cDNA Expression Profiles Research Articles

IMUP and GPRC5A: two newly identified risk score indicators in pancreatic ductal adenocarcinoma

BackgroundPancreatic cancer has been a threateningly lethal malignant tumor worldwide. Despite the promising survival improvement in other cancer types attributing to the fast development of molecular precise medicine, the current treatment situation of pancreatic cancer is still woefully challenging since its limited response to neither traditional radiotherapy and chemotherapy nor emerging immunotherapy. The study is to explore potential responsible genes during the development of pancreatic cancer, thus identifying promising gene indicators and probable drug targets.MethodsDifferent bioinformatic analysis were used to interpret the genetic events in pancreatic cancer development. Firstly, based on multiple cDNA microarray profiles from Gene Expression Omnibus (GEO) database, the genes with differently mRNA expression in cancer comparing to normal pancreatic tissues were identified, followed by being grouped based on the difference level. Then, GO and KEGG were performed to separately interpret the multiple groups of genes, and further Kaplan–Meier survival and Cox Regression analysis assisted us to scale down the candidate genes and select the potential key genes. Further, the basic physicochemical properties, the association with immune cells infiltration, mutation or other types variations besides expression gap in pancreatic cancer comparing to normal tissues of the selected key genes were analyzed. Moreover, the aberrant changed expression of key genes was validated by immunohistochemistry (IHC) experiment using local hospital tissue microarray samples and the clinical significance was explored based on TCGA clinical data.ResultsFirstly, a total of 22,491 genes were identified to express differently in cancer comparing to normal pancreatic tissues based on 5 cDNA expression profiles, and the difference of 487/22491 genes was over eightfold, and 55/487 genes were shared in multi profiles. Moreover, after genes interpretation which showed the > eightfold genes were mainly related to extracellular matrix structural constituent regulation, Kaplan–Meier survival and Cox-regression analysis were performed continually, and the result indicated that of the 55 extracellular locating genes, GPRC5A and IMUP were the only two independent prognostic indicators of pancreatic cancer. Further, detailed information of IMUP and GPRC5A were analyzed including their physicochemical properties, their expression and variation ratio and their association with immune cells infiltration in cancer, as well as the probable signaling pathways of genes regulation on pancreatic cancer development. Lastly, local IHC experiment performed on PAAD tissue array which was produced with 62 local hospital patients samples confirmed that GPRC5A and IMUP were abnormally up-regulated in pancreatic cancer, which directly associated with worse patients both overall (OS) and recurrence free survival (RFS).ConclusionsUsing multiple bioinformatic analysis as well as local hospital samples validation, we revealed that GPRC5A and IMUP expression were abnormally up-regulated in pancreatic cancer which associated statistical significantly with patients survival, and the genes’ biological features and clinical significance were also explored. However, more detailed experiments and clinical trials are obligatory to support their further potential drug-target role in clinical medical treatment.

Bioinformatic analysis identifying FGF1 gene as a new prognostic indicator in clear cell Renal Cell Carcinoma

BackgroundClear cell renal cell carcinoma (ccRCC) has been the commonest renal cell carcinoma (RCC). Although the disease classification, diagnosis and targeted therapy of RCC has been increasingly evolving attributing to the rapid development of current molecular pathology, the current clinical treatment situation is still challenging considering the comprehensive and progressively developing nature of malignant cancer. The study is to identify more potential responsible genes during the development of ccRCC using bioinformatic analysis, thus aiding more precise interpretation of the diseaseMethodsFirstly, different cDNA expression profiles from Gene Expression Omnibus (GEO) online database were used to screen the abnormal differently expressed genes (DEGs) between ccRCC and normal renal tissues. Then, based on the protein–protein interaction network (PPI) of all DEGs, the module analysis was performed to scale down the potential genes, and further survival analysis assisted our proceeding to the next step for selecting a credible key gene. Thirdly, immunohistochemistry (IHC) and quantitative real-time PCR (QPCR) were conducted to validate the expression change of the key gene in ccRCC comparing to normal tissues, meanwhile the prognostic value was verified using TCGA clinical data. Lastly, the potential biological function of the gene and signaling mechanism of gene regulating ccRCC development was preliminary explored.ResultsFour cDNA expression profiles were picked from GEO database based on the number of containing sample cases, and a total of 192 DEGs, including 39 up-regulated and 153 down-regulated genes were shared in four profiles. Based on the DEGs PPI network, four function modules were identified highlighting a FGF1 gene involving PI3K-AKT signaling pathway which was shared in 3/4 modules. Further, both the IHC performed with ccRCC tissue microarray which contained 104 local samples and QPCR conducted using 30 different samples confirmed that FGF1 was aberrant lost in ccRCC. And Kaplan–Meier overall survival analysis revealed that FGF1 gene loss was related to worse ccRCC patients survival. Lastly, the pathological clinical features of FGF1 gene and the probable biological functions and signaling pathways it involved were analyzed using TCGA clinical data.ConclusionsUsing bioinformatic analysis, we revealed that FGF1 expression was aberrant lost in ccRCC which statistical significantly correlated with patients overall survival, and the gene’s clinical features and potential biological functions were also explored. However, more detailed experiments and clinical trials are needed to support its potential drug-target role in clinical medical use.

