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Questions and Answers

What technique was used to assess the suppressive impact of ATRA on growth in tissue slices?

  • Immunohistochemical techniques (correct)
  • Western blotting
  • Flow cytometry
  • RT-PCR analysis

    • In the context of ATRA sensitivity, how were the findings categorized based on Ki-67 levels?

  • High, medium, and low responses (correct)
  • Positive, negative, and neutral responses
  • Active, dormant, and proliferative responses
  • Clinical, subclinical, and symptomatic responses

    • What was one of the primary results of the Gene Set Enrichment Analysis (GSEA) performed on the RNA-Seq data?

  • Up-regulation of the Interferon Response & Immune Regulation (correct)
  • Up-regulation of the Extracellular Matrix
  • Down-regulation of the Interferon Response
  • No significant pathway changes observed

    • Which analysis was applied to quantitatively assess the distribution of samples in the G-INT and G-DIFF groups?

    <p>Nearest Template Prediction Classification</p> Signup and view all the answers

    What was the primary marker assessed to evaluate the effectiveness of ATRA in reducing cell proliferation?

    <p>Ki-67</p> Signup and view all the answers

    How many instances indicated a reduction of Ki-67 levels as a response to ATRA?

    <p>9 instances</p> Signup and view all the answers

    What was the predominant profile observed in the 375 gastric carcinoma samples from TCGA?

    <p>G-INT profile</p> Signup and view all the answers

    What was the overall effect of ATRA on tumor growth as indicated by the results?

    <p>Reduces Ki-67 levels</p> Signup and view all the answers

    What is indicated by the ATRA-score in assessing cell-line sensitivity to ATRA treatment?

    <p>An ATRA-score above 0.55 indicates high sensitivity to ATRA.</p> Signup and view all the answers

    Which gene expression characteristics are primarily affected by ATRA treatment in sensitive cell-lines?

    <p>Down-regulation of c-myc target-genes.</p> Signup and view all the answers

    What does a Gene Set Enrichment Analysis (GSEA) aim to identify in the context of the study?

    <p>Pathways altered by the ATRA treatment in responsive cell-lines.</p> Signup and view all the answers

    Which of the following statements best describes the interactome associated with lipid metabolism in the study?

    <p>It includes 28 factors related to lipid metabolism.</p> Signup and view all the answers

    What correlation does the linear-regression r-values demonstrate in this study?

    <p>A direct correlation between ATRA-score and mRNA regulation.</p> Signup and view all the answers

    Which proteins are involved in the regulation of inflammation that are part of the network centered around IRF1?

    <p>CCL2, TLR3, USF1, BIRC3</p> Signup and view all the answers

    What is the total number of genes affected by ATRA in HGC-27 and RERF-GC-1B cells combined?

    <p>9,620</p> Signup and view all the answers

    What was the purpose of conducting RNA-seq investigations on the gastric carcinoma cell lines?

    <p>To identify gene networks involved in ATRA's effects</p> Signup and view all the answers

    How many cell lines tested are known to exhibit low sensitivity to ATRA?

    <p>5</p> Signup and view all the answers

    Which of the following refers to the characteristic grouping observed in the transcriptomic profiles of the gastric cancer cell lines?

    <p>Genomic Intestinal and Genomic Diffuse</p> Signup and view all the answers

    What was conducted to ensure transcriptomic coherence with the CCLE database?

    <p>Correlation analysis of control-treated cell lines</p> Signup and view all the answers

    Which gene, associated with epithelial polarization, is up-regulated while specific gene-products related to adhesion are down-regulated?

    <p>CYTH3</p> Signup and view all the answers

    Which component was NOT mentioned in the factors regulating pre-mRNA processing?

    <p>CPS1</p> Signup and view all the answers

    What is the ATRA-score of the G-DIFF cell line that displayed poor sensitivity to ATRA?

    <p>0.50</p> Signup and view all the answers

    What type of analysis was performed prior to measuring differential expression between treated and control cell lines?

    <p>Initial profiling for sample clustering</p> Signup and view all the answers

    Which of the following pathways is not associated with the proteins forming the largest network around IRF1?

    <p>Lipid metabolism</p> Signup and view all the answers

    Which factor from the third and fourth networks is primarily associated with metabolic regulation?

    <p>SIRT3</p> Signup and view all the answers

    What characterizes the interactome of down-regulated gene products identified in the study?

