the colon tissue of mice with acute colitis and colitis-associated cancer (CAC). We performed intersection of differentially expressed genes (DEGs), their functional annotation, reconstruction, and topology analysis of gene association networks, which, when combined with the text mining approach, revealed that a set of key overexpressed genes involved in the regulation of colitis (C3, Tyrobp, Mmp3, Mmp9, Timp1) and CAC (Timp1, Adam8, Mmp7, Mmp13) occupied hub positions within explored colitis- and CAC-related regulomes. Further validation of obtained data in murine models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS-stimulated CAC fully confirmed the association of revealed hub genes with inflammatory and malignant lesions of colon tissue and demonstrated that genes encoding matrix metalloproteinases (acute colitis: Mmp3, Mmp9; CAC: Mmp7, Mmp13) can be used as a novel prognostic signature for colorectal neoplasia in IBD. Finally, using publicly available transcriptomics data, translational bridge interconnecting of listed colitis/CAC-associated core genes with the pathogenesis of ulcerative colitis, Crohn's disease, and colorectal cancer in humans was identified. Taken together, a set of key genes playing a core function in colon inflammation and CAC was revealed, which can serve both as promising molecular markers and therapeutic targets to control IBD and IBD-associated colorectal neoplasia.

Canine transmissible venereal tumour established in immunodeficient mice reprograms the gene expression profiles associated with a favourable tumour microenvironment to enable cancer malignancy.

BACKGROUND: Canine transmissible venereal tumours (CTVTs) can cross the major histocompatibility complex barrier to spread among dogs. In addition to the transmissibility within canids, CTVTs are also known as a suitable model for investigating the tumour-host immunity interaction because dogs live with humans and experience the same environmental risk factors for tumourigenesis. Moreover, outbred dogs are more appropriate than inbred mice models for simulating the diversity of human cancer development. This study built a new model of CTVTs, known as MCTVTs, to further probe the shaping effects of immune stress on tumour development. For xenotransplantation, CTVTs were first injected and developed in immunodeficient mice (NOD.CB17-Prkdcscid/NcrCrl), defined as XCTVTs. The XCTVTs harvested from NOD/SCID mice were then inoculated and grown in beagles and named mouse xenotransplantation of CTVTs (MCTVTs). RESULTS: After the inoculation of CTVTs and MCTVTs into immune-competent beagle dogs separately, MCTVTs grew faster and metastasized more frequently than CTVTs did. Gene expression profiles in CTVTs and MCTVTs were analysed by cDNA microarray to reveal that MCTVTs expressed many tumour-promoting genes involved in chronic inflammation, chemotaxis, extracellular space modification, NF-kappa B pathways, and focal adhesion. Furthermore, several well-known tumour-associated biomarkers which could predict tumour progression were overexpressed in MCTVTs. CONCLUSIONS: This study demonstrated that defective host immunity can result in gene instability and enable transcriptome reprogramming within tumour cells. Fast tumour growth in beagle dogs and overexpression of tumour-associated biomarkers were found in a CTVT strain previously established in immunodeficient mice. In addition, dysregulated interaction of chronic inflammation, chemotaxis, and extracellular space modification were revealed to imply the possibly exacerbating mechanisms in the microenvironments of these tumours. In summary, this study offers a potential method to facilitate tumour progression and provide a niche for discovering tumour-associated biomarkers in cancer research.

Loss of Gene Information: Discrepancies between RNA Sequencing, cDNA Microarray, and qRT-PCR.

Molecular analyses of normal and diseased cells give insight into changes in gene expression and help in understanding the background of pathophysiological processes. Years after cDNA microarrays were established in research, RNA sequencing (RNA-seq) became a key method of quantitatively measuring the transcriptome. In this study, we compared the detection of genes by each of the transcriptome analysis methods: cDNA array, quantitative RT-PCR, and RNA-seq. As expected, we found differences in the gene expression profiles of the aforementioned techniques. Here, we present selected genes that exemplarily demonstrate the observed differences and calculations to reveal that a strong RNA secondary structure, as well as sample preparation, can affect RNA-seq. In summary, this study addresses an important issue with a strong impact on gene expression analysis in general. Therefore, we suggest that these findings need to be considered when dealing with data from transcriptome analyses.

