Effect of a Diet Enriched with Omega-6 and Omega-3 Fatty Acids on the Pig Liver Transcriptome
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Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences Department of Genomics and Biodiversity Doctoral Thesis Effect of a diet enriched with omega-6 and omega-3 fatty acids on the pig liver transcriptome Agnieszka Szostak Ph.D. thesis under the supervision of: Prof. dr hab. Mariusz Pierzchała Institute of Genetics and Animal Biotechnology Polish Academy of Sciences, Poland Dr Marinus F.W. te Pas Wageningen University and Research Centre The Netherlands Jastrzębiec, 2020 1 2 Acknowledgments The author would like to express her gratitude to Dr. hab Mariusz Pierzchała, Dr. Marinus Te Pas, and Prof. Dr. hab Chandra Shekhar Pareek for their critical reading and insights. This work was supported by grants from BIOFOOD – innovative, functional products of animal origin no. POIG.01.01.02-014-090/09 co-financed by the European Union from the European Regional Development Fund within the Innovative Economy Operational Programme 2007 – 2013, and by the National Science Centre, Poland (grant no. 2014/13/N/NZ9/00906). 3 4 Table of content Acknowledgments...................................................................................................................... 3 Abstract ...................................................................................................................................... 8 Streszczenie.............................................................................................................................. 10 1. Introduction .......................................................................................................................... 12 1.1. Dissertation‘s research hypothesis ................................................................................ 12 1.2. Review of the literature related to the doctoral dissertation.......................................... 13 1.2.1. Biochemistry and functions of polyunsaturated fatty acids (PUFAs) .................... 13 1.2.2. Omega-3 and omega-6 fatty acids as vital components of a diet ........................... 20 1.2.3. Supplementation of human and animal diet with PUFAs ...................................... 24 1.2.4. Pig as a model in biomedical research .................................................................... 25 1.2.5. Nutrigenomics – innovative methods in PUFAs studies ........................................ 25 2. Dissertations’ research objectives and goals........................................................................ 28 3. Materials and methods ......................................................................................................... 30 3.1. Feeding experiment including Polish Landrace purebred and Polish Landrace x Duroc crossbred............................................................................................................................... 30 3.1.1. Study design in the feeding experiment ................................................................. 30 3.1.2. Laboratory procedures in the feeding experiment .................................................. 35 3.1.3. Statistical analysis on fatty acids phenotypic data.................................................. 35 3.2. NGS based RNA-Seq experiment including Polish Landrace purebred (MiSeq Illumina platform) and Polish Landrace x Duroc crossbred (HiSeq Illumina platform) pigs. ........... 37 3.2.1. Study design ........................................................................................................... 37 3.2.2. Samples preparation in the NGS based RNA-Seq experiment ............................... 38 3.2.3. Laboratory procedures in the NGS based RNA-Seq approach .............................. 38 3.2.4. Bioinformatic analysis of RNA-Seq data of porcine liver transcriptome .............. 48 3.2.5. Validation of gene expression analysis results by Quantitative RT-PCR .............. 51 3.2.6. Statistical analysis................................................................................................... 60 4. Results .................................................................................................................................. 61 4.1. Feeding experiment including Polish Landrace purebred and Polish Landrace x Duroc crossbred pigs ....................................................................................................................... 61 4.1.1. Linseed and rapeseed oil supplementation reduces the hepatic omega-6/omega-3 ratio ................................................................................................................................... 61 5 4.2. NGS based RNA-Seq experiment of porcine liver representing Polish Landrace purebred and Polish Landrace x Duroc crossbred pigs ........................................................ 67 4.2.1. Preprocessing and post-processing of porcine liver RNA-Seq data representing Polish Landrace purebred and Polish Landrace x Duroc crossbred pigs ......................... 67 4.2.2. DEGs analysis of porcine liver by comparing the standard (control) diet versus experimental nutritional PUFAs diets within Polish Landrace purebred pigs (MiSeq RNA-Seq data) using CLC Genomics workbench ........................................................... 68 4.2.3. DEGs analysis of porcine liver by comparing the standard (control) diet versus experimental nutritional PUFAs diets within Polish Landrace x Duroc crossbred pigs (HiSeq RNA-Seq data) using CLC Genomics workbench ............................................... 77 4.2.4. DEGs analysis of porcine liver by comparing the Polish Landrace purebred pigs (MiSeq RNA-Seq data) and Polish Landrace x Duroc crossbred pigs (HiSeq RNA-Seq data) after experimental feeding with standard (control) diet using CLC Genomics workbench ........................................................................................................................ 86 4.2.5. DEGs analysis of porcine liver by comparing the Polish Landrace purebred pigs (MiSeq RNA-Seq data) and Polish Landrace x Duroc crossbred pigs (HiSeq RNA-Seq data) after experimental feeding with the supplementary healthy diet enriched with omega-6 and omega-3 fatty acids (PUFAs) diet using the CLC Genomics workbench .. 99 4.2.6. Overall comparisons of identified up- and down-regulated genes (Third filtering) ........................................................................................................................................ 121 4.3. Visualization of identified upregulated and downregulated DEGs in investigated purebred and crossbred pigs ............................................................................................... 141 4.3.1. Visualization of identified upregulated and downregulated DEGs in Volcano plot and in Heatmap ............................................................................................................... 141 4.4. Biological gene networks and pathways analysis of DEGs using Cytoscape ClueGO ............................................................................................................................................ 146 4.4.1. Identification of biological gene networks and pathways analysis by comparing the standard (control) diet versus supplementary healthy diet enriched with omega-6 and omega-3 fatty acids (PUFAs) in liver transcriptome Polish Landrace purebred pigs (First comparison). ................................................................................................................... 146 4.4.2. Identification of biological gene networks and pathways analysis by comparing the standard (control) diet versus supplementary healthy diet enriched with omega-6 and omega-3 fatty acids (PUFAs) in the liver transcriptome of Polish Landrace x Duroc crossbred pigs ................................................................................................................. 151 6 4.4.3. Identification of biological gene networks and pathways analysis by comparing the Polish Landrace purebred versus Polish Landrace x Duroc crossbred pigs liver transcriptome after experimental feeding with standard (control) diet .......................... 156 4.4.4. Identification of biological gene networks and pathways analysis by comparing the Polish Landrace purebred versus Polish Landrace x Duroc crossbred pigs liver transcriptome after experimental feeding with the diet enriched with omega-6 and omega-3 fatty acids (PUFAs) ......................................................................................... 161 4.5. Validation of RNA-Seq data by quantitative real-time PCR ...................................... 166 5. Discussion .......................................................................................................................... 168 5.1. Discussion on feeding experiment .............................................................................. 169 5.2. Discussion on NGS experiment .................................................................................. 170 6. Conclusions ........................................................................................................................ 180 7. References .......................................................................................................................... 181 7 Abstract Thesis title: Effect of a diet enriched with omega-6 and omega-3 fatty acids on the pig liver transcriptome Background: Omega-6 and omega-3 polyunsaturated fatty acids (PUFAs) have been recognized as molecules regulating a variety of functions in a cell. They