    <p>Proteins essential for cell attachment and movement</p> Signup and view all the answers

    Which aspect of gastric cancer did ATRA-sensitivity assessments focus on?

    <p>Gene expression modulation</p> Signup and view all the answers

    Among G-INT cell lines, which ones are indicative of high sensitivity to ATRA?

    <p>AGS, HuG1-N, and OCUM-1</p> Signup and view all the answers

    In the context of differential expression analysis, which sample type was used as a control?

    <p>DMSO control-treated cell lines</p> Signup and view all the answers

    What number of genes are up-regulated in response to ATRA within cell lines responsive to retinoids?

    <p>33</p> Signup and view all the answers

    Which gene-products are part of the second-largest interactome discussed in the context?

    <p>CDH17 and DSG3</p> Signup and view all the answers

    What was the correlation sought between the cell lines in the laboratory and the CCLE database?

    <p>Transcriptomic coherence</p> Signup and view all the answers

    Which gene from the networks includes a role in mediating mitochondrial homeostasis?

    <p>SIRT3</p> Signup and view all the answers

    What is the primary goal of developing a gene-expression model in the context of ATRA-sensitive gastric cancer patients?

    <p>To select patients who would respond positively to ATRA treatment.</p> Signup and view all the answers

    Which computational method was employed to identify transcripts correlated with ATRA-sensitivity?

    <p>RNA-seq analysis from the CCLE database.</p> Signup and view all the answers

    How many protein-coding mRNAs show a positive correlation with ATRA-sensitivity?

    <p>26</p> Signup and view all the answers

    What do the size differences of nodes in the interaction network indicate?

    <p>The centrality of each gene within the network.</p> Signup and view all the answers

    What is one of the roles of proteins in the most extensive network identified?

    <p>Participation in tissue development.</p> Signup and view all the answers

    What is a significant finding regarding the correlations between mRNAs and ATRA-sensitivity?

    <p>Distinct sets of mRNAs demonstrate positive and negative correlations.</p> Signup and view all the answers

    Which of the following is not part of the network associated with ATRA-sensitivity?

    <p>LRP5</p> Signup and view all the answers

    What process does the STRING database facilitate in relation to the identified genes?

    <p>Exploring interactions and networks of gene products.</p> Signup and view all the answers

    Which of the following components was part of the second network identified in the research?

    <p>COL6A1</p> Signup and view all the answers

    What is a primary consideration when establishing transcriptomic coherence with the CCLE database?

    <p>Using only control-treated cell lines for correlation analysis</p> Signup and view all the answers

    In a differential expression analysis, what is the role of the control sample treated with DMSO?

    <p>To provide a baseline for comparison against treated samples</p> Signup and view all the answers

    Which outcome is typically assessed during a Gene Set Enrichment Analysis (GSEA) related to ATRA sensitivity?

    <p>The identification of pathways correlated with treatment response</p> Signup and view all the answers

    What aspect of lipid metabolism is specifically regulated in the context of ATRA treatment?

    <p>The modulation of fatty acid oxidation pathways</p> Signup and view all the answers

    What characterizes the assessment of ATRA sensitivity among different gastric cancer cell lines?

    <p>It evaluates transcriptomic profiles and their correlation with treatment efficacy</p> Signup and view all the answers

    What is the relationship between ATRA sensitivity and the expression of c-myc target genes?

    <p>c-myc target genes are selectively decreased in ATRA-sensitive cell lines.</p> Signup and view all the answers

    Which of the following best describes the Gene Set Enrichment Analysis conducted on the RNA-Seq data?

    <p>It determines the pathways altered specifically by ATRA in responding cell lines.</p> Signup and view all the answers

    What does the ATRA-score signify in the context of ATRA treatment assessment?

    <p>The level of sensitivity of cell lines to ATRA treatment.</p> Signup and view all the answers

    What was the main focus of the differential expression analysis in this study?

    <p>To correlate the number of mRNAs with the ATRA-scores of various cell lines.</p> Signup and view all the answers

    Which outcome is primarily associated with the interaction network involved in lipid metabolism?

    <p>It includes several factors contributing to lipid metabolic pathways.</p> Signup and view all the answers

    How does the anti-proliferative effect of ATRA manifest at a molecular level?

    <p>By reducing the expression of genes regulating the G2M checkpoint.</p> Signup and view all the answers

    Which factor is NOT included in the interactome that regulates lipid metabolism?