Temporal analysis of mRNA expression profiles in Orientia infected C3HeB/FeJ mouse.

BACKGROUND: Scrub typhus causes up to 35% mortality if left untreated. One billion people living in the endemic regions are at risk. In spite of its heavy disease burden in some of the most populated areas in the world, there is no vaccine available. Although the disease can be effectively treated by proper antibiotics, timely and accurate diagnosis remains a challenge. Orientia tsutsugamushi infects a variety of mammalian cells in vitro and replicates in the cytoplasm of the infected cells. Microarray analysis has been used extensively to study host-pathogen interactions in in vitro models to understand pathogenesis. However there is a lack of in vivo studies. RESULTS: In this study, C3HeB/FeJ (C3H) mice were infected by O. tsutsugamushi via the intraperitoneal route and monitored gene expression at 10 different time points post infection. We observed two distinct types of expression profiles in the genes that we analyzed. There are two valleys (4-18 h and 2-4 days) with low number of differentially expressed genes (DEG) with three peaks with high number of DEG at 2 h, 1-day and 7-day post infection. Further analysis revealed that pathways like complement and coagulation cascade, and blood clotting cascade pathways showed significant global changes throughout entire time course. Real time quantitative Polymerase Chain Reaction (RT-qPCR) confirmed the change of expression for genes involved in complement and coagulation cascade. These results suggested dynamic regulation of the complement and coagulation cascades throughout most of the time post infection while some other specific pathways, such as fatty acid metabolism and tryptophan metabolism, are turned on or off at certain times post infection. CONCLUSIONS: The findings highlight the complex interconnection among all different biological pathways. It is conceivable that specific pathways such as cell growth control and cell development in the host are affected by Orientia in the initial phase of infection for Orientia to grow intracellularly. Once Orientia is replicating successfully inside the host as infection progresses, the infection could activate pathways involved in cellular immune responses to defend for host cell survival and try to eliminate the pathogen.

Molecular profiling of afatinib-resistant non-small cell lung cancer cells in vivo derived from mice.

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC patients with overexpressed or mutated epidermal growth factor receptor (EGFR) related to disease progression are treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Acquired drug resistance after TKI treatments has been a major focus for development of NSCLC therapies. This study aimed to establish afatinib-resistant cell lines from which afatinib resistance-associated genes are identified and the underlying mechanisms of multiple-TKI resistance in NSCLC can be further investigated. Nude mice bearing subcutaneous NSCLC HCC827 tumors were administered with afatinib at different dose intensities (5-100 mg/kg). We established three HCC827 sublines resistant to afatinib (IC50 > 1 µM) with cross-resistance to gefitinib (IC50 > 5 µM). cDNA microarray revealed several of these sublines shared 27 up- and 13 down-regulated genes. The mRNA expression of selective novel genes - such as transmembrane 4 L six family member 19 (TM4SF19), suppressor of cytokine signaling 2 (SOCS2), and quinolinate phosphoribosyltransferase (QPRT) - are responsive to afatinib treatments only at high concentrations. Furthermore, c-MET amplification and activations of a subset of tyrosine kinase receptors were observed in all three resistant cells. PHA665752, a c-MET inhibitor, remarkably increased the sensitivity of these resistant cells to afatinib (IC50 = 12-123 nM). We established afatinib-resistant lung cancer cell lines and here report genes associated with afatinib resistance in human NSCLC. These cell lines and the identified genes serve as useful investigational tools, prognostic biomarkers of TKI therapies, and promising molecule targets for development of human NSCLC therapeutics.

CXCL12 expression in intrahepatic cholangiocarcinoma is associated with metastasis and poor prognosis.