ANKRD22, a novel tumor microenvironment-induced mitochondrial protein promotes metabolic reprogramming of colorectal cancer cells.

Background: The leading cause of poor prognosis in colorectal cancer (CRC) is the presence of colorectal cancer-initiating cells (CCICs). The interplay between the tumor microenvironment (TME) and CRC cells induces reacquisition of initiating cell characteristics, but the underlying mechanisms remain elusive.Methods: Candidate molecules were screened by global differential cDNA expression profiles of CCICs, which were enriched from patient-derived tumor xenograft models. Luciferase reporters and chromatin immunoprecipitation assays were used to explore the mechanism of TME factors regulating the transcription of ANKRD22. The effects of Ankyrin repeat domain-containing protein 22 (ANKRD22) on energy metabolism were monitored by extracellular flux and 13C-based metabolic flux analysis. Mass spectrometry was used to identify the interacting partners of ANKRD22. Morphological changes of CCICs overexpressing ANKRD22 were observed by electron microscopy. The effects of ANKRD22 on mitochondrial lipid metabolism were analyzed by lipidomics.Results: We identified a novel nucleus-encoded mitochondrial membrane protein, ANKRD22, which was upregulated in CCICs. We found that ANKRD22 was induced by the p38/MAX pathway activated by different TME stimuli. As a key transcription factor, MAX promoted the transcription of ANKRD22. Expression of ANKRD22 promoted glycolysis associated with a decrease in ATP/ADP and an increase in AMP/ATP levels, which were related to its interaction with pyruvate dehydrogenase kinase isoform 1 (PDK1) and multiple subunits of ATP synthase. Further, in CCICs, ANKRD22 cooperated with the lipid transport protein, Extended Synaptotagmin-1 (E-Syt1), to transport excess lipids into mitochondria and reduced the number of mitochondria in an autophagy-independent manner, thus meeting the metabolic requirements of CCICs.Conclusion: ANKRD22 induced by TME promotes the metabolic reprogramming of CRC cells. Our study has identified ANKRD22/E-Syt1 as a potential target for eradicating CCICs.

Prognostic significance of TOP2A in non-small cell lung cancer revealed by bioinformatic analysis

BackgroundLung cancer has been a common malignant tumor with a leading cause of morbidity and mortality, current molecular targets are woefully lacking comparing to the highly progressive cancer. The study is designed to identify new prognostic predictors and potential gene targets based on bioinformatic analysis of Gene Expression Omnibus (GEO) database.MethodsFour cDNA expression profiles GSE19188, GSE101929, GSE18842 and GSE33532 were chosen from GEO database to analyze the differently expressed genes (DEGs) between non-small cell lung cancer (NSCLC) and normal lung tissues. After the DEGs functions were analyzed, the protein–protein interaction network (PPI) of DEGs were constructed, and the core gene in the network which has high connectivity degree with other genes was identified. We analyzed the association of the gene with the development of NSCLC as well as its prognosis. Lastly we explored the conceivable signaling mechanism of the gene regulation during the development of NSCLC.ResultsA total of 92 up regulated and 214 down regulated DEGs were shared in four cDNA expression profiles. Based on their PPI network, TOP2A was connected with most of other genes and was selected for further analysis. Kaplan–Meier overall survival analysis (OS) revealed that TOP2A was associated with worse NSCLC patients survival. And both GEPIA analysis and immunohistochemistry experiment (IHC) confirmed that TOP2A was aberrant gain of expression in cancer comparing to normal tissues. The clinical significance of TOP2A and probable signaling pathways it involved in were further explored, and a positive correlation between TOP2A and TPX2 expression was found in lung cancer tissues.ConclusionUsing bioinformatic analysis, we revealed that TOP2A could be adopted as a prognostic indicator of NSCLC and it potentially regulate cancer development through co-work with TPX2. However, more detailed experiments are needed to clarify its drug target role in clinical medical use.