    <p>EGFR</p> Signup and view all the answers

    What role does the linear-regression r-values serve in this study's analysis?

    <p>They establish the strength of the relationship between ATRA-scores and transcript regulation.</p> Signup and view all the answers

    Which aspect of gene regulation is primarily impacted by lifestyle and environmental factors as mentioned in the findings?

    <p>Epigenetic factors</p> Signup and view all the answers

    How many genes are typically reported to experience significant up-regulation in retinoid-sensitive cell lines when treated with ATRA?

    <p>6 genes</p> Signup and view all the answers

    Which two gene-products are specifically mentioned to interact within the second-largest network related to retinoid metabolism?

    <p>CYP26B1 and DHRS3</p> Signup and view all the answers

    In the context of differential expression analysis, which process is highlighted as a primary effect caused by ATRA treatment?

    <p>Decrease in gene expression levels</p> Signup and view all the answers

    What is the result of ATRA's regulation on the expression of AHNAK2 in all G-INT and G-DIFF cell lines?

    <p>AHNAK2 expression is down-regulated</p> Signup and view all the answers

    Which gene is involved in the interaction with IRF1 as part of the gene networks discussed?

    <p>CTSS</p> Signup and view all the answers

    Which of the following statements correctly describes the impact of ATRA on retinoid-resistant GSS cells?

    <p>ATRA leads to an increase in 137 genes and a decrease in 89 genes</p> Signup and view all the answers

    Which cellular process do the proteins forming the second-largest interactome primarily regulate?

    <p>Retinoid metabolism</p> Signup and view all the answers

    Which gene-product is identified as being common to the enhanced expression in both G-DIFF and G-INT cell lines due to ATRA?

    <p>IRF1</p> Signup and view all the answers

    What is the primary focus of the GSEA performed on the RNA-Seq data in relation to ATRA treatment?

    <p>Evaluating differential gene expression patterns</p> Signup and view all the answers

    What is a key purpose of the Gene Set Enrichment Analysis (GSEA) in the context of evaluating ATRA sensitivity?

    <p>To identify specific gene expressions that correlate with differential sensitivity to ATRA</p> Signup and view all the answers

    Which aspect characterizes transcriptomic coherence with the Cancer Cell Line Encyclopedia (CCLE) database?

    <p>Consistency in gene expression patterns between established lines and experimental models</p> Signup and view all the answers

    What analysis is critical before differentiating expression between treated and control cell lines?

    <p>Initial normalization of raw RNA-seq data</p> Signup and view all the answers

    In the context of ATRA-sensitivity assessment, which factors are primarily focused on?

    <p>Epigenetic modifications influencing gene expression</p> Signup and view all the answers

    Which of the following decisions would NOT be impacted by the results of differential expression analysis?

    <p>Identifying biomarkers for early cancer detection</p> Signup and view all the answers

    What critical aspect is often overlooked when studying lipid metabolism regulation in cancer?

    <p>Influence on sphingolipid metabolism specifically</p> Signup and view all the answers

    Which feature is typically not derived from a successful application of transcriptomic analysis?

    <p>Generation of a comprehensive profile of the natural killer cells</p> Signup and view all the answers

    Which method is least likely to contribute to the understanding of differential expression in relation to ATRA treatment?

    <p>Experimental validation using live cell imaging</p> Signup and view all the answers

    What is one major limitation when interpreting results from the STRING database regarding gene interactions?

    <p>The interactions may not represent all biological contexts</p> Signup and view all the answers

    What do alterations at the epigenetic level signify in the context of gastric cancer treatment sensitivity?

    <p>They may signify differences in responses to targeted therapies</p> Signup and view all the answers

    What role does ATRA have in the regulation of gene expression in gastric cancer cell lines?

    <p>It influences both up-regulation and down-regulation of various genes.</p> Signup and view all the answers

    Which cell line exhibited the highest observed sensitivity to ATRA based on the provided data?

    <p>MKN45</p> Signup and view all the answers

    In the context of lipid metabolism, what is a critical finding related to the down-regulated gene-products?

    <p>They play a significant role in lipid homeostasis regulation.</p> Signup and view all the answers

    What was the purpose of the Gene Set Enrichment Analysis (GSEA) conducted in this study?

    <p>To identify differential gene expression related to ATRA sensitivity.</p> Signup and view all the answers

    Which factor contributes to the complexity of the interactome regarding lipid homeostasis in the context of ATRA treatment?

