Analysis Of Differential Expression Profiles Research Articles

Integrating genome-wide association and transcriptome analysis to provide molecular insights into heterophylly and eco-adaptability in woody plants.

Heterophylly is regard as an important adaptive mechanism in response to different environments within plants. However, the genetic mechanisms responsible for heterophylly in woody plants are still poorly understood. Herein, the divergence of heterophyllous leaves was investigated at morphogenesis and using microdissection and physiological indexes in paper mulberry, and the genetic basis of heterophylly was further revealed combined with genome-wide association study (GWAS), transcriptome analysis and weighted gene coexpression network analysis (WGCNA). Our results revealed that the flavonoid content and antioxidant activity increased gradually from the entire leaf to the palmatisect leaf, while the hormone content and net photosynthetic rate decreased. Through GWAS and transcriptome analysis, a total of 98 candidate genes and 2338 differentially expressed genes associated with heterophylly were identified. Importantly, we uncovered critical variations in the candidate genes Bp07g0981 (WOX) and Bp07g0920 (HHO), along with significant differences in haplotypes and expression levels among heterophyllous leaves. Our results also suggested that the genes involved in hormone signaling pathways, antioxidant activity, and flavonoid metabolism might be closely related to the heterophylly of paper mulberry, which could account for the physiological data. Indeed, CR-wox mutant lines showed significant changes in leaf phenotypes, and differential expression profile analysis also highlighted the expression of genes related to phytohormones and transcription factors. Together, the genetic variations and candidate genes detected in this study provide novel insights into the genetic mechanism of heterophylly, and would improve the understanding of eco-adaptability in heterophyllous woody plants.

Paracrine effects of mir-210-3p on angiogenesis in hypoxia-treated c-kit-positive cardiac cells

Objective: Treatment with c-kit-positive cardiac cells (CPCs) has been shown to improve the prognosis of ischemic heart disease. MicroRNAs (miRNAs) confer protection by enhancing the cardiac repair process, but their specific functional mechanisms remain unclear. This study aimed to screen for differentially expressed miRNAs in CPCs under hypoxia and explore their effects on the function of CPCs. Methods: We harvested CPCs from C57 adult mice and later performed a high-throughput miRNA sequencing for differential expression profiling analysis. Subsequently, we intervened with the differentially expressed gene miR-210-3p in CPCs and detected changes in the secretion of angiogenesis-related factors through a protein-chip analysis. Finally, we applied CPC supernatants of different groups as conditioned medium to treat mouse cardiac microvascular endothelial cells (CMECs) and further investigated the functional effects of miR-210-3p on c-kit+CPCs under ischemia and hypoxia conditions. Results: The miR-210-3p was highly increased in hypoxia-treated CPCs. Protein-chip detection revealed that CPCs expressed cytokines such as FGF basic, angiogenin, and vascular endothelial growth factor (VEGF) and that hypoxia enhanced their release. Silencing miR-210-3p resulted in a reduction in the release of these angiogenesis-related factors. In addition, the conditioned medium of hypoxia-treated CPCs promoted the proliferation, migration, and tube-forming capabilities of CMECs. In contrast, the conditioned media of CPCs with silenced miR-210-3p after hypoxia decreased the proliferation, migration, and tube-forming ability of CMEC. Conclusions: The CPCs exert proangiogenic effects via paracrine pathways mediated by miR-210-3p. Upregulation of miR-210-3p in hypoxia-treated CPCs may enhance their paracrine function by regulating the secretion of angiogenic factors, thereby promoting angiogenesis in ischemic heart disease.

Analysis of Differential Expression Profiles of Liver Cancer Cell Proteins After Treatment with Bile Acid

The pathogenesis of liver cancer has not been fully elucidated yet. Bile acids are components of bile, which are inorganic substances and regulate tumor progression. However, the differential expression profile of liver cancer cell proteins after bile acid treatment remains unclear. Human hepatoma cell line SMMC7721 was cultured and randomly assigned into control group and bile acid group followed by measuring the protein expression profile by protein fingerprinting. SMMC7721 cells were transfected with UGT2B or UGT2B, followed by analysis of UGT2B expression, cell proliferation, apoptosis, migration and PI3K/AKT signaling protein expression. The most obvious proteins with an increased expression after bile acid treatment were UGT2B, AAP, APLP2, LAPTM4B, NCOA4 with UGT2B being the most significant one. Overexpression of UGT2B decreased cell proliferation, promoted cell apoptosis, downregulated migration ability and AKT phosphorylation (P <0.05). UGT2B siRNA transfection significantly down-regulated UGT2B expression, promoted cell proliferation, decreased apoptosis rate, increased migration ability and AKT phosphorylation (P <0.05). In conclusion, bile acid can alter the protein expressions of liver cancer cells, with UGT2B being changed most obviously. UGT2B can affect liver cancer cell behaviors via modulating PI3K/AKT signaling.