Identification of potential biomarkers of lung adenocarcinoma brain metastases via microarray analysis of cDNA expression profiles.

Brain metastases originating from lung adenocarcinoma (LAD) occur frequently. The aim of the current study was to assess potential biomarkers for the prognosis of lung adenocarcinoma brain metastasis (LAD-BM) through the analysis of gene expression microarrays. The current study downloaded two gene expression datasets, GSE14108 and GSE10245, from the Gene Expression Omnibus database. From GSE14108 and GSE10245, 19 LAD-BM samples and 40 primary LAD samples were selected for analysis. To identify the differentially expressed genes (DEGs), the current study compared the two sample groups, using the limma R package. Subsequently, pathway enrichment analysis was conducted using the Cluster Profiler R package, and the construction of the protein-protein interaction (PPI) network was executed utilizing the Search Tool for the Retrieval of Interacting Genes database. The microRNA-target network was built using the TargetScore R package. Then, these networks were established and visualized using Cytoscape software. An array of 463 DEGs was identified in the LAD-BM samples, including 256 upregulated and 207 downregulated genes. Based on functional term enrichment analysis using the Gene Ontology database and signaling pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes database, it was identified that the overlapping DEGs were primarily involved in chemokine-associated signal transduction, which may mediate lung cancer cell metastasis to the brain. Chemokine ligand 2, lysozyme, matrix metalloproteinase-2 (MMP-2), lysyl oxidase (LOX) and granzyme B were identified as potential biomarkers according to a topological analysis of the PPI networks. Two notable nodes, MMP-2 and LOX, appeared in the PPI network and were key points in the microRNA-target network, as they were regulated by hsa-let-7d. Many DEGs and microRNAs were regarded as prognostic biomarkers for lung adenocarcinoma metastasis in the current study. These DEGs were primarily associated with chemokine-mediated signaling pathways. In addition, MMP-2 and LOX were predicted to be targets of hsa-let-7d.

The cDNA Structures and Expression Profile of the Ascorbate Peroxidase Gene Family During Drought Stress in Wild Watermelon

Ascorbate peroxidase (APX) plays an important role in detoxifying reactive oxygen species under environmental stress. Although previous work in drought-tolerant wild watermelon has shown an increase in chloroplast APX enzyme activity under drought, molecular entities of APX have remained uncharacterized. In this study, structure and transcriptional regulation of the APX gene family in watermelon were characterized. Five APX genes, designated as CLAPX1 to CLAPX5, were identified from watermelon genome. The mRNA alternative splicing was suggested for CLAPX5, which generated two distinct deduced amino acid sequences at their C-terminus, in resemblance to a reported alternative splicing of chloroplast APXs in pumpkin. This observation suggests that two isoenzymes for stromal and thylakoid-bound APXs may be generated from the CLAPX5 gene. Phylogenetic analysis classified CLAPX isoenzymes into three clades, i.e., chloroplast, microbody, and cytosolic. Physiological analyses of wild watermelon under drought showed a decline in stomatal conductance and CO2 assimilation rate, and a significant increase in the enzyme activities of both chloroplast and cytosolic APXs. Profiles of mRNA abundance during drought were markedly different among CLAPX genes, suggesting distinct transcriptional regulation for the APX isoenzymes. Up-regulation of CLAPX5-I and CLAPX5-II was observed at the early phase of drought stress, which was temporally correlated with the observed increase in chloroplast APX enzyme activity, suggesting that transcriptional up-regulation of the CLAPX5 gene may contribute to the fortification of chloroplast APX activity under drought. Our study has provided an insight into the functional significance of the CLAPX gene family in the drought tolerance mechanism in this plant.

PO-296 Elucidating the potential role of CD109 as a biomarker for cancer stem-like cells in cervical cancer