    <p>The presence of both inflammatory and antigen presentation-related proteins.</p> Signup and view all the answers

    What common trend was observed in the expression of genes across the G-INT cell lines in relation to ATRA treatment?

    <p>A mixture of up-regulation and down-regulation occurred.</p> Signup and view all the answers

    In relation to epithelial polarization, which gene's up-regulation is significant during ATRA treatment?

    <p>CYTH3</p> Signup and view all the answers

    What was discovered about the differential expression of genes in HGC-27 and RERF-GC-1B cells compared to LMSU and GCIY cells?

    <p>Fewer genes are up- or down-regulated in HGC-27 compared to LMSU cells.</p> Signup and view all the answers

    Which gene product is particularly mentioned as part of the second-largest interactome related to ATRA effects?

    <p>DSG3</p> Signup and view all the answers

    How many genes are indicated as being significantly affected by ATRA in LMSU cells?

    <p>1,886</p> Signup and view all the answers

    Which process may be enhanced due to the decrease in expression of specific gene-products linked to lipid metabolism regulation?

    <p>Epithelial polarization</p> Signup and view all the answers

    Which cell-line exhibited the highest sensitivity to ATRA among those tested?

    <p>AGS</p> Signup and view all the answers

    What is a significant characteristic of the interactome that contains down-regulated gene-products?

    <p>It contains proteins linked to cell adhesion and motility.</p> Signup and view all the answers

    Which gene is identified as up-regulated and previously associated with the enhancement of epithelial polarization?

    <p>CYTH3</p> Signup and view all the answers

    What does the differential expression analysis indicate when comparing ATRA-treated to control cells?

    <p>Redirection of gene expression profiles</p> Signup and view all the answers

    How many genes showed up-regulation in cell lines responsive to retinoids after ATRA treatment?

    <p>33 genes</p> Signup and view all the answers

    Which statistical approach helps in establishing transcriptomic coherence with external databases?

    <p>Linear regression analysis</p> Signup and view all the answers

    In the STRING analysis, what do the distinct networks formed by up-regulated genes suggest?

    <p>Gene collaboration in metabolic pathways</p> Signup and view all the answers

    Which gene product is part of the network involved in inflammation regulation around IRF1?

    <p>USF1</p> Signup and view all the answers

    What is the main purpose of the Gene Set Enrichment Analysis conducted on the collected RNA-Seq data?

    <p>To identify enriched pathways related to ATRA sensitivity</p> Signup and view all the answers

    Which proteins are primarily involved in lipid metabolism according to the analysis?

    <p>PCK1, NR1H3, PCSK9, GAL3ST1</p> Signup and view all the answers

    In assessing ATRA sensitivity, which ATRA-score range indicates high sensitivity in cell-lines?

    <p>ATRA-score &gt; 0.55</p> Signup and view all the answers

    What is critical for establishing transcriptomic coherence with external databases in the study?

    <p>Validating RNA-seq results through additional methods</p> Signup and view all the answers

    Which statement regarding the differential expression analysis of ATRA-treated cell-lines is accurate?

    <p>The analysis indicates unique gene expression patterns across different cell-lines.</p> Signup and view all the answers

    What was the primary aim of conducting Gene Set Enrichment Analysis (GSEA) in the context of ATRA treatment?

    <p>To identify pathways altered by ATRA in responsive cell-lines</p> Signup and view all the answers

    Which of the following proteins is NOT known to be part of the interactome associated with lipid metabolism as discussed in the content?

    <p>SRRM1</p> Signup and view all the answers

    What is the primary consideration for establishing transcriptomic coherence with the CCLE database?

    <p>The correlation of baseline expression with control-treated cell lines</p> Signup and view all the answers

    Which of the following represents a significant aspect of ATRA-sensitivity assessments in gastric cancer cell lines?

    <p>The identification of retinoid-responsive cell lines</p> Signup and view all the answers

    Which analysis is conducted before performing differential expression analysis between treated and control samples?

    <p>Initial profiling of sample clustering</p> Signup and view all the answers

    In the context of Gene Set Enrichment Analysis (GSEA) regarding ATRA sensitivity, which outcome is primarily assessed?

    <p>Differential expression of specific genomic signatures</p> Signup and view all the answers

    What type of genes are typically up-regulated within cell lines responsive to ATRA treatment?