Comprehensive Analysis of Differential Expression Profiles of Long Noncoding RNAs with Associated Co-expression and Competing Endogenous RNA Networks in the Hippocampus of Patients with Alzheimer's Disease.

Alzheimer's disease (AD) is still one of the major threats to human health. Although a satisfactory treatment for AD has not yet been discovered, it is necessary to continue to search for novel approaches to deal with this insidious and debilitating disease. Although numerous studies have shown that long non-coding RNA (lncRNA) occupy a significant role in a variety of diseases, their roles in AD remain unclear. Using data analysis to explore the role of lncRNA in the course of AD, to further our understanding of AD, and to look forward to finding a new breakthrough for the treatment of AD. We downloaded and screened expression data of the hippocampal regions of patients with AD from the Gene Expression Omnibus database. We generated lncRNA-miRNA-mRNA networks based on the competing endogenous RNA (ceRNA) hypothesis, and according to gene expression level, we constructed a coding-noncoding co-expression (CNC) network and then executed cis- and trans-regulation analyses. Through comprehensive and systematic analyses, we found that lncRNAs MALAT1, OIP5-AS1, LINC00657, and lnc-NUMB-1 regulated the expression of the key AD pathogenic genes APP, PSEN1, BACE1; and that these lncRNAs may promote the distribution of β-amyloid (Aβ protein) in the brain through exosomes. In addition, lncRNAs were found to adjust viral transcriptional expression, thereby further supporting viral pathogenesis for AD. The lncRNAs MALAT1, OIP5-AS1, LINC00657, and lnc-NUMB-1 that are present in the hippocampus of AD patients exert an important influence on the development of this disease.

Comprehensive analysis of differential expression profiles via transcriptome sequencing in SH-SY5Y cells infected with CV-A16.

Coxsackievirus A16 (CV-A16) is one of the viruses that is most frequently associated with hand-foot-and-mouth disease (HFMD). Previous studies have shown that CV-A16 infections are mostly self-limiting, but in recent years, it has been gradually found that CV-A16 infections can also induce neurological complications and eventually cause death in children with HFMD. Moreover, no curative drugs or preventative vaccines have been developed for CV-A16 infection. Therefore, it is particularly important to investigate the mechanism of CV-A16 infection-induced neuropathy. In the current study, transcriptome sequencing technology was used to identify changes in the transcriptome of SH-SY5Y cells infected with CV-A16, which might hide the mechanism of CV-A16-induced neuropathology. The transcriptome profiling showed that 82,406,974, 108,652,260 and 97,753,565 clean reads were obtained in the Control, CV-A16-12 h and CV-A16-24 h groups, respectively. And it was further detected that a total of 136 and 161 differentially expressed genes in CV-A16-12 h and CV-A16-24 h groups, respectively, when compared with Control group. Then, to explore the mechanism of CV-A16 infection, we focused on the common differentially expressed genes at different time points of CV-A16 infection and found that there were 34 differentially expressed genes based on which clustering analysis and functional category enrichment analysis were performed. The results indicated that changes in oxidation levels were particularly evident in the GO term analysis, while only the "Gonadotropin-releasing hormone receptor pathway" was enriched in the KEGG pathway analysis, which might be closely related to the neurotoxicity caused by CV-A16 infection. Meanwhile, the ID2 closely related to nervous system has been demonstrated to be increased during CV-A16 infection. Additionally, the data on differentially expressed non-protein-coding genes of different types within the transcriptome sequencing results were analyzed, and it was speculated that these dysregulated non-protein-coding genes played a pivotal role in CV-A16 infection. Ultimately, qRT-PCR was utilized to validate the transcriptome sequencing findings, and the results of qRT-PCR were in agreement with the transcriptome sequencing data. In conclusion, transcriptome profiling was carried out to analyze response of SH-SY5Y cells to CV-A16 infection. And our findings provide important information to elucidate the possible molecular mechanisms which were linked to the neuropathogenesis of CV-A16 infection.