IntroductionCervical cancer is a common genital tract cancer. Radiotherapy is the mainstay of management for advanced cervical cancer. Response to radiation varies widely which may be explained by the existence of cancer stem-like cells (CSCs). Since CSCs is implicated in cervical cancer and demonstrated a high degree of resistance to radiation, the identification of novel CSC markers could be critical to specifically target the cervical CSC. In our pilot study, we established attached and spheroid cells from primary cervical tumour tissue. Multiple ‘stemness’ genes were detected in spheroid cells which indicated primary cervical cancer tissue harboured CSCs population. cDNA microarray analysis was performed to compare cDNA expression profile and CD109 was significantly up-regulated in spheroid cells. Therefore, we hypothesise that CD109 may serve as a potential cervical CSCs marker.Material and methodsFlow cytometry was performed to analyse the CD109 expression and isolate the CD109 positive and negative sub-populations in cervical cancer cells. Following the sorting, cell proliferation and migration assay were performed. In order to investigate the effect on the blockade of CD109, SiHa, Caski and C4-1 cells were transfected with CD109 siRNAs. XTT assay, migration and invasion assay and spheroid formation assay were performed. Immunohistochemistry (IHC) was performed for the detection of CD109 expression in cervical cancer tissue microarray (TMA).Results and discussionsThe post-sorted CD109 (+) cells grew remarkably faster and have stronger migration capability than CD109(-) cells in Caski and C4-1. The CD109 knockdown cells with siRNA exhibited a slower cell growth, decreasing migration and self-renewal ability, as compared with the control group in SiHa, Caski and C4-1 cells. IHC of TMA indicated that CD109 was highly expressed in cancer cases than that in normal/benign cases.ConclusionCD109 increased cell proliferation rate and migration ability in post-sorted cervical cancer cell lines Caski and C4-1. On the contrary, CD109 knockdown reduced cell growth, migration and self-renewal capability in cervical cancer SiHa, Caski and C4-1 cells. Cervical carcinoma showed high expression of CD109 protein by IHC. Further in vivo and in vitro functional assays are essential to characterise the CD109-positive sub-population in cervical cancer which may provide more information of cervical CSCs-related properties and resistance of radiation therapy, with the underlying molecular mechanism involved.

The full-length cDNA cloning and expression profiles of 14-3-3 genes from the mud crab Scylla paramamosain Estampador, 1949

Two full-length cDNA sequences of14-3-3genes were isolated from the mud crab,Scylla paramamosainEstampador, 1949, using RT-PCR and RACE (rapid-amplification of cDNA ends). Sequence analysis indicates that both14-3-3genes contain an open reading frame of 744 bp with a deduced 247-amino-acid protein. The gene and protein sequences of Sp14-3-3genes show 96 and 97% identity, respectively, and both Sp14-3-3cluster together with other animal 14-3-3 proteins in phylogenetic tree analysis. Tissue specific expression analysis reveals that both Sp14-3-3genes are ubiquitously expressed, however, their expression patterns are different from each other. Expressions of both Sp14-3-3genes are sensitive to salinity decrease, even a 5 ppt drop from 30 ppt to 25 ppt, indicating that Sp14-3-3genes are involved in osmoregulation. Our observations may contribute to a better understanding of the molecular and functional evolution of the14-3-3family in both crustaceans and (in)vertebrates as a whole.

Abstract 1487: Identification of oral carcinoma miRNA signature and control OSCC tumorigenesis through Wnt/β-catenin signaling

Abstract Aberrant microRNAs (miRNAs) expressions are often happened in cancer. In order to identify miRNAs altered in oral carcinogenesis, we investigated miRNAs and cDNA expression profiles in 40 pair-wise oral squamous cell carcinoma (OSCC) specimens. Eighty-four miRNAs were differentially expressed in the OSCC specimens compared to the matched tissue. Interestingly, 19 down-regulated miRNAs are clustered in DLK-1/Meg3 imprinted domain of chromosome 14q32.2 region. Loss of 14q32.2-miRNAs expression also raised the 14q32.2-miRNA targets expression in OSCC. We identified one target protein, Wnt-7b, was highly expressed in OSCC and regulated by miR-329 and -410. Wnt-7b increased the Wnt/β-catenin signaling activity and promoted OSCC tumor progression. Furthermore we also identify the regulation mechanism that down-regulated 14q32.2 miRNAs. Hyper-methylation in meg-3 differential methylated region (DMR) was confirmed by bisulfate sequencing. The hyper-methylation of meg-3 DMR was also induced by betel nuts extract exposure. In summary, we identified the expression signature of aberrant miRNAs in OSCC and provide a novel molecular insight how betel quid contribute to oral carcinogenesis through the epigenetic silencing of tumor suppressor miRNAs targeting the Wnt/β-catenin signaling pathway. Note: This abstract was not presented at the meeting. Citation Format: Wei-Min Chang, Yuan-Ming Hsu, Sung-Tau Chou, Yi-Shing Shieh, Michael Hsiao, Jenn-Ren Hsiao, Jang-Yang Chang, Shine-Gwo Shiah. Identification of oral carcinoma miRNA signature and control OSCC tumorigenesis through Wnt/β-catenin signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1487. doi:10.1158/1538-7445.AM2014-1487

CDNA cloning and expression profile analysis of an ATP-binding cassette transporter in the hepatopancreas and intestine of shrimp Fenneropenaeus chinensis