    <p>Genes involved in retinoid signaling pathways</p> Signup and view all the answers

    Which factor is commonly explored when analyzing the relationship between ATRA sensitivity and lipid metabolism in gastric cancer cell lines?

    <p>Impact of retinoids on cholesterol metabolism</p> Signup and view all the answers

    Which approach is taken to ensure the accuracy of expression data when analyzing ATRA's effects on cell lines?

    <p>Performing correlation analyses with baseline expression data</p> Signup and view all the answers

    In terms of transcriptomic clustering, how many gastric cancer cell lines were categorized as part of the Genomic Intestinal (G-INT) group?

    <p>8</p> Signup and view all the answers

    What is a key characteristic of the two distinct transcriptomic profiles identified in the study?

    <p>They align with classifications established by previous research</p> Signup and view all the answers

    Which macro-cluster is down-regulated in response to ATRA treatment according to the findings?

    <p>Extracellular Matrix (EMC) and Cell Interaction</p> Signup and view all the answers

    What is a primary feature assessed in comparing ATRA-sensitivity among gastric cancer cell lines?

    <p>Expression of c-myc target genes</p> Signup and view all the answers

    In the context of establishing transcriptomic coherence, which is a crucial consideration?

    <p>Expression Levels of Specific Genes</p> Signup and view all the answers

    What type of analysis reveals the differential gene expression results as part of ATRA validation?

    <p>RNA-Seq Analysis</p> Signup and view all the answers

    Which outcome is typically measured during a Gene Set Enrichment Analysis (GSEA) related to ATRA sensitivity?

    <p>Statistical Significance of Pathway Enrichment</p> Signup and view all the answers

    What analysis method is used to quantitatively categorize samples into G-INT and G-DIFF groups?

    <p>Nearest Template Prediction Classification (NTP)</p> Signup and view all the answers

    Which component is NOT typically evaluated during differential expression analysis in ATRA studies?

    <p>Patient Demographics</p> Signup and view all the answers

    What is a crucial aspect of lipid metabolism regulation in the context of ATRA sensitivity assessments?

    <p>Fatty Acid Synthesis Inhibition</p> Signup and view all the answers

    Which protein is NOT associated with the most extensive network identified in relation to ATRA sensitivity?

    <p>COL6A1</p> Signup and view all the answers

    What correlation pattern was observed among protein-coding mRNAs in relation to ATRA-sensitivity?

    <p>Some mRNAs showed a mix of positive and negative correlations.</p> Signup and view all the answers

    Which aspect of lipid metabolism regulation is principally highlighted in the context of ATRA treatment?

    <p>Integration of gene expression with metabolic pathways.</p> Signup and view all the answers

    Which factor is NOT considered essential when establishing transcriptomic coherence with the CCLE database?

    <p>Selection of appropriate control samples.</p> Signup and view all the answers

    In the context of differential expression analysis, what is the objective regarding ATRA treatment?

    <p>To measure the expression differences between treated and control cell lines.</p> Signup and view all the answers

    Which outcome is a primary focus of the Gene Set Enrichment Analysis conducted related to ATRA-sensitivity?

    <p>Determination of gene networks associated with metabolic pathways.</p> Signup and view all the answers

    Which specific pattern in gene expression correlates positively with ATRA-sensitivity in gastric cancer?

    <p>High-Basal-Expression-Levels.</p> Signup and view all the answers

    Which pathway component does NOT feature in the most extensive gene interaction network identified?

    <p>Myogenesis pathway</p> Signup and view all the answers

    In relation to gene interactions, what does the node size in the network signify?

    <p>The number of interconnections the gene has.</p> Signup and view all the answers

    What is the main aim of developing a gene-expression model for ATRA-sensitive gastric cancer patients?

    <p>To predict individual responses to treatment.</p> Signup and view all the answers

    Study Notes

    ATRA-Sensitivity Classification

    • Gene Set Variation Analysis (GSVA) used to classify gastric cancer samples based on ATRA responsiveness.
    • Samples categorized into three response levels: low, medium, high.
    • Combination of clustering results from G-INT and G-DIFF profiles observed after 48 hours of treatment with either vehicle (DMSO) or ATRA.
    • Ki-67 proliferation marker assessment revealed ATRA reduced Ki-67 levels in nine cases: five from G-DIFF, three from G-INT, and one unspecified.