Intrahepatic cholangiocarcinoma is a rare malignant biliary neoplasm that causes a poor prognosis even after curative hepatectomy. Liver metastasis is the major recurrence pattern of intrahepatic cholangiocarcinoma; therefore, the prevention of liver metastasis is a desirable objective. The aim of this study is to identify gene(s) related to liver metastasis of intrahepatic cholangiocarcinoma and to examine the inhibitory effects on metastasis of intrahepatic cholangiocarcinoma by controlling such gene(s). We collected 3 pairs of intrahepatic cholangiocarcinoma frozen samples, and 36 pairs (primary and metastatic lesions) of intrahepatic cholangiocarcinoma formalin-fixed paraffin-embedded samples, from patients who underwent surgical resection at hospitals related to the Kyushu Study Group of Liver Surgery between 2002 and 2016. We carried out cDNA microarray analyses and immunohistochemistry to identify candidate genes, and evaluated one of them as a therapeutic target using human cholangiocarcinoma cell lines. We identified 4 genes related to liver metastasis using cDNA microarray, and found that CXCL12 was the only gene whose expression was significantly higher in liver metastasis than in primary intrahepatic cholangiocarcinoma by immunohistochemistry (P = .003). In prognosis, patients in the high CXCL12 group showed a significantly poor prognosis in disease-free (P < .0001) and overall survival (P = .0004). By knockdown of CXCL12, we could significantly suppress the invasive and migratory capabilities of 2 human cholangiocarcinoma cell lines. Therefore, CXCL12 might be associated with metastasis and poor prognosis in intrahepatic cholangiocarcinoma.

Genome-wide analysis of spatiotemporal gene expression patterns during floral organ development in Brassica rapa.

Flowering is a key agronomic trait that directly influences crop yield and quality and serves as a model system for elucidating the molecular basis that controls successful reproduction, adaptation, and diversification of flowering plants. Adequate knowledge of continuous series of expression data from the floral transition to maturation is lacking in Brassica rapa. To unravel the genome expression associated with the development of early small floral buds (< 2 mm; FB2), early large floral buds (2-4 mm; FB4), stamens (STs) and carpels (CPs), transcriptome profiling was carried out with a Br300K oligo microarray. The results showed that at least 6848 known nonredundant genes (30% of the genes of the Br300K) were differentially expressed during the floral transition from vegetative tissues to maturation. Functional annotation of the differentially expressed genes (DEGs) (fold change ≥ 5) by comparison with a close relative, Arabidopsis thaliana, revealed 6552 unigenes (4579 upregulated; 1973 downregulated), including 131 Brassica-specific and 116 functionally known floral Arabidopsis homologs. Additionally, 1723, 236 and 232 DEGs were preferentially expressed in the tissues of STs, FB2, and CPs. These DEGs also included 43 transcription factors, mainly AP2/ERF-ERF, NAC, MADS-MIKC, C2H2, bHLH, and WRKY members. The differential gene expression during flower development induced dramatic changes in activities related to metabolic processes (23.7%), cellular (22.7%) processes, responses to the stimuli (7.5%) and reproduction (1%). A relatively large number of DEGs were observed in STs and were overrepresented by photosynthesis-related activities. Subsequent analysis via semiquantitative RT-PCR, histological analysis performed with in situ hybridization of BrLTP1 and transgenic reporter lines (BrLTP promoter::GUS) of B. rapa ssp. pekinensis supported the spatiotemporal expression patterns. Together, these results suggest that a temporally and spatially regulated process of the selective expression of distinct fractions of the same genome leads to the development of floral organs. Interestingly, most of the differentially expressed floral transcripts were located on chromosomes 3 and 9. This study generated a genome expression atlas of the early floral transition to maturation that represented the flowering regulatory elements of Brassica rapa.

Exposure of Human Gastric Cells to Oxidized Lipids Stimulates Pathways of Amino Acid Biosynthesis on a Genomic and Metabolomic Level.