ATP-binding cassette (ABC) transporters are integral membrane proteins which carry diverse substrates across biological membranes. In this study, we report the cloning of a full-length, 2122-bp cDNA sequence encoding a gene of the ABC transporter subfamily G (ABCG) from the shrimp Fenneropenaeus chinensis. The deduced protein, FcABCG, contains 633 amino acid residues with a theoretical molecular weight of 70.57kDa. Phylogenetic analysis showed that the FcABCG clusters with the shrimp Litopenaeus vannamei ABCG in the same clade and is more closely related to the insect ABC transporter/white proteins than to the ABCG2 of the fish Salmo salar. High-level mRNA expression of FcABCG was detected in the tissues of hepatopancreas and intestine using a real-time quantitative reverse transcriptase PCR. Furthermore, exposure to alkaline pH stress conditions and norfloxacin diets result in the activation of the FcABCG gene in F. chinensis. These results suggest that the ABCG transporter of F. chinensis might play a role in coping high pH stress and in exporting xenobiotics.

The full length cDNA cloning and expression profile of 14-3-3 gene from the mud crab( Scylla paramamosain)

Full-length cDNA sequence of 14-3-3 gene was isolated from the eyestalk of mud crab Scylla paramamosain through RT-PCR and RACE.Sequence analysis indicated that 14-3-3 gene had an open reading frame of 744 bp encoding 247aa of 28.086 ku and pI at 4.675.The amino acid sequences of 14-3-3 gene possessed 95%,93%,92% identity with the 14-3-3 genes of Penaeus monodon,Fenneropenaeus merguiensis,Megachile rotundata respectively.14-3-3 protein firstly clustered with 14-3-3 proteins of Penaeus monodon and Fenneropenaeus merguiensis in the phylogenetic analysis.The expression of 14-3-3 gene in tissues was analyzed by Real-Time PCR,the result showed that 14-3-3 gene was most expressed in hepatopancreas and muscle,then in eyestalk,intestinal,heart and gill,the lest in stomach.After 24h’s stress in salinity,the expression of 14-3-3 gene increased great significantly(P0.01) whatever the salinity reduced(5) or raise(15,20,25,30).The more the salinity changed,the more the 14-3-3 gene expressed.These results will serve further studies on functions and regulation mechanism of 14-3-3 genes.

Identification of porcine polycystic kidney disease 1 ( PKD1) gene: Molecular cloning, expression profile, and implication in disease model

The polycystic kidney disease 1 ( PKD1) gene, which accounts for ~ 85% of human autosomal dominant polycystic kidney disease (ADPKD) cases, has been extensively studied in human and mouse. Much information about the pathogenesis of and treatments for ADPKD has been gained from the use of mouse models. However, because mouse models pose some limitations, further studies in other model systems are needed to investigate the biological basis of ADPKD. The pig is regarded as an important biomedical model. Thus, we isolated a pig PKD1 homolog and characterized its cDNA sequence, genomic structure, expression profile, alternative splicing, methylation status, protein characteristics, and immunohistochemical features in both neonatal and adult pigs. The pig PKD1 cDNA is 14,209 bp long and encodes a 4305-residue polypeptide. The genomic sequence of PKD1 is ~ 50 kb with 46 exons. An alternative splice acceptor site was identified in intron 9. PKD1 is expressed in all tissues tested in both neonatal and adult pigs and exhibits a developmentally regulated expression pattern. Western blotting revealed that the molecular mass of polycystin-1 is ~ 460 kDa, but its expression level is relatively low. Immunohistochemical study of the kidneys shows that polycystin-1 is mainly expressed in the tubular epithelia. Bisulfite methylation analysis of CpG islands in the promoter region does not show a direct correlation between methylation status and expression level among different tissues/cells. The cloning and characterization of pig PKD1 indicates that the pig and human genes are highly similar in length of genomic and cDNA sequences, genomic structure and context, expression patterns, conserved transcription factor binding sites, and the molecular mass of the encoded polycystin-1. These data support our current understanding of PKD1, and suggest that the pig is an ideal candidate for development of an ADPKD disease model.