Analysis of differential expression profile of miRNA in peripheral blood of patients with lung cancer.

PurposeTo identify potential molecular targets for lung cancer intervention and diagnosis, we analyzed the differential miRNA expression of peripheral blood between lung cancer patients and healthy controls.MethodsThree pairs of cases’ and controls’ peripheral blood samples were evaluated for miRNA expression by microarray. 12 miRNAs were selected for RT‐PCR validation and target genes prediction. In addition, 4 miRNAs were selected for future validation by RT‐PCR in a large sample of 145 cases and 55 frequency‐matched healthy controls.ResultsA total of 338 differentially expressed miRNAs were screened and identified by microarray. According to the fold changes, the top ten upregulated miRNAs were hsa‐miR‐124‐3p, hsa‐miR‐379‐5p, hsa‐miR‐3655, hsa‐miR‐450b‐5p, hsa‐miR‐29a‐5p, hsa‐miR‐200a‐3p, hsa‐miR‐542‐3p, hsa‐miR‐138‐5p, hsa‐miR‐219a‐2‐3p, and hsa‐miR‐4701‐3p, and the top ten downregulated miRNAs were hsa‐miR‐34c‐5p, hsa‐miR‐135a‐5p, hsa‐miR‐132‐3p, hsa‐miR‐3178, hsa‐miR‐4449, hsa‐miR‐4999‐3p, hsa‐miR‐1246, hsa‐miR‐4424, hsa‐miR‐1252‐5p, and hsa‐miR‐24‐2‐5p. RT‐PCR verification of the 12 miRNAs revealed that 5 of 8 upregulated miRNAs, 2 of 4 downregulated miRNAs showed a significant difference between the cases and controls (P < .05). A large number of target genes and their functional set showed overlapping among the 453 predicted target genes of the 12 miRNAs (P < .01). RT‐PCR in the large sample confirmed the significant differential expression level of hsa‐miR‐29a‐5p, hsa‐miR‐135a‐5p, hsa‐miR‐542‐3p, and hsa‐miR‐4491 between cases and controls (P < .05), and three of these microRNA, except hsa‐miR‐29a‐5p, were significant after Bonferroni correction for adjustment of multiple comparisons.ConclusionThere was a significant difference in miRNAs expression in the peripheral blood between lung cancer patients and healthy controls, and 4 miRNAs were validated by a large‐size sample.

Differential expression profile analysis of cisplatin‑regulated miRNAs in a human gastric cancer cell line.

Cisplatin, one of the most commonly used drugs in combination chemotherapy, is an effective anti‑tumor agent widely used for diverse tumor types. MicroRNAs (miRNAs/miRs) are involved in the occurrence, development, diagnosis and treatment of cancer. Therefore, the aim of the current study was to explore whether cisplatin exerts anticancer effects by causing differential expression of miRNAs in human gastric cancer cells. The human gastric cancer cell line NCI‑N87 was cultured with a certain dose of cisplatin and high‑throughput sequencing combined with reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was performed to detect cisplatin‑regulated miRNAs. miRNAs upregulated and downregulated following cisplatin exposure were analyzed. High‑throughput sequencing revealed 33 upregulated and 16 downregulated miRNAs. A total of five significantly upregulated and five significantly downregulated miRNAs were identified by RT‑qPCR. The expression levels of hsa‑miR‑1246 and hsa‑miR‑892b were consistent with the results obtained from high‑throughput sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway clustering of cisplatin‑regulated miRNAs revealed that the miRNAs regulated genes involved in several biological processes and signaling pathways. The results obtained in the current study suggested that cisplatin may exert an important anticancer effect in gastric cancer via complex biological processes and signaling pathways.

Differential expression profile analysis of PSTK-regulated mRNAs in podocytes.