The Western diet is characterized by a high consumption of heat-treated fats and oils. During deep-frying processes, vegetable oils are subjected to high temperatures which result in the formation of lipid peroxidation products. Dietary intake of oxidized vegetable oils has been associated with various biological effects, whereas knowledge about the effects of structurally-characterized lipid peroxidation products and their possible absorption into the body is scarce. This study investigates the impact of linoleic acid, one of the most abundant polyunsaturated fatty acids in vegetable oils, and its primary and secondary peroxidation products, 13-HpODE and hexanal, on genomic and metabolomic pathways in human gastric cells (HGT-1) in culture. The genomic and metabolomic approach was preceded by an up-to-six-hour exposure study applying 100 µM of each test compound to the apical compartment in order to quantitate the compounds' recovery at the basolateral side. Exposure of HGT-1 cells to either 100 µM linoleic acid or 100 µM 13-HpODE resulted in the formation of approximately 1 µM of the corresponding hydroxy fatty acid, 13-HODE, in the basolateral compartment, whereas a mean concentration of 0.20 ± 0.13 µM hexanal was quantitated after an equivalent application of 100 µM hexanal. An integrated genomic and metabolomic pathway analysis revealed an impact of the linoleic acid peroxidation products, 13-HpODE and hexanal, primarily on pathways related to amino acid biosynthesis (p < 0.05), indicating that peroxidation of linoleic acid plays an important role in the regulation of intracellular amino acid biosynthesis.

The constitutive differential transcriptome of a brain circuit for vocal learning.

BACKGROUND: The ability to imitate the vocalizations of other organisms, a trait known as vocal learning, is shared by only a few organisms, including humans, where it subserves the acquisition of speech and language, and 3 groups of birds. In songbirds, vocal learning requires the coordinated activity of a set of specialized brain nuclei referred to as the song control system. Recent efforts have revealed some of the genes that are expressed in these vocal nuclei, however a thorough characterization of the transcriptional specializations of this system is still missing. We conducted a rigorous and comprehensive analysis of microarrays, and conducted a separate analysis of 380 genes by in situ hybridizations in order to identify molecular specializations of the major nuclei of the song system of zebra finches (Taeniopygia guttata), a songbird species. RESULTS: Our efforts identified more than 3300 genes that are differentially regulated in one or more vocal nuclei of adult male birds compared to the adjacent brain regions. Bioinformatics analyses provided insights into the possible involvement of these genes in molecular pathways such as cellular morphogenesis, intrinsic cellular excitability, neurotransmission and neuromodulation, axonal guidance and cela-to-cell interactions, and cell survival, which are known to strongly influence the functional properties of the song system. Moreover, an in-depth analysis of specific gene families with known involvement in regulating the development and physiological properties of neuronal circuits provides further insights into possible modulators of the song system. CONCLUSION: Our study represents one of the most comprehensive molecular characterizations of a brain circuit that evolved to facilitate a learned behavior in a vertebrate. The data provide novel insights into possible molecular determinants of the functional properties of the song control circuitry. It also provides lists of compelling targets for pharmacological and genetic manipulations to elucidate the molecular regulation of song behavior and vocal learning.

cDNA microarray analysis of human keratinocytes cells of patients submitted to chemoradiotherapy and oral photobiomodulation therapy: pilot study.

Oral mucositis is an acute toxicity that occurs in patients submitted to chemoradiotherapy to treat head and neck squamous cell carcinoma. In this study, we evaluated differences in gene expression in the keratinocytes of the oral mucosa of patients treated with photobiomodulation therapy and tried to associate the molecular mechanisms with clinical findings. From June 2009 to December 2010, 27 patients were included in a randomized double-blind pilot study. Buccal smears from 13 patients were obtained at days 1 and 10 of chemoradiotherapy, and overall gene expression of samples from both dates were analyzed by complementary DNA (cDNA) microarray. In addition, samples from other 14 patients were also collected at D1 and D10 of chemoradiotherapy for subsequent validation of cDNA microarray findings by qPCR. The expression array analysis identified 105 upregulated and 60 downregulated genes in our post-treatment samples when compared with controls. Among the upregulated genes with the highest fold change, it was interesting to observe the presence of genes related to keratinocyte differentiation. Among downregulated genes were observed genes related to cytotoxicity and immune response. The results indicate that genes known to be induced during differentiation of human epidermal keratinocytes were upregulated while genes associated with cytotoxicity and immune response were downregulated in the laser group. These results support previous clinical findings indicating that the lower incidence of oral mucositis associated with photobiomodulation therapy might be correlated to the activation of genes involved in keratinocyte differentiation.

