Computational study of dietary phytochemicals in disease prevention
Creators: name: Sargsyan, Davit
Publisher: name: No Publisher Supplied
Year: 2024

description: Chapter 1 provides the background for my research. It discusses the general idea of omics data and its applications in biomedical research. Specifically, how and why we collected and analyzed omics data while studying benefits of dietary phytochemicals tested in preclinical setting. The effects produced by these compounds in vitro and in vivo were associated with specific changes at the molecular level. The chapter describes several key technologies employed in our studies such as quantitative polymerase chain reaction (qPCR), microarrays and Next Generation Sequencing (NGS). The chapter also summarizes computational methods associated with these technologies.Chapter 2 contains the results from our published study of transcriptomic changes produced by SETD7 knockdown (KD) and PEITC dietary phytochemical treatment in prostate cancer cells. Microarrays were used to analyze the cells’ transcriptomes. We identified 651 differentially expressed genes. Specifically, we found that in SETD7 KD cells a tumor suppressor gene RARRES3 that codes for the retinoic acid receptor responder protein 3 and an antioxidant gene SOD2 that codes for superoxidative dismutase 2 enzyme were up-regulated 28.06-fold and 5.66-fold, respectively. The KD also significantly affected several canonical pathways such as Death Receptor Signaling, TNFR2 Signaling and Acute Phase Response Signaling pathways. At the same time, PEITC downregulated TIMP1, TNKS, HLA-DRB1, GNAI2, and H6PD genes by more than 2.3-fold, and upregulated RBBP4, CNOT7, EXT2, PTK2, PHB, and RAB11B genes by at least 2.3-fold in the wild type (WT) cells compared to the DMSO-treated (control) SETD7 KD cells. Key results from the microarray analyses were validated by qPCR confirming that PCDHB13, CD24, SDC4, GADD45B, SACS, RAB31, and GH2 were upregulated in the KD versus the WT group but decreased in the KD PEITC versus KD group. Additionally, ID1 and LXN were downregulated in the KD versus the WT group but upregulated in the KD PEITC versus KD group. qPCR also confirmed that RRTM1, FCRL6, THSD7A, APOM, ARMC12, TKTL1, and ZFP235 expressions increased in PEITC-treated WT cells, whereas SETD7 KD prevented this induction. TPM3, OR9A2, DCAF4L1, CBLN2, and TAS2r19 were all downregulated in the PEITC-treated WT cells but SETD7 KD prevented this suppression.Chapter 3 is a study of DNA methylome and transcriptome alterations in high glucose (HG) induced diabetic nephropathy in the mouse kidney mesangial cells and the protective effect of Tanshinone IIA (TIIA) treatment. After a 5-day HG treatment, RNA and DNA were extracted from the cells, sequenced and analyzed. Previously, we observed at least 2-fold increase of intracellular ROS damage in HG compared to the control while TIIA-treated cells were protected from the damage. We identified 213 differentially expressed genes with log2-fold changes greater than 0.3. 124 genes were downregulated in the HG-treated cells but not in the HG+TIIA. At the same time, 89 genes were downregulated by HG treatment, but the trend was reversed by TIIA treatment. Next, we examined CpG methylation profiles of the identified genes. Our main hypothesis was that hypermethylation of CpG clusters in the gene promoter region in DNA should lead to decreased amount of RNA for the same genes. Conversely, hypomethylated promoters may result in higher levels of the gene expressions although other factors can prevent the transcription, hence, we expected this association to be less obvious in the data. We identified several candidate genes and were able to validate that FGL2 and NMU genes’ promoter regions became hypermethylated after HG treatment, but TIIA treatment prevented hypermethylation. The opposite trend was found and validated in GULO and KCNIP2 as HG treatment resulted in demethylation of promoters’ CpGs while TIIA-treated cells did not exhibit it.Chapter 4 is a study of triterpenoid corosolic acid (CA) modulating global CpG methylation and transcriptome of tumor