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Thesis advisor: Vassilios Bezzerides / The aim of this study was to utilize a massively parallel reporter assay (MPRA) to identify organ-specific microRNA (miRNA) target sequences to refine the timing and expression of transgene expression for gene therapy. We previously had developed a cardiac gene therapy for Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) using a systemically delivered adeno-associated virus (AAV9) vector. We hypothesized that incorporation of organ specific miRNA target sites into our vector construct could improve our therapy’s tissue specificity due to the ability of miRNAs to silence transgene expression. Initially, we attempted to incorporate mir-124 target sequences into our vector to detarget the brain. Although these initial attempts were unsuccessful, the study allowed us to develop a protocol to test the effectiveness of miRNA target sequences. Thereafter, we developed a method to screen thousands of putative miRNA target sequences simultaneously. In this study, target sequences of miRNAs specific to the heart, brain and liver were incorporated into a plasmid library. This plasmid library was subsequently made into AAV and injected into mice from a CPVT transgenic line. Total DNA and RNA was later extracted from the target organs, converted into genomic DNA (gDNA) and complementary DNA (cDNA) libraries respectively, and sent for amplicon sequencing. We analyzed the results using Comparative Microbiome Analysis 2.0 software (CoMA) and a custom python script to count the occurrence of each specified barcode per sample. In doing so, we showed that the miRNA suppression mechanism is not only effective but also organ specific. Furthermore, we developed a second script to create a combinatorial library from a set list of miRNA target sequences enabling us to efficiently test thousands of target sequence combinations at once. In doing so, we will be able to identify effective miRNA target sequence combinations to further improve gene therapy specificity. / Thesis (BS) — Boston College, 2021. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Cancer is the second leading cause of death in females. According to the American Cancer Society, there are 327,660 new cases in breast and gynecological cancers estimated in 2014, placing emphasis on the need for cancer prevention and new cancer treatment strategies. One important approach to cancer prevention involves phytochemicals, biologically active compounds derived from plants. A variety of studies on the impact of dietary compounds found in cruciferous vegetables, green tea, and spices like curry and black pepper have revealed epigenetic changes in female cancers. Thus, an important emerging topic comprises epigenetic changes due to the modulation of noncoding RNA levels. Since it has been shown that non-coding RNAs such as microRNAs and long non-coding RNAs are aberrantly expressed in cancer, and furthermore are linked to distinct cancer phenotypes, understanding the effects of dietary compounds and supplements on the epigenetic modulator non-coding RNA is of great interest. This article reviews the current findings on nutrition-induced changes in breast and gynecological cancers at the non-coding RNA level.

Glioblastoma is the most common malignant brain tumor, accounting for 17% of all primary brain tumors in the United States. Despite the available surgical, radiation, and chemical therapeutic options, the invasive and infiltrative nature of the tumor render current treatment options minimally effective. Recent reports have identified multiple regulators of glioblastoma progression and invasiveness. It has been demonstrated that ADAM12, A Disintegrin And Metalloproteinase encoded by ADAM12 gene, is over-expressed in glioblastoma and directly correlated with tumor proliferation. Additionally, dysregulation of the Notch signaling pathway has been implicated in the pathogenesis of many gliomas. Lastly, an evolving role of microRNAs, small noncoding RNAs, in carcinogenesis is progressively growing. A recent study has identified ADAM12 as a notch-related gene, and another demonstrated that inhibition of notch signaling decreased glioblastoma recurrence. However the mechanisms of regulation are still unknown. In this study, we hypothesize that direct downregulation of microRNA-29, downstream of over-expression of notch enhances glioblastoma malignancy through upregulation of ADAM12. Although our data demonstrate upregulation of Notch1, its downstream target HES1, and ADAM12 in U87MG glioblastoma cell line. Expression of the cleaved intracellular Notch1 was not detected. Furthermore, we were unable to demonstrate an inhibitory effect of ɣ-secretase inhibitor on Notch signaling, likely reflecting the requirement for modifying culturing conditions or detection in our assays. Furthermore, miR-29 was detected in glioblastoma cells. The expression of miR-29 was further elevated by ɣ-secretase inhibitor treatment, suggesting a role for Notch1 inhibition on miR-29 expression. Although no conclusive results are shown in our work, a role of Notch1 through miR-29 is implicated in the pathogenesis of glioblastoma pathogenesis warranting further investigation into the role downstream target genes in the Notch signaling pathway.