The Effect of MMP-13, MMP-12, and AMBN on Gingival Enlargement and Root Deformation In a New Type of Gingival Fibromatosis.

This case compared gene-expression between a new type of idiopathic gingival fibromatosis (IGF) and normal gingiva, to clarify the nature of the gingival overgrowth and dental anomaly. A 6-year-old girl with generalized gingival overgrowth and root deformations was diagnosed with IGF. Gene expression profiles were compared between normal gingiva (N=9) and one IGF gingiva using cDNA microarray. Genes related to regulation of cell proliferation and proteolytic degradation were expressed strongly in IGF. MMP-13 and MMP-12 expression were 120 times and 96 times lower in IGF, respectively, whereas AMBN expression was 79 times higher. RT-PCR and immunohistochemical staining supported the microarray results. Reduced proteolytic activity due to low MMP-13 and MMP-12 expression appears to be a potential mechanism for gingival overgrowth. Genetic investigations, such as expression levels of MMP-13, MMP-12, and AMBN, may enable classification of a new syndrome characterized by gingival enlargement with abnormal root development.

Differentially expressed genes and gene networks involved in pig ovarian follicular atresia.

Ovarian folliculogenesis corresponds to the development of follicles leading to either ovulation or degeneration, this latter process being called atresia. Even if atresia involves apoptosis, its mechanism is not well understood. The objective of this study was to analyze global gene expression in pig granulosa cells of ovarian follicles during atresia. The transcriptome analysis was performed on a 9,216 cDNA microarray to identify gene networks and candidate genes involved in pig ovarian follicular atresia. We found 1,684 significantly regulated genes to be differentially regulated between small healthy follicles and small atretic follicles. Among them, 287 genes had a fold-change higher than two between the two follicle groups. Eleven genes (DKK3, GADD45A, CAMTA2, CCDC80, DAPK2, ECSIT, MSMB, NUPR1, RUNX2, SAMD4A, and ZNF628) having a fold-change higher than five between groups could likely serve as markers of follicular atresia. Moreover, automatic confrontation of deregulated genes with literature data highlighted 93 genes as regulatory candidates of pig granulosa cell atresia. Among these genes known to be inhibitors of apoptosis, stimulators of apoptosis, or tumor suppressors INHBB, HNF4, CLU, different interleukins (IL5, IL24), TNF-associated receptor (TNFR1), and cytochrome-c oxidase (COX) were suggested as playing an important role in porcine atresia. The present study also enlists key upstream regulators in follicle atresia based on our results and on a literature review. The novel gene candidates and gene networks identified in the current study lead to a better understanding of the molecular regulation of ovarian follicular atresia.

Gene expression and plant hormone levels in two contrasting rice genotypes responding to brown planthopper infestation.