    Validation through RNA-Seq Analysis

    • Gene Set Enrichment Analysis (GSEA) performed to validate RNA-Seq findings in gastric cancer cell lines and xenografts.
    • Major macro-cluster up-regulated: Interferon Response & Immune Regulation.
    • Major macro-cluster down-regulated: Extracellular Matrix (EMC) and Cell Interaction.

    TCGA Analysis of Gastric Cancer Patients

    • Nearest Template Prediction Classification (NTP) indicated a dominant G-INT profile among 375 gastric carcinoma samples.
    • Importance of developing gene-expression models for identifying patients likely to respond positively to ATRA.

    Gene-Expression Patterns and ATRA-Sensitivity

    • Correlation between specific mRNA expression levels and ATRA-sensitivity established using computational methods.
    • Identification of 26 mRNAs positively correlated and 16 mRNAs negatively correlated with ATRA sensitivity.
    • STRING database analysis revealed four protein interaction networks formed by these genes.

    Network Analysis

    • Largest network includes 28 components related to tissue development and WNT pathway (e.g., PITX2, PAX9).
    • Second network focuses on myogenesis and collagen homeostasis.
    • Additional networks regulate pre-mRNA processing and metabolic/mitochondrial homeostasis.

    Transcriptomic Characterization of Gastric-Cancer Cell Lines

    • Transcriptomic profiles of 13 gastric cancer cell lines classified into two groups: G-INT and G-DIFF consistent with Tan et al.'s classification.
    • Validation of transcriptomic coherence with CCLE database enhances study reliability.
    • ATRA treatment observed to inhibit growth in G-INT neoplastic cells, associated with lipid homeostasis regulation.

    Differential Gene Expression Analysis

    • High ATRA-sensitivity correlates with G-INT cell lines (ATRA-score > 0.55) and low sensitivity in G-DIFF cell lines (ATRA-score < 0.55).
    • RNA-seq experiments conducted after 48-hour treatment with ATRA showed correlation between transcript expressions and ATRA-scores.
    • GSEA conducted indicated that ATRA reduces expression of genes regulated by the G2M checkpoint and E2F transcription factor.

    Pathways Affected by ATRA

    • ATRA treatment decreased expression of c-myc target genes, inhibiting cell proliferation.
    • Notable interactome for lipid metabolism includes numerous factors such as MOGAT3, DGAT2, and PCK1, highlighting ATRA's role in lipid metabolic pathway alterations.

    Differential Expression Analysis and ATRA Treatment Effects

    • ATRA sensitivity classified as high (ATRA-score > 0.55) in seven cell lines; low sensitivity (ATRA-score < 0.55) in six cell lines.
    • RNA-seq experiments involved treating each cell line with either a vehicle or ATRA (1.0 µM) for 48 hours, prior to growth suppression.
    • Linear regression analyses reveal a significant correlation between ATRA-scores and mRNA fluctuations in response to ATRA.
    • Gene Set Enrichment Analysis (GSEA) performed to identify altered pathways with the HALLMARK dataset.

    Anti-Proliferative Mechanism of ATRA

    • ATRA reduces gene expression linked to the G2M-checkpoint and E2F transcription-factor, inhibiting cell proliferation.
    • Specific down-regulation of c-myc target genes observed in sensitive cell lines.
    • Noteworthy interactome with 28 lipid metabolism-associated components, including MOGAT3, DGAT2, and CYP family members.
    • Smaller networks highlight other lipid metabolism regulators such as SPTLC3 and DEGS2.
    • In retinoid-sensitive G-DIFF cell lines, four specific genes (CYP26B1, RARB, DHRS3, TINAGL1) are significantly up-regulated.

    ATRA Effects on G-INT and G-DIFF Cell Lines

    • ATRA increases expression of six genes (IRF1, DHRS3, CTSS, PSMB10, CYP26B1, TINAGL1) in both G-DIFF and G-INT cell lines.
    • Significant decrease in expression of AHNAK2 noted across all G-INT/G-DIFF cell lines.
    • Interaction established between CTSS and PSMB10 with IRF1, and between CYP26B1 and DHRS3.

    Role of Epigenetic Factors

    • ATRA modulates epigenetic factors by influencing DNA methylation and histone modifications.
    • The interactome of down-regulated gene products related to cell adhesion and motility might enhance epithelial polarization.
    • Investigated five G-INT cell lines with low ATRA-sensitivity while also studying GSS, the only G-DIFF cell line with poor sensitivity.