This study aimed to elucidate the precise mechanisms underlying the protective effects of phosphoseryl-tRNA kinase (PSTK) against cisplatin-induced podocyte injury. PSTK overexpression and knockdown vectors were generated and transfected into murine podocyte cells-5. PSTK levels were measured, and transcriptome sequencing was conducted. Differential expression analysis was performed to identify messenger RNAs (mRNAs) that were positively and negatively correlated with PSTK. We selected 10 candidate genes identified via real-time quantitative polymerase chain reaction and Western blot analysis for further analysis. As expected, PSTK levels were significantly higher in PSTK-overexpressing podocytes and significantly lower in PSTK-knockdown podocytes. PSTK overexpression resulted in the upregulation of 122 genes and downregulation of 372 genes in podocytes. On the other hand, PSTK knockdown resulted in the upregulation of 231 genes and downregulation of 445 genes. Furthermore, the analysis revealed that 11 genes were positively correlated with PSTK, whereas 20 genes were negatively correlated with PSTK. The obtained PSTK-regulated genes were primarily involved in molecular function, biological process, and cellular component, as well as the angiogenesis pathway. The Wnt family member 10A levels were significantly higher after PSTK overexpression, but were significantly lower after PSTK knockdown. In addition, Na+/K+ ATPase subunit α-2 and matrix metalloproteinase 9 levels were significantly downregulated after PSTK overexpression, but significantly upregulated upon PSTK knockdown. Cell proliferation was significantly increased upon PSTK overexpression, but significantly decreased upon PSTK suppression. The results of this study not only identified several significant PSTK-regulated genes for further validation, but also provided insights into the mechanisms underlying the protective effects of PSTK on podocytes.

Comprehensive analysis of differential expression profiles of mRNAs and lncRNAs and identification of a 14-lncRNA prognostic signature for patients with colon adenocarcinoma

The objective of this study was to identify potentially significant genes and long non-coding RNAs (lncRNAs) in colon cancer for a panel of lncRNA signatures that could be used as prognostic markers for colon adenocarcinoma (COAD) based on the data from The Cancer Genome Atlas (TCGA). RNA-seq V2 exon data of COAD were downloaded from the TCGA data portal for 285tumor samples and 41normal tissue samples adjacent to tumors. Differentially expressed mRNAs and lncRNAs were identified. A functional enrichment analysis of differentially expressed mRNAs was performed, followed by protein-protein interaction (PPI) network construction and significant module selection. Additionally, the regulatory relationships in differentially expressed mRNAs and lncRNAs were assessed, and an lncRNA-lncRNA co-regulation and functional synergistic analysis were performed. Furthermore, the risk score model and Cox regression analysis based on the expression levels of lncRNAs were used to develop a prognostic lncRNA signature. A total of 976 differentially expressed mRNAs and 169 differentially expressed lncRNAs were identified. MDFI and MEOX2 were the PPI network hubs. We found these lncRNAs to be mainly involved in vascular smooth muscle contraction and the cGMP-PKG signaling pathway. Several lncRNA-lncRNA pairs had co-regulatory relationships or functional synergistic effects, including BVES-AS1/MYLK-AS1, ADAMTS9-AS1/MYLK-AS1 and FENDRR/MYLK-AS1. The differential expression profile analysis of four candidate lncRNAs (MYLK-AS1, BVES-AS1, ADAMTS9-AS1, and FENDRR) in COAD tumors were confirmed by reverse transcription-quantitative PCR. Moreover, this study identified a 14-lncRNA signature that could predict the survival for COAD patients.

Comprehensive analysis of differential expression profiles reveals potential biomarkers associated with the cell cycle and regulated by p53 in human small cell lung cancer.

Small cell lung cancer (SCLC) is the subtype of lung cancer with the highest degree of malignancy and the lowest degree of differentiation. The purpose of this study was to investigate the molecular mechanisms of SCLC using bioinformatics analysis, and to provide new ideas for the early diagnosis and targeted therapy of SCLC. Microarray data were downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) in SCLC were compared with the normal lung samples and identified. Gene Ontology (GO) function and pathway analysis of DEGs was performed through the DAVID database. Furthermore, microarray data was analyzed by using the clustering analysis tool GoMiner. Protein-protein interaction (PPI) networks of DEGs were constructed using the STRING online database. Protein expression was determined from the Human Protein Atlas, and SCLC gene expression was determined using Oncomine. In total, 153 DEGs were obtained. Functional enrichment analysis suggested that the majority of DEGs were associated with the cell cycle. CCNB1, CCNB2, MAD2L1 and CDK1 were identified to contribute to the progression of SCLC through combined use of GO, Kyoto Encyclopedia of Genes and Genomes enrichment analysis and a PPI network. mRNA and protein expression were also validated in an integrative database. The present study indicated that the formation of SCLC may be associated with cell cycle regulation. In addition, the four crucial genes CCNB1, CCNB2, MAD2L1 and CDK1, which are downstream of p53, may have important roles in the occurrence and progression of SCLC, and thus may be promising potential biomarkers and therapeutic targets.