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression via inhibiting mRNA translation or degrading mRNA. Since the discovery of miRNAs, dysregulated miRNAs have been identified in human patients with various diseases. Moreover, the role of miRNAs in biological processes, including immune homeostasis and autoimmunity pathogenesis, has been widely investigated. Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease that causes systemic damage to multiple organs and is characterized by the production of pathogenic autoimmune antibodies. In previous work in my lab, a set of commonly upregulated miRNAs in splenic lymphocytes of three lupus-prone mouse models was identified, including the miR-183-96-182 cluster (miR-183C) and miRNAs located at DLK1-DIO3 region. The work presented in this dissertation focuses on comparing the dysregulation pattern of miRNAs from different cell sources of lupus-prone mice and investigating the potential role of miR-183C in the pathogenesis of SLE and inflammation. The first goal was to test whether dysregulated miRNAs initially identified in the spleen of MRL/lpr mice, a standard model for SLE, is also reflected in the peripheral blood mononuclear cells (PBMCs) as PBMC is the primary source of lymphocytes in human patients. In MRL/lpr mice, we found that dysregulated miRNAs in PBMCs were overall comparable to those identified in the splenic lymphocytes. Further, comparing dysregulated miRNAs between mice and humans showed a similarity in the dysregulation of miRNAs in PBMCs of murine and human lupus. Among the upregulated miRNAs, the expression of three miRNAs of miR-183C was found to be commonly upregulated. To investigate the role of miR-183C, we developed miR-183C in CD2+ cells of C57BL/6 Faslpr/lpr (miR-183C-/-B6/lpr) mice. In miR-183C-/-B6/lpr mice, we observed a significantly reduced level of anti-dsDNA in the serum and IgG immunocomplex deposition in the kidney. Importantly, in vitro inhibition of miR-183C in activated splenic lymphocytes led to reduced production of the proinflammatory cytokine, IFN, and Foxo1, a transcription factor that is a target of miR-183C miRNAs. I also tested for miRNA changes in C57BL/6 Faslpr/lpr mice with conditional deletion of Early Growth Response-2 (EGR2) (Egr2-/- B6/lpr), another knockout mouse developed in our laboratory. Egr2 has recently been shown to regulate immunity and autoimmunity and play a role in lupus. An unexpected observation is that Egr2-/-B6/lpr mice had significantly reduced expression of a group of lupus-related miRNAs that are located at the genomic imprinted DLK1-DIO3 locus. Given that the upregulation of DLK1-DIO3 miRNAs in lupus is subjected to DNA methylation regulation and that the epigenetic regulatory role of EGR2 is emerging in recent studies, reduced representative bisulfite sequencing (RRBS) was performed to evaluate the methylation changes induced by Egr2 deletion. Global DNA hypomethylation and methylation changes at specific sites at DLK1-DIO3 region were noticed in CD4+ T cells of Egr2-/-B6/lpr mice. Overall, our research suggested a therapeutic effect of inhibiting the miR-183C expression on SLE. The interplay between epigenetic factors could help expand the possibility of controlling epigenetic regulators in autoimmune disease treatment. / Doctor of Philosophy / Systemic lupus erythematosus (SLE) is an autoimmune disease that causes damage to multiple organs. Same with other autoimmune diseases, the exacerbated immune reaction to self-antigen and auto-reactive adaptive immune cells were described in SLE. Currently, the treatment of lupus mainly uses immunosuppressive drugs to inhibit the global immune reaction. Thus, the innovative drug is desperately needed for SLE patients. MicroRNAs (miRNAs) are small RNAs that inhibit the expression of genes by binding to mRNAs in a complimentary manner. Since the discovery of the first microRNA, the pivotal role of microRNAs in immunity and autoimmunity was vigorously investigated. Our lab was the first to describe a set of miRNAs that are commonly upregulated in three murine lupus models. Among these miRNAs, miR-183, miR-96, and miR-182 belong to the miR-183-96-182 cluster (miR-183C). The aim of the study in this dissertation focused on illuminating the dysregulation pattern of miRNAs in different cell sources in the murine lupus model and the role of miR-183C in the pathogenesis of SLE. We found that miRNAs are similarly dysregulated in peripheral blood mononuclear cells and splenic lymphocytes of MRL/lpr mice. Then we conditionally knocked out the miR-183C in B6/lpr mice and investigated the effect of miR-183C loss on the pathogenesis of autoimmunity. Importantly, we found that the deletion of miR-183C led to a reduced production level of autoantibodies and ameliorated the deposition of immune complexes in the kidney. Moreover, the production of proinflammatory cytokines of splenic lymphocytes was regulated by miR-183C as well. Besides miR-183C, I also investigated the effect of early growth response 2 (EGR2), a transcription factor, on the expression of a set of lupus-related miRNAs and the methylation change at the genome location of these miRNAs. In summary, miR-183C can be a potential therapeutic target for lupus treatment while clinical human studies are needed to better clarify the effectiveness and efficiency.

