DIPLOMARBEIT Titel der Diplomarbeit “Identification of unknown epigenetically regulated genes in non-small cell lung cancer” Durchgeführt an der Medizinischen Universität Wien Univ. Klinik für Innere Med. I/Klinische Abteilung für Onkologie Verfasserin Valerie Nadine Babinsky angestrebter akademischer Grad Magistra der Naturwissenschaften (Mag.rer.nat.) Wien, 2011 Studienkennzahl lt. Studienblatt: A 441 Studienrichtung lt. Studienblatt: Mikrobiologie und Genetik Betreuer: Ao.Prof. Dr. Wolfgang Mikulits Table of Content 1 TABLE OF CONTENT 1 Table of Content ............................................................................................................ 2 2 Acknowledgment / Danksagung ..................................................................................... 5 3 Introduction ................................................................................................................... 6 4 Aim of study .................................................................................................................. 7 5 Background .................................................................................................................... 9 5.1 Epigenetics: Histone protein modifications and DNA methylation ......................................... 9 5.1.1 Histone protein modifications ............................................................................................... 10 5.1.2 DNA methylation ................................................................................................................... 11 5.1.2.1 CpG islands ............................................................................................................................. 12 5.1.2.2 CpG methylation .................................................................................................................... 13 5.1.2.3 DNA methyltransferases ........................................................................................................ 14 5.2 Effects of DNA methylation on gene transcription ................................................................ 16 5.3 Epigenetic changes are reversible ......................................................................................... 18 5.4 Lung cancer ............................................................................................................................ 21 5.4.1 DNA methylation as molecular biomarker in NSCLC ............................................................. 23 5.5 DNA methylation analyses ..................................................................................................... 23 5.5.1 Sodium bisulfite treatment .................................................................................................... 23 5.5.2 PCR based techniques for analyzing DNA methylation ......................................................... 24 5.5.3 DNA methylation analysis on a whole genome level ............................................................. 26 5.6 DNA methylation and its impact on NSCLC ........................................................................... 27 6 Material and Methods.................................................................................................. 31 6.1 Cell culture ............................................................................................................................. 31 6.1.1 Cell passaging ......................................................................................................................... 31 6.1.2 Cell freezing............................................................................................................................ 32 6.1.3 Cell de-freezing ...................................................................................................................... 32 6.1.4 Incubation of cells with 5-aza-2’-deoxycytidine .................................................................... 32 6.2 Nucleic acid isolation ............................................................................................................. 33 6.2.1 Isolation of genomic DNA ...................................................................................................... 33 6.2.2 Isolation of total RNA ............................................................................................................. 34 6.2.3 RNAse treatment of extracted DNA ....................................................................................... 35 6.3 Methylation-specific PCR (MSP) ............................................................................................ 35 2 Valerie Babinsky 0405551 Table of Content 6.3.1 Sodium bisulfite treatment .................................................................................................... 35 6.3.2 SssI CpG-methyltransferase treatment of genomic DNA ...................................................... 35 6.3.3 Primer design ......................................................................................................................... 36 6.3.4 MSP analysis........................................................................................................................... 39 6.4 Expression microarray analysis .............................................................................................. 40 6.4.1 RNA clean-up with the MinElute Kit (Qiagen) ....................................................................... 40 6.4.2 Double strand cDNA synthesis ............................................................................................... 40 6.4.3 Double strand cDNA clean-up ................................................................................................ 41 6.4.4 In vitro transcription .............................................................................................................. 41 6.4.5 cRNA clean-up ........................................................................................................................ 42 6.4.6 cRNA fragmentation .............................................................................................................. 42 6.4.7 Microarray preparation and hybridization ............................................................................ 43 6.4.8 Washing and scanning of microarrays ................................................................................... 43 6.5 Methylated DNA immunoprecipitation (MeDIP)-chip analysis ............................................. 45 6.5.1 Fragmentation of genomic DNA ............................................................................................ 45 6.5.2 Methylated DNA immunoprecipitation (MeDIP) ................................................................... 45 6.5.3 Whole genome amplification (WGA) ..................................................................................... 47 6.5.3.1 Step 1 of WGA ........................................................................................................................ 47 6.5.3.2 Step 2 of WGA ........................................................................................................................ 48 6.5.4 Clean-up of amplified DNA .................................................................................................... 48 6.5.5 Test for enrichment efficiency by quantitative real-time PCR .............................................. 49 6.5.6 MeDIP-chip analysis ............................................................................................................... 50 6.5.7 Statistical analysis of microarray data ................................................................................... 51 6.6 Buffer ingredients .................................................................................................................. 53 7 Results ......................................................................................................................... 54 7.1 Genome-wide DNA methylation analysis of NSCLC cell lines ................................................ 54 7.1.1 Gene expression analysis of the NSCLC cell lines A549, NCI-H1993 and NCI-H2073 before and after treatment with Aza-dC and/or TSA ........................................................................ 54 7.1.1.1 Aza-dC induced genes ............................................................................................................ 55 7.1.1.2 TSA induced genes ................................................................................................................. 56 7.1.1.3 TSA and Aza-dC treated cells ................................................................................................. 58 7.1.1.4 Identification of cancer-associated genes ............................................................................. 63 3 Valerie Babinsky 0405551 Table of Content 7.1.1.5 Single gene DNA methylation analysis of selected genes in tissue samples of NSCLC patients (n=10) ..................................................................................................................................... 64 7.1.2 Establishing MeDIP-chip ........................................................................................................ 65 7.1.2.1 MeDIP-chip analysis ............................................................................................................... 72 7.1.2.2 Functional analysis of tumor-specifically methylated genes ................................................