BACKGROUND: The brown planthopper (BPH; Nilaparvata lugens Stål) is a destructive piercing-sucking insect pest of rice. The plant hormones salicylic acid (SA) and jasmonic acid (JA) play important roles in plant-pest interactions. Many isolated rice genes that modulate BPH resistance are involved in the metabolism or signaling pathways of SA, JA and ethylene. 'Rathu Heenati' (RH) is a rice cultivar with a high-level, broad-spectrum resistance to all BPH biotypes. Here, RH was used as the research material, while a BPH-susceptible rice cultivar 'Taichung Native 1' (TN1) was the control. A cDNA microarray analysis illuminated the resistance response at the genome level of RH under BPH infestation. The levels of SA and JA in RH and TN1 seedlings after BPH infestation were also determined. RESULTS: The expression pattern clustering indicated that 1467 differential probe sets may be associated with constitutive resistance and 67 with the BPH infestation-responsive resistance of RH. A Venn diagram analysis revealed 192 RH-specific and BPH-inducible probe sets. Finally, 23 BPH resistance-related gene candidates were selected based on the expression pattern clustering and Venn diagram analysis. In RH, the SA content significantly increased and the JA content significantly decreased after BPH infestation, with the former occurring prior to the latter. In RH, the differential genes in the SA pathway were synthesis-related and were up-regulated after BPH infestation. The differential genes in the JA pathway were also up-regulated. They were jasmonate ZIM-domain transcription factors, which are important negative regulators of the JA pathway. Comparatively, genes involved in the ET pathway were less affected by a BPH infestation in RH. DNA sequence analysis revealed that most BPH infestation-inducible genes may be regulated by the genetic background in a trans-acting manner, instead of by their promoters. CONCLUSIONS: We profiled the analysis of the global gene expression in RH and TN1 under BPH infestation, together with changes in the SA and JA levels. SA plays a leading role in the resistance response of rice to BPH. Our results will aid in understanding the molecular basis of RH's BPH resistance and facilitate the identification of new resistance-related genes for breeding BPH-resistant rice varieties.

Musashi-1 is the candidate of the regulator of hair cell progenitors during inner ear regeneration.

BACKGROUND: Hair cell loss in the cochlea is caused by ototoxic drugs, aging, and environmental stresses and could potentially lead to devastating pathophysiological effects. In adult mammals, hair cell loss is irreversible and may result in hearing and balance deficits. In contrast, nonmammalian vertebrates, including birds, can regenerate hair cells through differentiation of supporting cells and restore inner ear function, suggesting that hair cell progenitors are present in the population of supporting cells. RESULTS: In the present study, we aimed to identify novel genes related to regeneration in the chicken utricle by gene expression profiling of supporting cell and hair cell populations obtained by laser capture microdissection. The volcano plot identified 408 differentially expressed genes (twofold change, p = 0.05, Benjamini-Hochberg multiple testing correction), 175 of which were well annotated. Among these genes, we focused on Musashi-1 (MSI1), a marker of neural stem cells involved in Notch signaling, and the downstream genes in the Notch pathway. Higher expression of these genes in supporting cells compared with that in hair cells was confirmed by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry analysis demonstrated that MSI1 was mainly localized at the basal side of the supporting cell layer in normal chick utricles. During the regeneration period following aminoglycoside antibiotic-induced damage of chicken utricles, the expression levels of MSI1, hairy and enhancer of split-5, and cyclin D1 were increased, and BrdU labeling indicated that cell proliferation was enhanced. CONCLUSIONS: The findings of this study suggested that MSI1 played an important role in the proliferation of supporting cells in the inner ear during normal and damaged conditions and could be a potential therapeutic target in the treatment of vestibular defects.

Sulforaphane-induced apoptosis in human leukemia HL-60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray.

Sulforaphane (SFN), one of the isothiocyanates, is a biologically active compound extracted from cruciferous vegetables, and has been shown to induce cytotoxic effects on many human cancer cells including human leukemia cells. However, the exact molecular mechanism and altered gene expression associated with apoptosis is unclear. In this study, we investigated SFN-induced cytotoxic effects and whether or not they went through cell-cycle arrest and induction of apoptosis and further examined molecular mechanism and altered gene expression in human leukemia HL-60 cells. Cell viability, cell-cycle distribution, sub-G1 (apoptosis), reactive oxygen species (ROS) and Ca2+ production, levels of mitochondrial membrane potential (ΔΨm ), and caspase-3, -8, and -9 activities were assayed by flow cytometry. Apoptosis-associated proteins levels and gene expressions were examined by Western blotting and cDNA microarray assays, respectively. Results indicated that SFN decreased viable cells, induced G2/M phase arrest and apoptosis based on sub-G1 phase development. Furthermore, SFN increased ROS and Ca2+ production and decreased the levels of ΔΨm and activated caspase-3, -8, and -9 activities in HL-60 cells. SFN significantly upregulated the expression of BAX, Bid, Fas, Fas-L, caspase-8, Endo G, AIF, and cytochrome c, and inhibited the antiapoptotic proteins such as Bcl-x and XIAP, that is associated with apoptosis. We also used cDNA microarray to confirm several gene expressions such as caspase -8, -3, -4, -6, and -7 that are affected by SFN. Those results indicated that SFN induced apoptosis in HL-60 cells via Fas- and mitochondria-dependent pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 311-328, 2017.