The full length cDNA cloning and expression profile of prophenoloxidase of <I>Fenneropenaeus chinensis</I>

Chinese shrimp (Fenneropenaeus chinensis) is one of the most economically valuable and widely cultured species in China. During the last decade its development has been seriously affected on a regular basis by the outbreak of viral and bacterial diseases. To analyze the immune defense mechanisms of shrimp F.chinensis,a prophenoloxidase gene (FCproPO) was cloned from haemocytes of Chinese shrimp by Rapid Amplification Complementary DNA Ends (RACE) method. The full length cDNA of FCproPO consisted of 2 311 bp with a 2 061 bp Open Reading Frame(ORF),which encoded 687 amino acids,and the predicted molecular mass was 78.71 ku. It showed 84% and 71% homology with those of Litopenaeus vannamei and Marsupenaeus japonicas. The Realtime-PCR results showed that the expression level of FCproPO in haemoeytes was highest among seven studied tissues.Sequence analysis showed FCproPO contained two conserved copper-binding sites. Phylogenetic analysis revealed that FCproPO and prophenoloxidase from L. vannamei,M. japonicus and P. monodon were in the same phylogenetic branches. Realtime-PCR analysis showed that the expression of FCproPO was up-regulated distinctly in the hepatopanereas of shrimp when they were challenged by Vibrio anguillarum and white spot syndrome virus (WSSV). FCproPO showed different expression profiles in the hepatopancreas during V. anguillarum or WSSV virus infection.The result implied that FCproPO might play an important role in the shrimp immune system.

MicroRNA-155 Modulates Transforming Growth Factor-β Signaling In Chronic Lymphocytic Leukemia through Targeting of Casein Kinase γ Isoform 2

AbstractAbstract 3584Chronic lymphocytic leukemia (CLL) is typically characterized by defects in programmed cell death rather than alterations in cell cycle regulation. Transforming growth factor β (TGFβ), a ubiquitously expressed growth factor, regulates multiple normal cellular responses including proliferation, differentiation, migration and apoptosis. Loss of growth inhibition by TGFβ is thought to contribute to the development and progression of a variety of tumors including CLL (DeCoteau et al., PNAS 1997). Approximately 40% of patients contain mutations in the signal sequence of TGFβ receptor 1 (TBR-1) in the form of substitutions or deletions (Schiemann et al., Cancer Detect Prev 2004). In the wild type form, the signal sequence contains a nine alanine stretch, which if truncated has been shown to impair signaling through the receptor and specifically, a truncated, six alanine form is associated with increased cancer risk (Pasche et al., Cancer Res 1999). TGFβ signaling can regulate expression of micoRNAs (miRNA), which are ~22 nucleotide-long RNA gene regulators. Deregulated miRNA expression has been implicated in tumorigenesis, including CLL. Several miRNAs have been shown to be over-expressed in CLL as compared to normal B cells (Fulci et al., Blood 2007). This includes miR-155, which is part of a 13-miRNA signature that has prognostic implications, including a shorter need-for-treatment interval (Calin et al., N Engl J Med 2005). Interestingly, miR-155 has been shown to be upregulated by TGFβ in murine mammary gland cells (Kong et al., Mol Cell Biol 2008). The goals of our study are to investigate the link between TGFβ signaling and miR-155 in CLL and to determine how the interaction between the two may contribute to the pathogenesis of CLL. Here we show that miR-155 is in fact upregulated by TGFβ in mouse splenic B cells and in human peripheral blood B cells. In CLL, miR-155 expression inversely correlates with the proportion of CLL cells harboring signal sequence mutation in TBR-1, consistent with miR155 regulation by TGFβ in vivo. To understand the role of TGFβ-induced miR-155 in CLL pathobiology, identification of specific target genes in the context of this disease is essential. To this end, we compared the gene (cDNA) expression profile between CLL with high miR-155 vs. low miR-155 expression and identified putative miR-155 target genes by selecting those genes that are differentially expressed in SAM analysis with lower expression in the high miR-155 group, and which harbor predicted miR-155 binding sites in their 3' untranslated region (UTR). Based on this algorithm, we have identified casein kinase 1 gamma 2 (CSK1γ2) as a target for miR155 in CLL. CSK1γ2 is a negative modulator of the TGFβ signaling pathway by targeting the phosphorylated form of SMAD3 for degradation (Guo et al., Oncogene 2008). MiR-155 represses luciferase reporter gene expression by specific binding to the miR-155 site in the CSK1γ2 3'UTR. In addition, we found that CSK1γ2 itself is upregulated in B cells upon TGFβ stimulation, and treatment of human B cells with PNA miR-155 inhibitor (Fabani et al., Nucleic Acids Research 2010) further increases CSK1γ2 mRNA levels. Surprisingly, comparison of CSK1γ2 protein levels between CLLs with high or low miR-155 by Western blotting revealed higher CSK1γ2 protein expression despite lower CSK1γ2 mRNA levels, suggesting that miR-155 may enhance CSK1γ2 translation in CLL cells and implying an intriguing regulatory interaction between miR-155 and CSK1γ2. In summary, our data indicates that the variation of miR-155 seen in CLL is primarily a function of TGFβ signaling activity. Moreover, miR-155 is an important player in a complex auto-regulatory network in TGFβ signaling by fine-tuning the negative feedback mechanism on TGFβ signaling mediated by CSK1γ2. In CLL cells harboring TBR-1 with wild-type signal sequence, higher miR-155 levels may help modulate the TGFβ signaling activity to a level optimal for the survival or other pathobiological functions of CLL. Furthermore, since CLL cells are predominantly non-proliferating, our findings that miR-155 may enhance translation of CSK1γ2 provide support to the model of cell cycle dependence of microRNA functions (Vasudevan et al., Cell Cycle 2008). Disclosures:No relevant conflicts of interest to declare.