promotor 12-O-tetradecanoyl- phorbol-13-acetate (TPA) induced mouse epidermal cells. JB6 P+ cells were challenged with TPA and treated with CA for 5 days. DNA and RNA were isolated and sequenced. Genes differentially expressed in the positive control (TPA only) but not in the treatment (TPA+CA) were identified, and their promoter regions analyzed for differential methylation. Out of these, genes for which promoters’ CpG methylation profiles were associated with RNA expressions were further selected for validation using qPCR. Genes such Prolactin-2C2 (Prl2c2) growth and angiogenesis factor, Sprr2h (small proline-rich protein 2) known to be regulated by IL6/STAT3 signaling, Mrc1 (Macrophage mannose receptor1), Dkk2 (Dickkopf-related protein 2) which is WNT signaling pathway inhibitor, NOS2 (Nitric oxide synthase 2) and CYP27B1 (cytochrome P450 family 27 subfamily B member1) were found to be differentially modulated in TPA+CA treatment as compared to TPA only. Smad-3 (signal transducers for transforming growth factor beta (TGF-B) superfamily), Tasp1 gene (endopeptidase which needs for the maintenance of HOX genes), Uri1 (prefoldin like chaperone protein), and Nsg2 (Neuronal vesicle trafficking-associated protein 2) were hypermethylated in TPA treatment while hypomethylated in TPA+CA. Madd (MAP kinase activating death domain), Dusp22 (Dual specificity protein phosphatase 22), and Rassf (tumor suppressor gene family, Ras-association domain family) genes were hypomethylated in TPA but hypermethylated in TPA+CA. Major canonical pathways affected by the treatments included cell cycle regulatory, cancer and cell differentiation, apoptosis, signaling kinases associated pathways such as p53 signaling, Erk signaling, and TGF beta signaling pathway. Chapter 5 explores epigenomic and transcriptomic of mouse kidney mesangial cells challenged with high glucose (HG) and treated with carotenoid fucoxanthin (FX). We identified several differentially expressed genes and examined the association of RNA expression levels with methylation changes in the promoter regions of those genes. Specifically, we detected that KCNIP2, MAK, CNN1, ARHGDIB and TMEFF1 where downregulated while having one or more hypermethylated CpG clusters. Top canonical pathways affected by FX treatment were multiple signaling pathways/kinases; Toll-like receptor 7/8 pathways and a Hedgehog signaling pathway. CYP2E1signaling, which is thought to activate protoxins, the adenylate cyclase inhibitory pathway, the Hyaluronan metabolism pathway, and the PKA mediated phosphorylation of CREB protein pathway were upregulated by HG and attenuated by FX. Protein kinase A signaling which is involved in metabolism, cytoskeleton dynamics, and regulation of gene expression was downregulated by HG and FX accentuated its expression.Chapter 6 is a study of mouse gut microbiomes. In a series of three experiments, we explored the effects of Nrf2 gene using a knock-out mouse model, gut inflammation induced by DSS, and PEITC and cranberry enriched diets. Our results indicated that Nrf2 KO was significantly associated with higher diversity and richness of the microbial communities compared to WT. Inflammation negatively affected these communities but PEITC and cranberry diets had protective effect. Firmicutes to Bacteroidetes ratio (F/B), an indirect indicator of inflammatory processes in the guts, confirmed the protective effect of these dietary supplements. We further examined microbiome profile differences between the treatment groups and identified several bacterial phyla, classes, families and OTUs with significantly differentiated relative abundance in the samples. Additionally, we examined metabolic profiles of the microbiomes. As expected, the dietary supplements increased production of many microbial metabolites which, in turn, moved the animal guts condition toward homeostasis. Finally, in Chapter 7 I provided a conclusion for my research and discussed future prospective including improved study designs and more advanced computational techniques that would further advance our understanding of the effects of dietary phytochemicals on human health.