Myocardial ischemia/reperfusion (I/R) injury is a clinically fatal disease, caused by restoring myocardial blood supply after a period of ischemia or hypoxia. However, the underlying mechanism remains unclear. Recently, increasing evidence reveal that microRNAs (miRs) participate in myocardial I/R injury. This study aimed to investigate whether miR-128-1-5p contributed to cardiomyocyte apoptosis induced by myocardial I/R injury. Here, we showed that the expression of miR-128-1-5p was decreased in mice following myocardial I/R injury. Down-regulation of miR-128-1-5p was also showed in H9c2 cardiomyocytes after hypoxia/reoxygenation (H/R), and in neonatal rat cardiomyocytes (NRCMs) with H2O2 treatment. Importantly, we found that overexpression of miR-128-1-5p ameliorates cardiomyocyte apoptosis both in H9c2 cardiomyocytes and NRCMs. Moreover, we also found that growth arrest DNA damage-inducible gene 45 gamma (Gadd45g) is identified as a direct target of miR-128-1-5p, which negatively regulated Gadd45g expression. Additionally, silencing of Gadd45g inhibits cardiomyocyte apoptosis in H9c2 cardiomyocytes and NRCMs. These results reveal a novel mechanism by which miR-128-1-5p regulates Gadd45g-mediated cardiomyocyte apoptosis in myocardial I/R injury.

Indiana University-Purdue University Indianapolis (IUPUI) / Cytochrome P450 2D6 (CYP2D6) is an important drug metabolizing enzyme that is involved in the metabolism of 20-25% of commonly prescribed drugs. There is interindividual variability in CYP2D6 enzyme activity and this leads to compromised metabolism of many drugs. Genetic and environmental factors explain only a part of the interindividual variability; the other factors that contribute to this variability are largely unknown. Hence, it becomes important to study CYP2D6 to understand the endogenous and exogenous factors that control its activity. The specific objective of this research was to determine the contribution of genetic and epigenetic factors in the regulation of CYP2D6 expression and activity. The specific aims were: (1) to identify the common CYP2D6 polymorphisms in Vietnamese and Filipino women with breast cancer and evaluate its association with plasma concentrations of endoxifen (an active metabolite of the breast cancer therapeutic drug, tamoxifen); (2) to identify the CYP2D6 copy number variations (CNVs) in these women and evaluate their association with endoxifen concentration; and (3) to identify microRNAs (miRNAs) that regulate the expression of CYP2D6 directly or indirectly. The results of this study indicated that: (1) in Vietnamese and Filipino women, the reduced function allele CYP2D6*10 was frequent (~55%) and it was significantly associated with reduced endoxifen concentration; (2) in these women, only 39% carried two copies of the CYP2D6 gene, the rest had a genomic imbalance for CYP2D6, primarily involving the CYP2D6(*36)n-*10 allele. However, carrying multiple copies of CYP2D6*36 allele did not significantly affect CYP2D6 activity, suggesting that multiple copies of a gene does not always translate to additive effects; and (3) microRNAs were identified to target HNF4A, a transcriptional factor that regulates CYP2D6 expression. These miRNAs are likely to play an important role in the indirect regulation of CYP2D6. Taken together, these results emphasize on the role of polymorphisms, CNVs and possibly miRNAs in the regulation of CYP2D6. These clinically important biomarkers will help to improve the efficacy and reduce the side effects of many CYP2D6 substrate drugs and thus contribute to personalization of drug therapy.

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