Efficient replication systems for hepatitis C virus using a new human hepatoma cell line

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a serious global health problem. Cell culture-based persistent HCV RNA replication systems and infectious HCV production systems are widely used in HCV research. However, persistent HCV production systems have been developed only for HuH-7 hepatoma cells. Here we found a new human hepatoma cell line, Li23, that enables persistent HCV production and anti-HCV reagent assay. Li23's cDNA expression profile differed from HuH-7's, although the two cells had similar liver-specific expression profiles. We used HCV RNA with a specific combination of adaptive mutations to develop an HCV replicon system and genome-length HCV RNA replicating systems including a reporter assay system. Finally, Li23-derived cells persistently produced infectious virus of an HCV strain. Li23-derived cells are potentially useful for understanding the HCV life cycle and for finding antiviral targets.

Eukaryotic transcriptomics in silico: Optimizing cDNA-AFLP efficiency

BackgroundComplementary-DNA based amplified fragment length polymorphism (cDNA-AFLP) is a commonly used tool for assessing the genetic regulation of traits through the correlation of trait expression with cDNA expression profiles. In spite of the frequent application of this method, studies on the optimization of the cDNA-AFLP assay design are rare and have typically been taxonomically restricted. Here, we model cDNA-AFLPs on all 92 eukaryotic species for which cDNA pools are currently available, using all combinations of eight restriction enzymes standard in cDNA-AFLP screens.ResultsIn silco simulations reveal that cDNA pool coverage is largely determined by the choice of individual restriction enzymes and that, through the choice of optimal enzyme combinations, coverage can be increased from <40% to 75% without changing the underlying experimental design. We find evidence of phylogenetic signal in the coverage data, which is largely mediated by organismal GC content. There is nonetheless a high degree of consistency in cDNA pool coverage for particular enzyme combinations, indicating that our recommendations should be applicable to most eukaryotic systems. We also explore the relationship between the average observed fragment number per selective AFLP-PCR reaction and the size of the underlying cDNA pool, and show how AFLP experiments can be used to estimate the number of genes expressed in a target tissue.ConclusionThe insights gained from in silico screening of cDNA-AFLPs from a broad sampling of eukaryotes provide a set of guidelines that should help to substantially increase the efficiency of future cDNA-AFLP experiments in eukaryotes. In silico simulations also suggest a novel use of cDNA-AFLP screens to determine the number of transcripts expressed in a target tissue, an application that should be invaluable as next-generation sequencing technologies are adapted for differential display.

Linkage of cDNA expression profiles of mesencephalic dopaminergic neurons to a genome-wide in situ hybridization database

Midbrain dopaminergic neurons are involved in control of emotion, motivation and motor behavior. The loss of one of the subpopulations, substantia nigra pars compacta, is the pathological hallmark of one of the most prominent neurological disorders, Parkinson's disease. Several groups have looked at the molecular identity of midbrain dopaminergic neurons and have suggested the gene expression profile of these neurons. Here, after determining the efficiency of each screen, we provide a linked database of the genes, expressed in this neuronal population, by combining and comparing the results of six previous studies and verification of expression of each gene in dopaminergic neurons, using the collection of in situ hybridization in the Allen Brain Atlas.