    Gene Expression Impact of ATRA

    • ATRA affects expression in various cell lines: HGC-27 (6,063 genes), RERF-GC-1B (5,557 genes), and smaller impacts on LMSU (1,886 genes) and GCIY (914 genes).
    • In ATRA-responsive cell lines, 33 genes up-regulated while 10 genes down-regulated.
    • STRING analysis of up-regulated genes forms three distinct networks, with the largest surrounding IRF1, comprised of inflammation and antigen presentation factors.

    Transcriptomic Characterization

    • Initial clustering assessment of cell line samples indicated two transcriptomic profiles: Genomic Intestinal (G-INT) and Genomic Diffuse (G-DIFF).
    • Validation of transcriptomic coherence with the CCLE database was performed using control-treated (DMSO) cell lines to ensure reliability in the cell line panel.

    ATRA-Sensitivity Classification

    • Gene Set Variation Analysis (GSVA) used to classify gastric cancer samples based on ATRA responsiveness.
    • Samples categorized into three response levels: low, medium, high.
    • Combination of clustering results from G-INT and G-DIFF profiles observed after 48 hours of treatment with either vehicle (DMSO) or ATRA.
    • Ki-67 proliferation marker assessment revealed ATRA reduced Ki-67 levels in nine cases: five from G-DIFF, three from G-INT, and one unspecified.

    Validation through RNA-Seq Analysis

    • Gene Set Enrichment Analysis (GSEA) performed to validate RNA-Seq findings in gastric cancer cell lines and xenografts.
    • Major macro-cluster up-regulated: Interferon Response & Immune Regulation.
    • Major macro-cluster down-regulated: Extracellular Matrix (EMC) and Cell Interaction.

    TCGA Analysis of Gastric Cancer Patients

    • Nearest Template Prediction Classification (NTP) indicated a dominant G-INT profile among 375 gastric carcinoma samples.
    • Importance of developing gene-expression models for identifying patients likely to respond positively to ATRA.

    Gene-Expression Patterns and ATRA-Sensitivity

    • Correlation between specific mRNA expression levels and ATRA-sensitivity established using computational methods.
    • Identification of 26 mRNAs positively correlated and 16 mRNAs negatively correlated with ATRA sensitivity.
    • STRING database analysis revealed four protein interaction networks formed by these genes.

    Network Analysis

    • Largest network includes 28 components related to tissue development and WNT pathway (e.g., PITX2, PAX9).
    • Second network focuses on myogenesis and collagen homeostasis.
    • Additional networks regulate pre-mRNA processing and metabolic/mitochondrial homeostasis.

    Transcriptomic Characterization of Gastric-Cancer Cell Lines

    • Transcriptomic profiles of 13 gastric cancer cell lines classified into two groups: G-INT and G-DIFF consistent with Tan et al.'s classification.
    • Validation of transcriptomic coherence with CCLE database enhances study reliability.
    • ATRA treatment observed to inhibit growth in G-INT neoplastic cells, associated with lipid homeostasis regulation.

    Differential Gene Expression Analysis

    • High ATRA-sensitivity correlates with G-INT cell lines (ATRA-score > 0.55) and low sensitivity in G-DIFF cell lines (ATRA-score < 0.55).
    • RNA-seq experiments conducted after 48-hour treatment with ATRA showed correlation between transcript expressions and ATRA-scores.
    • GSEA conducted indicated that ATRA reduces expression of genes regulated by the G2M checkpoint and E2F transcription factor.

    Pathways Affected by ATRA

    • ATRA treatment decreased expression of c-myc target genes, inhibiting cell proliferation.
    • Notable interactome for lipid metabolism includes numerous factors such as MOGAT3, DGAT2, and PCK1, highlighting ATRA's role in lipid metabolic pathway alterations.

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    Description

    Summary Overview of the Outcomes:

    This chapter explores the responsiveness of gastric cancer cells to All-trans Retinoic Acid (ATRA), highlighting its capacity to limit cell proliferation. The first goal is to categorize 37 gastric-cancer cell-lines into separate subgroups using the gene expression patterns obtained from the Cancer Cell Line Encyclopedia's RNA-sequencing data. In addition, the study involves evaluating ATRA-sensitivity in a significant number of these cell lines. Furthermore, we investigate the role of RARα in the anti-proliferative effects of ATRA and the genetic networks linked to ATRA sensitivity. Moreover, our research includes the assessment of ATRA's ability to limit growth in certain cell lines and tissue samples obtained from patients.

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