Systematic Analysis of Differential Expression Profile in Rheumatoid Arthritis Chondrocytes Using Next-Generation Sequencing and Bioinformatics Approaches.

Cartilage destruction in rheumatoid arthritis (RA) occurs primarily in the pannus-cartilage interface. The close contact of the synovium-cartilage interface implicates crosstalk between synovial fibroblasts and chondrocytes. The aim of this study is to explore the differentially expressed genes and novel microRNA regulations potentially implicated in the dysregulated cartilage homeostasis in joint destruction of RA. Total RNAs were extracted from human primary cultured normal and RA chondrocytes for RNA and small RNA expression profiling using next-generation sequencing. Using systematic bioinformatics analyses, we identified 463 differentially expressed genes in RA chondrocytes were enriched in biological functions related to altered cell cycle process, inflammatory response and hypoxic stimulation. Moreover, fibroblast growth factor 9 (FGF9), kynureninase (KYNU), and regulator of cell cycle (RGCC) were among the top dysregulated genes identified to be potentially affected in the RA joint microenvironment, having similar expression patterns observed in arrays of clinical RA synovial tissues from the Gene Expression Omnibus database. Additionally, among the 31 differentially expressed microRNAs and 10 candidate genes with potential microRNA-mRNA interactions in RA chondrocytes, the novel miR-140-3p-FGF9 interaction was validated in different microRNA prediction databases, and proposed to participate in the pathogenesis of joint destruction through dysregulated cell growth in RA. The findings provide new perspectives for target genes in the management of cartilage destruction in RA.

Transcriptome and Differential Expression Profiling Analysis of the Mechanism of Ca2+ Regulation in Peanut (Arachis hypogaea) Pod Development.

Calcium not only serves as a necessary nutrient for plant growth but also acts as a ubiquitous central hub in a large number of signaling pathways. Free Ca2+ deficiency in the soil may cause early embryo abortion, which eventually led to abnormal development of peanut pod during the harvest season. To understand the mechanisms of Ca2+ regulation in pod development, transcriptome analysis of peanut gynophores and pods was performed by comparing the treatments between free Ca2+ sufficiency and free Ca2+ deficiency using Illumina HiSeq™ 2000. 9,903,082,800 nt bases are generated totally. After assembly, the average length of 102,819 unigenes is 999 nt, N50 is 1,782 nt. RNA-seq based gene expression profilings showed a large number of genes at the transcriptional level changed significantly between the aerial pegs and underground swelling pods under free Ca2+ sufficienct or deficiency treatments, respectively. Genes encoding key members of Ca2+ signaling transduction pathway, enzymes for hormone metabolism, cell division and growth, transcriptional factor as well as embryo development were highlighted. This information provides useful information for our further study. The results of digital gene expression (DGE) indicated that exogenous calcium might contribute to the development of peanut pod through its signal transduction pathway, meanwhile, promote the normal transition of the gynophores to the reproductive development.

Differential expression profile analysis of lncRNA UCA1α regulated mRNAs in bladder cancer.

Urothelial carcinoma associated 1α (UCA1α) is a novel long non-coding RNA (lncRNA) that regulates bladder cancer proliferation, migration, and invasion. The target genes of UCA1α have, however, not been identified. To address this, a pCDNA3.1(+)-UCA1α over-expression vector was transfected into UM-UC-2 bladder cancer cells. Genes differentially expressed between pCDNA3.1(+)-UCA1α and pCDNA3.1(+) transfected cell were then detected by microarray and bioinformatics analysis. A total of 71 differentially expressed genes were identified, including 52 up-regulated genes and 19 down-regulated genes. As expected, the lncRNA UCA1α expression level was significantly increased when compared to that of pCDNA3.1(+) transfected cells. The five most significantly up-regulated and five most significantly down-regulated genes were selected, and their expression levels were also assessed by real time quantitative polymerase chain reaction and Western blot. The mRNA and protein expression levels of FOXI3 and GSTA3 were found to be significantly increased, and those of MED18 and TEX101 were found to be significantly decreased. Gene ontology (GO) clustering identified several significant biological processes, cellular components, and molecular functions, associated with lncRNA UCA1α over-expression. The differentially expressed genes were involved in several significant pathways as shown by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway clustering. Cell proliferation activity was significantly increased following overexpression of lncRNA UCA1α increasing over culture time. The present study identifies, for the first time, potential target genes for lncRNA UCA1α in bladder cancer, and provides a significant reference for studying the role of lncRNA UCA1α in bladder cancer.