Insulin‐like growth factor‐I of pejerrey, Odontesthes bonariensis: cDNA characterization, tissue distribution and expression profiles after growth hormone administration

The liver production of the insulin-like growth factor-I (IGF-I) is a key factor in the endocrine control of body growth by a growth hormone. As pejerrey Odontesthes bonariensis has been reported as a fish with low growth rates in captivity, basic research on this respect is needed in order to understand it. In this context, the pejerrey IGF-I cDNA was cloned and its hepatic expression was examined in fish after recombinant pejerrey growth hormone (pjGHr) administration. The full length of IGF-I transcript showed a high sequence similarity to other teleost sequences. The tissue distribution analysis by reverse transcriptase polymerase chain reaction in adult fish revealed that IGF-I expressed ubiquitously with the highest mRNA levels in the liver, posterior intestine and brain. No alternative IGF-I mRNA was found in the liver, as it was reported for other teleosts. IGF-I transcript was measured in the liver after pjGHr in vivo stimulation by means of quantitative real-time polymerase chain reaction assays. A dose-dependent response of IGF-I mRNA was observed after pjGHr administration, and reached a six-fold IGF-I maximum increase over control group when 2.5 microg pjGH/g-body weight (bw) was injected. Temporal analysis of hepatic IGF-I mRNA level showed that administration of a single dose of pjGHr into juvenile pejerrey resulted in a significant increase (P <0.02) 9 hours post-injection (hpi). These results add novel information on the nucleotide sequence of IGF-I in Atheriniformes and demonstrate that pjGHr could promote a dramatic response in liver, increasing the IGF-I mRNA level.

Surface-enhanced laser desorption/ionization time-of-flight proteomic profiling of breast carcinomas identifies clinicopathologically relevant groups of patients similar to previously defined clusters from cDNA expression

IntroductionMicroarray-based gene expression profiling represents a major breakthrough for understanding the molecular complexity of breast cancer. cDNA expression profiles cannot detect changes in activities that arise from post-translational modifications, however, and therefore do not provide a complete picture of all biologically important changes that occur in tumors. Additional opportunities to identify and/or validate molecular signatures of breast carcinomas are provided by proteomic approaches. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) offers high-throughput protein profiling, leading to extraction of protein array data, calling for effective and appropriate use of bioinformatics and statistical tools.MethodsWhole tissue lysates of 105 breast carcinomas were analyzed on IMAC 30 ProteinChip Arrays (Bio-Rad, Hercules, CA, USA) using the ProteinChip Reader Model PBS IIc (Bio-Rad) and Ciphergen ProteinChip software (Bio-Rad, Hercules, CA, USA). Cluster analysis of protein spectra was performed to identify protein patterns potentially related to established clinicopathological variables and/or tumor markers.ResultsUnsupervised hierarchical clustering of 130 peaks detected in spectra from breast cancer tissue lysates provided six clusters of peaks and five groups of patients differing significantly in tumor type, nuclear grade, presence of hormonal receptors, mucin 1 and cytokeratin 5/6 or cytokeratin 14. These tumor groups resembled closely luminal types A and B, basal and HER2-like carcinomas.ConclusionOur results show similar clustering of tumors to those provided by cDNA expression profiles of breast carcinomas. This fact testifies the validity of the SELDI-TOF MS proteomic approach in such a type of study. As SELDI-TOF MS provides different information from cDNA expression profiles, the results suggest the technique's potential to supplement and expand our knowledge of breast cancer, to identify novel biomarkers and to produce clinically useful classifications of breast carcinomas.

Differential Immunoreactivity of p16 in Leiomyosarcomas and Leiomyoma Variants

Several studies have now examined the cDNA expression profiles of healthy myometrium, leiomyomas (LM), and leiomyosarcomas (LMS). This has produced a list of candidate genes that might be useful tools for distinguishing these entities from each other. The potential candidates identified from this body of research include insulinlike growth factor 1, h-caldesmon, cytokeratin 18, and the cyclin-dependent kinase 4 inhibitor, p16. To determine whether the immunohistochemical expression of these proteins could aid in the diagnosis of LMS and LM variants, we constructed a tissue microarray consisting of cases of healthy myometrium (n = 10), LM (not otherwise specified and variants; n = 47), and LMS (n = 8), and then measured the immunoreactivity of each of these proteins. The cases were scored on the basis of staining intensity (weak, moderate, or strong) and extent (focal or diffuse) and were assigned a final score from 0 to +3. Immunostaining for p16 was statistically stronger in LMS than in LM and its subtypes (P < 0.001). Specifically, the p16 immunostaining score in LMS cases (n = 8) was at least +2, whereas the p16 immunostaining scores in all LM cases (n = 47) were either 0 (n = 35) or +1 (n = 12). The expression of the remaining antibodies did not show a statistically significant difference between the 2 groups. Furthermore, none of the markers studied showed any differences among the LM variants. The results of this study confirm the overexpression of p16 in LMS and suggest that p16 can serve as a reliable immunohistochemical marker in distinguishing uterine LMS from LM and its benign variants.