Casticin induced apoptotic cell death and altered associated gene expression in human colon cancer colo 205 cells.

Casticin, a polymethoxyflavone, derived from natural plant Fructus Viticis exhibits biological activities including anti-cancer characteristics. The anti-cancer and alter gene expression of casticin on human colon cancer cells and the underlying mechanisms were investigated. Flow cytometric assay was used to measure viable cell, cell cycle and sub-G1 phase, reactive oxygen species (ROS) and Ca2+ productions, level of mitochondria membrane potential (ΔΨm ) and caspase activity. Western blotting assay was used to detect expression of protein level associated with cell death. Casticin induced cell morphological changes, decreased cell viability and induced G2/M phase arrest in colo 205 cells. Casticin increased ROS production but decreased the levels of ΔΨm , and Ca2+ , increased caspase-3, -8, and -9 activities. The cDNA microarray indicated that some of the cell cycle associated genes were down-regulated such as cyclin-dependent kinase inhibitor 1A (CDKN1A) (p21, Cip1) and p21 protein (Cdc42/Rac)-activated kinase 3 (PAK3). TNF receptor-associated protein 1 (TRAP1), CREB1 (cAMP responsive element binding protein 1) and cyclin-dependent kinase inhibitor 1B (CDKN1B) (p27, Kip1) genes were increased but matrix metallopeptidase 2 (MMP-2), toll-like receptor 4 (TLR4), PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, bet), and CaMK4 (calcium/calmodulin-dependent protein kinase IV) genes were inhibited. Results suggest that casticin induced cell apoptosis via the activation of the caspase- and/or mitochondria-dependent signaling cascade, the accumulation of ROS and altered associated gene expressions in colo 205 human colon cancer cells.

Temporal course of gene expression during motor memory formation in primary motor cortex of rats.

Motor learning is associated with plastic reorganization of neural networks in primary motor cortex (M1) that depends on changes in gene expression. Here, we investigate the temporal profile of these changes during motor memory formation in response to a skilled reaching task in rats. mRNA-levels were measured 1h, 7h and 24h after the end of a training session using microarray technique. To assure learning specificity, trained animals were compared to a control group. In response to motor learning, genes are sequentially regulated with high time-point specificity and a shift from initial suppression to later activation. The majority of regulated genes can be linked to learning-related plasticity. In the gene-expression cascade following motor learning, three different steps can be defined: (1) an initial suppression of genes influencing gene transcription. (2) Expression of genes that support translation of mRNA in defined compartments. (3) Expression of genes that immediately mediates plastic changes. Gene expression peaks after 24h - this is a much slower time-course when compared to hippocampus-dependent learning, where peaks of gene-expression can be observed 6-12h after training ended.

Evidence for embryonic stem-like signature and epithelial-mesenchymal transition features in the spheroid cells derived from lung adenocarcinoma.