MiR-33a inhibits cell proliferation and invasion by targeting CAND1 in lung cancer.

Lung cancer continues to be one of the top five causes of cancer-related mortality. This study aims to identify down- and upregulated miRNAs and mRNA which can be used as potential biomarkers and/or therapeutic targets for lung cancer. Integrated analysis of differential expression profiles of miRNA and mRNA in lung cancer was performed by searching Gene Expression Omnibus datasets. Based on miRNA expression profiles, direct mRNA targets of miRNAs with experimental support were identified through miRTarBase. The levels of representative miRNAs and mRNAs were confirmed through qualitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). The miR-33a was decreased in non-small cell lung cancer (NSCLC) tissues compared with the para-carcinoma tissues, whereas its target mRNA of cullin-associated NEDD8-dissociated protein 1 (CAND1) was increased in NSCLC tissues. Further research has shown that miR-33a can inhibit lung cancer cell proliferation, cell cycle progression, and migration by targeting CAND1. Moreover, the CAND1 knockout lung cancer cells showed similar results as cells transfected with miR-33a mimic. These results suggested that the data mining based on online databases was an effective method in finding novel target in cancer research, and the miR-33a and CAND1 played an important role in lung cancer proliferation and cell migration.

Abstract 5933: Cancer-associated fibroblasts (CAFs) release exosomal microRNAs that dictate an aggressive phenotype in breast cancer

Abstract Cancer-associated fibroblasts (CAFs), the major components of the tumor microenvironment may drive tumor progression. Exosomes mediate horizontal transfer of miRs, mRNAs and proteins, thus affecting breast cancer progression. Differential expression profile analysis identified three miRs increased in exosomes from CAFs as compared from normal fibroblasts. Immunofluorescence indicated that exosomes may be transferred from CAFs to breast cancer cells, releasing their cargo miRs. Breast cancer cells exposed to CAF exosomes or transfected with those miRs exhibited a significant increased capacity to form mammospheres, increased stem cell and epithelial-mesenchymal transition (EMT) markers, and anchorage-independent cell growth. These effects were reverted by transfection with anti-miRs. Similarly to CAF exosomes, normal fibroblast exosomes transfected with CAFs-miRs promoted the stemness and EMT phenotype of breast cancer cells. Thus, we provided evidence of the role of CAF exosomes and their miRs in the induction of the stemness and EMT phenotype in different breast cancer cell lines. Note: This abstract was not presented at the meeting. Citation Format: Gerolama Condorelli, Elvira Donnarumma, Danilo Fiore, Martina Nappa, Giuseppina Roscigno, Assunta Adamo, Margherita Iaboni, Valentina Russo, Alessandra Affinito, Cristina Quintavalle, Anna Rienzo, Salvatore Piscuoglio, Renato Thomas. Cancer-associated fibroblasts (CAFs) release exosomal microRNAs that dictate an aggressive phenotype in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5933. doi:10.1158/1538-7445.AM2017-5933

Differential expression profile analysis of DNA damage repair genes in CD133+/CD133- colorectal cancer cells.

The present study examined differential expression levels of DNA damage repair genes in COLO 205 colorectal cancer cells, with the aim of identifying novel biomarkers for the molecular diagnosis and treatment of colorectal cancer. COLO 205-derived cell spheres were cultured in serum-free medium supplemented with cell factors, and CD133+/CD133- cells were subsequently sorted using an indirect CD133 microbead kit. In vitro differentiation and tumorigenicity assays in BABA/c nude mice were performed to determine whether the CD133+ cells also possessed stem cell characteristics, in addition to the COLO 205 and CD133- cells. RNA sequencing was employed for the analysis of differential gene expression levels at the mRNA level, which was determined using reverse transcription-quantitative polymerase chain reaction. The mRNA expression levels of 43 genes varied in all three types of colon cancer cells (false discovery rate ≤0.05; fold change ≥2). Of these 43 genes, 30 were differentially expressed (8 upregulated and 22 downregulated) in the COLO 205 cells, as compared with the CD133- cells, and 6 genes (all downregulated) were differentially expressed in the COLO 205 cells, as compared with CD133+ cells. A total of 18 genes (10 upregulated and 8 downregulated) were differentially expressed in the CD133- cells, as compared with the CD133+ cells. By contrast, 6 genes were downregulated and none were upregulated in the CD133+ cells compared with the COLO 205 cells. These findings suggest that CD133+ cells may possess the same DNA repair capacity as COLO 205 cells. Heterogeneity in the expression profile of DNA damage repair genes was observed in COLO 205 cells, and COLO 205-derived CD133- cells and CD133+ cells may therefore provide a reference for molecular diagnosis, therapeutic target selection and determination of the treatment and prognosis for colorectal cancer.

Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer.

Cancer-associated fibroblasts (CAFs) are the major components of the tumor microenvironment. They may drive tumor progression, although the mechanisms involved are still poorly understood. Exosomes have emerged as important mediators of intercellular communication in cancer. They mediate horizontal transfer of microRNAs (miRs), mRNAs and proteins, thus affecting breast cancer progression. Differential expression profile analysis identified three miRs (miRs -21, -378e, and -143) increased in exosomes from CAFs as compared from normal fibroblasts. Immunofluorescence indicated that exosomes may be transferred from CAFs to breast cancer cells, releasing their cargo miRs. Breast cancer cells (BT549, MDA-MB-231, and T47D lines) exposed to CAF exosomes or transfected with those miRs exhibited a significant increased capacity to form mammospheres, increased stem cell and epithelial-mesenchymal transition (EMT) markers, and anchorage-independent cell growth. These effects were reverted by transfection with anti-miRs. Similarly to CAF exosomes, normal fibroblast exosomes transfected with miRs -21, -378e, and -143 promoted the stemness and EMT phenotype of breast cancer cells. Thus, we provided evidence for the first time of the role of CAF exosomes and their miRs in the induction of the stemness and EMT phenotype in different breast cancer cell lines. Indeed, CAFs strongly promote the development of an aggressive breast cancer cell phenotype.

Go protein subunit Goα and the secretory process of the natriuretic peptide hormones ANF and BNP.

Expression of the G protein subunit Goα has been shown to be prominent in the atria of the rat heart and to be significantly associated with atrial natriuretic factor (ANF)-containing atrial-specific secretory granules by immunocytochemistry. In addition, differential expression profile analysis using oligonucleotide arrays has shown that the Goα isoform 1 (Goα1) is 2.3-fold more abundant in the atria than it is in the ventricles. In the present report, we show protein-protein interaction between Goα and ANF by yeast two-hybrid and by immunoprecipitation. A cardiac conditional Goα knockout model developed for the present study showed a 90% decrease in Goα expression and decreased atrial expression and ANF and brain natriuretic peptides (BNP) content. Expression of chromogranin A, a specific atrial granule core constituent, was not affected. Morphometric assessment of atrial tissue showed a very significant decrease in atrial-specific granule density as well as granule core electron density. Atrial electrical activity was not affected. The results obtained are compatible with the suggestion that Goα plays a role in ANF sorting during intracellular vectorial transport and with the presence of a mechanism that preserves the molar relationship between cellular ANF and BNP stores in the face of the decreased production of these hormones.

Identification of characteristic TRB V usage in HBV-associated HCC by using differential expression profiling analysis

Liver cancer is one of the most common cancers worldwide. CDR3 sequencing-based immune repertoire can be closely associated with cancer prognosis and development. Identifying the specific interaction between the TCR and cellular antigens is important for developing novel immunotherapeutic approaches for the treatment of cancer. The rearranged TCRβ loci amplified using Vβ- and Jβ-specific primers by multi-PCR and sequenced using high-throughput sequencing (HTS) in liver cancers were compared with those of T cells from healthy adult peripheral blood and from adjacent liver tissue. The T-cell repertoires within each tumor show strong similarity to one another but are distinct from those of the circulating T-cell repertoire. In addition, our results demonstrate that there are significant differences in the T-cell repertoires of HCC (hepatocellular carcinoma), ICC (intrahepatic cholangiocarcinoma), and MHC (mixed hepatocellular and cholangiocellular carcinoma). Furthermore, we found that the highly expanded clone (HEC) ratio in blood samples from liver cancer patients differed significantly from those in the blood of healthy adults and hepatitis patients (p < 0.001). The above results suggest that comparison of the T-cell repertoires of tissue and blood could be used to distinguish liver cancer patients from healthy adults and from hepatitis patients. In the future, the diversity of CDR3 sequences in liver cancer may prove to be a useful and novel biomarker for detecting aggressive tumors with high invasive or metastatic capacity.

