Final project-kbakshy
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The document describes a study that analyzed gene expression data from ovarian carcinoma cell lines treated with and without the chemotherapy drug cisplatin. The study aimed to explore the connection between cellular responses to cisplatin and epithelial-mesenchymal transition (EMT). Gene expression microarrays were used to analyze 171 samples and identify differentially expressed genes between epithelial-like and mesenchymal-like cell lines. Statistical tests identified over 6500 differentially expressed genes. Classification analysis correctly predicted the epithelial or mesenchymal status of 70 test samples based on their gene expression profiles. Top genes found to be upregulated or downregulated in epithelial versus mesenchymal cell lines are involved in processes like cell adhesion, migration and proliferation
![Introduction
• Expression data from 46 cultured human ovarian
carcinoma cell lines with and without Cisplatin
treatment
• Array: A-AFFY-141 - Affymetrix GeneChip Human
Gene 1.0 ST Array [HuGene-1_0-st-v1] (GPL6244)
• Technology type: in situ oligonucleotide
• Experiment type: transcription profiling by array
• Samples: 171
• NCBI GEO accession no. GSE47856
(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GS
E47856)
Epithelial-mesenchymal status renders differential responses to cisplatin in ovarian cancer. Miow QH,
Tan TZ, Ye J, Lau JA, Yokomizo T, Thiery JP, Mori S. , Europe PMC 24858042](https://image.slidesharecdn.com/8f64d2bd-ee54-4e16-873e-125d5195cfe6-150330225631-conversion-gate01/85/Final-project-kbakshy-2-320.jpg)
Final project-kbakshy
- 1. MICROARRAYS AND DATA ANALYSIS FINAL PROJECT Kiranmayee Bakshy 08/19/2014
- 2. Introduction • Expression data from 46 cultured human ovarian carcinoma cell lines with and without Cisplatin treatment • Array: A-AFFY-141 - Affymetrix GeneChip Human Gene 1.0 ST Array [HuGene-1_0-st-v1] (GPL6244) • Technology type: in situ oligonucleotide • Experiment type: transcription profiling by array • Samples: 171 • NCBI GEO accession no. GSE47856 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GS E47856) Epithelial-mesenchymal status renders differential responses to cisplatin in ovarian cancer. Miow QH, Tan TZ, Ye J, Lau JA, Yokomizo T, Thiery JP, Mori S. , Europe PMC 24858042
- 3. Background • Chemo-resistance to platinum in anti-cancer drugs such as cisplatin is critical in the treatment of cancer. • Epithelial-mesenchymal transition (EMT) is linked with the drug resistance as a contributing mechanism. • The current study is designed to explore the connection between cellular responses to cisplatin with EMT in ovarian cancer. • Expression microarrays were utilized to estimate the EMT status as a binary phenotype • Various bioassays such as cell number, proliferation rate and apoptosis were conducted to quantify phenotypic responses to Cisplatin treatment.
- 4. Data Analysis pipeline Load raw CEL files into R and normalize using RMA Outlier analysis (CV vs mean plot, Hierarchical clustering dendrogram and Average correlation plot) Run statistical tests and fold change to select differentially expressed genes Dimensionality reduction/clustering (PCA) Classification (QDA) Report top 5 up- regulated and down- regulated gene names and their functions Dataset: Total no. of samples: 171 Total no. of probesets: 33297 Annotation classes: Epithelial-like: 86 samples Mesenchymal-like: 85 samples
- 7. Outlier-GSM1160845 Outlier analysis The outlier - GSM1160845 was removed from the data matrix 13707 genes that have low expression values (mean < 5) were also deleted
- 8. Statistical analysis Student’s t-test and fold change No. of probes with p-value: < 0.05 9507 < 0.01 7074 < 0.05/no. of probes 2133 Linear fold change: Min. -8.651798 Max. 33.05966 Threshold for selecting differentially expressed genes: p-value < 0.05/no. of probes and fold change > log2(2)
- 9. Visualization of differentially expressed genes 656 differentially expressed genes were selected from the analysis based on the threshold
- 10. Dimensionality reduction of differentially expressed genes Around 50% of variability in data can be explained by the first two eigenfunctions of PCA Principle component analysis
- 11. Spectral k-means clustering of 50 random differentially expressed genes Spectral k-means clustering is useful in this case as the variability can be best summarized in a few eigenfunctions.
- 12. Classification – Quadratic Discriminant Analysis Epithelial -like Mesenchymal- like Epithelial-like 36 0 Mesenchymal- like 0 34 Confusion matrix from QDA predicted membership Actualmembership Training set: 50 epithelial-like 50 mesenchymal-like Test set: 36 epithelial-like 34 mesenchymal-like QDA was performed on the first three components of principle component analysis of training set. QDA predicted all the samples of the test set correctly
- 13. AFFYMETRIX_EX ON_GENE_ID GENE NAME GENE SYMBOL FUNCTION 8102792 protocadherin 18 PCDH18 Potential calcium-dependent cell- adhesion protein 7899167 lin-28 homolog(C. elegans) LIN28A Acts as a 'translational enhancer', driving specific mRNAs to polysomes and thus increasing the efficiency of protein synthesis 7906878 discoidin domain receptor tyrosine kinase-2 DDR2 This tyrosine kinase receptor for fibrillar collagen mediates fibroblast migration and proliferation 7906900* discoidin domain receptor tyrosine kinase 2 DDR2 This tyrosine kinase receptor for fibrillar collagen mediates fibroblast migration and proliferation 7926368 vimentin VIM class-III intermediate filaments found in mesenchymal cells DAVID functional annotations of top 5 discriminant genes (Negative) * Unmapped in DAVID; information obtained from NetAffx No pathways or GO information was suggested by DAVID.
- 14. AFFYMETRIX_EX ON_GENE_ID GENE NAME GENE SYMBOL FUNCTION 8026490 urothelial cancer associated-1 UCA1 role in bladder cancer progression and embryonic development 8041853 epithelial cell adhesion molecule EpCAM carcinoma-associated antigen EpCAM up regulates c-myc and induces cell proliferation 8098439 epithelial cell adhesion molecule EpCAM carcinoma-associated antigen EpCAM up regulates c-myc and induces cell proliferation 8147351 mal, T-cell differentiation protein-2 MAL-2 Member of the machinery of polarized transport 8148040 epithelial splicing regulatory protein- 1 Esrp-1 mRNA splicing factor that regulates the formation of epithelial cell-specific isoforms DAVID functional annotations of top 5 discriminant genes (Positive) No pathways or GO information was suggested by DAVID.
- 15. Conclusions: • The outlier observed in this dataset was GSM1160845 which is a mesenchymal-like ovarian cancer cell line treated with Cisplatin. • 656 out of 19590 genes were selected as differentially expressed genes based on the threshold. • The QDA classification model trained using 100 samples predicted the classes of test set with 70 samples successfully. • All the top 5 positively and negatively regulated genes obtained in this analysis are involved in cellular processes such as cell adhesion, migration, proliferation and protein synthesis. • The authors have reported an epithelial gene set consisting of known epithelial cell markers such as DDR1, KRT8, KRT18, CDH1, CDH3, CLDN3, CLDN4 and EPCAM, and a mesenchymal gene set consisting of known mesenchymal cell markers ZEB1, CDH2, VIM and TWIST1.