Identification of the cellular and molecular aspects of lung cancer stem cells (LCSCs) that are suggested to be the main culprit of tumor initiation, maintenance, drug resistance, and relapse is a prerequisite for targeted therapy of lung cancer. In the current study, LCSCs subpopulation of A549 cells was enriched, and after characterization of the spheroid cells, complementary DNA (cDNA) microarray analysis was applied to identify differentially expressed genes (DEGs) between the spheroid and parental cells. Microarray results were validated using quantitative real-time reverse transcription-PCR (qRT-PCR), flow cytometry, and western blotting. Our results showed that spheroid cells had higher clonogenic potential, up-regulation of stemness gene Sox2, loss of CD44 expression, and gain of CD24 expression compared to parental cells. Among a total of 160 genes that were differentially expressed between the spheroid cells and the parental cells, 104 genes were up-regulated and 56 genes were down-regulated. Analysis of cDNA microarray revealed an embryonic stem cell-like signature and over-expression of epithelial-mesenchymal transition (EMT)-associated genes in the spheroid cells. cDNA microarray results were validated at the gene expression level using qRT-PCR, and further validation was performed at the protein level by flow cytometry and western blotting. The embryonic stem cell-like signature in the spheroid cells supports two important notions: maintenance of CSCs phenotype by dedifferentiating mechanisms activated through oncogenic pathways and the origination of CSCs from embryonic stem cells (ESCs). PI3/AKT3, as the most common up-regulated pathway, and other pathways related to aggressive tumor behavior and EMT process can confer to the spheroid cells' high potential for metastasis and distant seeding.

The milk-derived fusion peptide, ACFP, suppresses the growth of primary human ovarian cancer cells by regulating apoptotic gene expression and signaling pathways.

BACKGROUND: ACFP is an anti-cancer fusion peptide derived from bovine milk protein. This study was to investigate the anti-cancer function and underlying mechanisms of ACFP in ovarian cancer. METHODS: Fresh ovarian tumor tissues were collected from 53 patients who underwent initial debulking surgery, and primary cancer cells were cultured. Normal ovarian surface epithelium cells (NOSECs), isolated from 7 patients who underwent surgery for uterine fibromas, were used as normal control tissue. Anti-viabilities of ACFP were assessed by WST-1 (water-soluble tetrazolium 1), and apoptosis was measured using a flow cytometry-based assay. Gene expression profiles of ovarian cancer cells treated with ACFP were generated by cDNA microarray, and the expression of apoptotic-specific genes, such as bcl-xl, bax, akt, caspase-3, CDC25C and cyclinB1, was assessed by real time PCR and western blot analysis. RESULTS: Treatment with ACFP inhibited the viability and promoted apoptosis of primary ovarian cancer cells but exhibited little or no cytotoxicity toward normal primary ovarian cells. Mechanistically, the anti-cancer effects of ACFP in ovarian cells were shown to occur partially via changes in gene expression and related signal pathways. Gene expression profiling highlighted that ACFP treatment in ovarian cancer cells repressed the expression of bcl-xl, akt, CDC25C and cyclinB1 and promoted the expression of bax and caspase-3 in a time- and dose-dependent manner. CONCLUSIONS: Our results suggest that ACFP may represent a potential therapeutic agent for ovarian cancer that functions by altering the expression and signaling of cancer-related pathways in ovarian cancer cells.

Altered gene expression in the lower respiratory tract of Car6 (-/-) mice.

From birth, the respiratory tract mucosa is exposed to various chemical, physical, and microbiological stress factors. Efficient defense mechanisms and strictly regulated renewal systems in the mucosa are thus required. Carbonic anhydrase VI (CA VI) is the only secreted isoenzyme of the α-CA gene family. It is transported in high concentrations in saliva and milk into the alimentary tract where it contributes to optimal pH homeostasis. Earlier study of transcriptomic responses of Car6 (-/-) mice has shown changes in the response to oxidative stress and brown fat cell differentiation in the submandibular gland. It has been suggested that CA VI delivered to the mucosal surface of the bronchiolar epithelium is an essential factor in defense and renewal of the lining epithelium. In this study, the transcriptional effects of CA VI deficiency were investigated in both trachea and lung of Car6 (-/-) mice using a cDNA microarray analysis. Functional clustering of the results indicated significant changes of gene transcription in the lower airways. The altered biological processes included antigen transport by M-cells, potassium transport, muscle contraction, and thyroid hormone synthesis. Immunohistochemical staining confirmed the absence of CA VI in the submandibular gland of Car6 (-/-) mice. Immunostaining of the trachea and lung samples revealed no differences between the knockout and wild type groups nor were any morphological changes observed. The present findings can help us to recognize novel functions for CA VI-one of the major protein constituents of saliva and milk.