Abstract A59: MDA-MB-231 stem cell subpopulation upregulates KRAB domain-containing zinc-finger gene family to hold undifferentiated ground state

Abstract Introduction: Breast cancer cell lines frequently contain a sub-population of stem cells (SC). MDA-MB-231 is a model of basal-like breast cancer triple negative (TN) lineage widespread used in laboratories across the world. It contains a stem cell subpopulation, however little is known about the molecular mechanisms underlying undifferentiated ground state regulation in this type of cell. Aberrations in epigenetic processes such as histone (de)methylation may cause cancer. The largest family of zinc-finger transcription factors is composed of the Krüpell-associated box (KRAB) domain containing proteins. They do not have intrinsic enzymatic activity, instead, this transcription factor family recruits chromatin remodelers and histone-modifying enzymes to regulate gene transcription, generally acting as repressor proteins. Underlying molecular mechanisms for aggressiveness of basal-like/TN breast cancer stem cell remains to be elucidated. Objective: To investigate the molecular basis of MDA-MB-231 sub-populations (progenitor and differentiated cells), in order to obtain and compare the genome expression profile of stem cell and of differentiated subpopulations and identify gene networks regulating distinct differentiation states. Experimental Procedures: Cells were cultured in T75 flasks, suspended in Aldefluor assay buffer and incubated for 30 minutes for staining. Heterogeneity of aldehyde dehydrogenase expression in MDA-MB-231 sub-populations was revealed by Aldefluor flow cytometry-based assay. Separated cell sub-populations were harvested, had their RNA extracted (TRIzol), purified (silica columns plus DNase), measured (Nanodrop) and analyzed (Bioanalyzer). RNA samples were submitted to Agilent whole genome (8X60K) microarray experiments (one-color), according to manufacturer's instructions. cDNA samples from each experimental group (Aldefluor-positive cells, Aldefluor-negative cells and overall population - control) were loaded into a genome array. Experiments were performed in triplicates. The analysis of differential expression profiles were performed by GX11.5 GeneSpring software. Results: Fluorescence activated cell sorting (FACS) assay detected 4% of the overall MDA-MB-231 cell population as Aldefluor positive and 50% as non-Aldefluor positive. Microarray results analyzed with GeneSpring software demonstrate that among 337 genes upregulated (fold change ≥ 2.0, p≤ 0.001) exclusively in the stem cell-like subpopulation. Gene Ontology (GO) terms allowed annotation of these 337 upregulated genes as, molecular function (61.7.0%), cellular component (46.2) and biological process (2.1%). Among sixty-four molecular function related genes, twety-two (34.4%) are zinc-finger proteins and eighteen of them are genes coding for KRAB domain-containing zinc-finger proteins (ZNF688, ZNF419, ZNF397, ZNF821, ZNF12, ZNF280C, ZNF821, ZNF239, ZNF468, ZNF702P, ZNF496, ZNF205, ZNF829, ZNF345, ZNF526, ZNF329, ZNF561 and ZNF562). HDAC5 is downregulated 3.13 times in stem cell-like subpopulation when compared to Aldefluor negative sub-population. Conclusions: Our results show that the upregulation of the Krüpell family of zinc finger proteins might be part of the genome signature that distincts MDA-MB-231 stem cell sub-population from its progeny. It is known that HDACs have an important role in mammary tumor cell growth control. KRAB-containing zinc-finger proteins are involved in transcriptional regulation by recruiting histone modifiers and chromatin remodelers like, DNMT3a, HDAC5, JARID2 and Activation Protein 1 (AP1), in order to repress gene transcription. This being the case, zinc-finger proteins are linked to epigenetic processes and can be selectively targeted to regulate gene expression and determine cell fate. Citation Format: Samuel Marcos Ribeiro de Noronha, Carlos Fernandes Baptista, Werica Bernardo, Ismael Dale Cotrim Guerreiro Silva, Silvana Aparecida Alves Correa-Noronha. MDA-MB-231 stem cell subpopulation upregulates KRAB domain-containing zinc-finger gene family to hold undifferentiated ground state. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A59.