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microRNA , Cancer Epigenetics and Bioinformatics

SPRING 2019 BIO307. microRNA , Cancer Epigenetics and Bioinformatics. Jasmin Šutković 2 9th May 2019. Outline. Introduction Micro RNA s Epigenetic DNA Methylation In silico study – Bioinformatics Examples.

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microRNA , Cancer Epigenetics and Bioinformatics

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  1. SPRING 2019 BIO307 microRNA, Cancer Epigenetics and Bioinformatics Jasmin Šutković 29th May 2019

  2. Outline • Introduction • Micro RNA s • Epigenetic • DNA Methylation • In silico study – Bioinformatics • Examples

  3. Epigenetic  refers to heritable changes in gene expression (active versus inactive genes), that does not involve changes to the underlying DNA sequence; a change in phenotype without a change in genotype. Or “Epigenetics” refers to covalent modification of DNA, protein, or RNA, resulting in changes to the function and/or regulation of these molecules, without altering their primary DNA sequences.

  4. Micro RNA molecules MicroRNAs (miRNAs) comprise species of short noncoding RNA (18-25 bp)that regulate gene expression post-transcriptionally. Recent studies have demonstrated that epigenetic mechanisms, including DNA methylation and histone modification, not only regulate the expression of protein-encoding genes, but also miRNAs, such as let-7a, miR-9, miR-34a, miR-124, miR- 137, miR-148 and miR-203.

  5. microRNA processing Picture taken from: http://discovermagazine.com/~/media/import/images/0/2/6/micrornadiag.jpg

  6. DNA methylation • Epigenetic factor that occurs by covalent addition of a methyl group (CH3) on the 5’ position of cytosine. • Altersthe expression of genes in cells as cells divide and differentiate from embryonic stem cells into specific tissues. • This event typically occurs in CpG dinucleotide content where60% to90% of all CpGs are methylated in mammals(Varley et al., 2013). • Regulation of microRNA expression is influenced by DNA methylation and abnormal methylation is playing an important role in the tumorgenesis(Suzuki et al., 2012).

  7. DNA methylation Picture taken from: https://drjanephilpott.wordpress.com/tag/dna-methylation/

  8. It is a discipline that represents a marriage between biotechnology and computer technologies and has evolved the convergence of advances in each of these fields. • Today bioinformatics is a field that encompasses all aspects of the application of computer technologies to biological data. Computers are used to organize, link, analyze and visualize complex sets of biological data. What is bioinformatics ?

  9. In silico study – Bioinformatics • Using compitutional based methods to analyse biological data • Protein structure and function prediction • Genome wide screening for epigenetic factors • Currently several gene banks are publicly available for micrRNA screening and methylation analysis • Correlations can be found between genes, noncoding RNA and methylation • Predictictions often match with in vitro studies

  10. Methods The canger genome atlas – (TCGA) • aimed to analyze the genomic features through sequencing of about 30 different types of cancers • TCGA is a joint project (27 instititutes) • https://tcga-data.nci.nih.gov/tcga/ - platform for researchers to search, download, and analyze data sets (methylation data, expression of genes, SNPs)

  11. Functional annotation of genes Functional annotation of genes The Database for Annotation, Visualization and Integrated Discovery (DAVID)- v6.7- assigns functions to particular genes ! Famous software- more then 10000 citied papers, some papers were published in many journals of Nature !!! Link :http://david.abcc.ncifcrf.gov/

  12. KEGG KEGG Koyoto Encyclopedia of Genes and Genomes (KEGG) – to annotate functions of large scale molecular databases generated by genome sequencing and other high-throughput experimental technologies Used published databases of genes or proteins to generate pathway diagrams… Link : http://www.genome.jp/kegg/

  13. Example • Cancer related in silico search for genes, microRNA

  14. Experimental Design and Methods • Purchase several non-small- cell lung cancer (NSCLC) cell lines • Growing them under standard conditions in CO2 incubator • Do polymerase chain reaction (qRT-PCR). • To investigate Expression of miR-10a and PhoGDI2 in cell lines • Isolate RNA • qRT-PCR assays for detection of miR-10a and PhoGDI2 expression using ready kits • To check the PhoGDI2 mRNA expression, total RNA will be reverse transcribed by RT-PCR • To verify integrity of PhoGDI2 expression, GAPDH gene can be used as an internal endogenous control - GAPDH is a housekeeping gene commonly used as a reference for quantification of gene expression. • The relative levels of PhoGDI2 mRNA can be calculated using the 2-Δct method.

  15. 3. Effect of DNA methylation • To confirm the effect of DNA methylation on miR-10a expression • Inhibit methyltion in cells by 5-Aza-CdR - DNA methyltransferase inhibitor, can be detected by qRTPCR ( to check if methylation has reduced the gene expression or not) • CpG island methylationcan be assessed by bisulfitesequencing- to determine the patterns of methylation . • 4. Verification of miR-10a connection to RhoGDI2 expression • luciferase reporter vector – clononing of the the wild-type 3’ untranslated region (UTR) of RhoGDI2 into the firefly luciferase gene (pLuc) of pCDNA vector. – to measure or track expression of the cloned gene. • New plasmid will be cotransfected into the cell lines and treated with Lipofectamine 2000reagent – used to increase the transfection efficiency of the plasmid

  16. 5. To investigate cell proliferation affects by miR-10a and RhOGDI2 • Cell Proliferation Reagent Kit I (MTT) (Roche Applied Science) for cell viability. • MiR-10a inhibitors cells and pCDNA/miR-10a will be transfected to our cells and allowed to grow in well plates. Appropriate negative controls will used. Cell proliferation will be documented every 24 h following the manufacturer’s protocol • Todetermine whether RhoGDI2 could also affect the cell proliferation we will do taget knock down of RhoDGI2 expression using RNAi in our cell lines. • RNA interference (RNAi) is a means of silencing genes by way of mRNA degradation

  17. Expected Result and Potential Problems • Expexted results are in vitro correlation of RhoDGI2 and miR-10a • This study will help us to understand the biological roles ofthe molecules involved in lung cancers suppression and lead us to find potential therapeutic target for the lung cancer. • Potential Problems: • Besides miR-10a, we may have other microRNA interaction with RhoGDI2,

  18. Oncogenomics

  19. Oncogenomics (Cancer Genomics) • Oncogenomics is a relatively new sub-field of genomics that applies high throughput technologies to characterize genes associated with cancer. • Cancer is a genetic disease caused by accumulation of mutations to DNA leading to unrestrained cell proliferation and neoplasm formation. • The goal of oncogenomics is to identify new oncogenes or tumor suppressor genesthat may provide new insights into cancer diagnosis, predicting clinical outcome of cancers, and new targets for cancer therapies. • The success of targeted cancer therapies such as Gleevec, Herceptin, and Avastinraised the hope for oncogenomics to elucidate new targets for cancer treatment.

  20. Overall Goals of Oncogenomics

  21. Current Technology being used in Oncogenomics

  22. Databases for Cancer Research • Cancer Genome Project is an initiative to map out all the somatic intragenic mutations in cancer. • COSMIC is a resource • Oncomine has compiled data from cancer transcriptome profiles. • IntOGen integrates multidimensional human oncogenomic data classified by tissue type using the ICD-O terms. • International Cancer Genome Consortium is so far the biggest project to collect human cancer genome data. The data is accessible through the ICGC website.

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