Integrated microRNA and mRNA expression profile analysis of tumor-associated macrophages after exposure to single-dose irradiation

[[abstract]]Background: Radiotherapy (RT) is a common approach that accounts for nearly 50% of cancer patient treatment and has the potential for long-term tumor control. Recently, we published a research article on gene expression profiling of tumor-associated macrophages (TAM) that were exposed to ionizing radiation (IR). Single-dose irradiation of tumors could initiate differentially expressed genes in TAM as a time series from irradiated tumors that are associated with the immune response. It is also well known that human cancers are associated with microRNA (miRNA) alterations that are involved in cancer progression. However, the role of miRNA on TAM after exposure to irradiation remains unclear. Results: In this study, miRNA expression profiles from microarrays were used to identify the key miRNAs and correlating pathways involved in the role of TAMs in tumor progression and recurrence after RT. Using a mouse tumor model, we identified miRNA pattern changes over time in response to irradiation. Based on our results, we hypothesize that miRNA expression in the irradiated tumor may be used as a distinguishing marker to indicate the best time for therapeutic intervention to prevent tumor recurrence after RT. Conclusions: We established a murine model irradiated with a single dose of 25 Gy that could initiate temporal changes in the expression of miRNAs associated with cell proliferation and the immune response, as evidenced by macrophages directly extracted from irradiated tumors after two weeks of IR. Statistical analyses were conducted by comparing the miRNA expression in macrophages from non-irradiated versus irradiated tumors. Thus, our study could lead to a better understanding of the function of miRNA expressions, which changed temporally in an irradiated tumor microenvironment

Epitranscriptomic regulation in breast cancer and PCB-induced liver disease.

Post-transcriptional RNA modifications including N6-methyladenosine (m6A) regulate mRNA stability, splicing, and translation. My research examined m6A in two disease models: breast cancer (BCa) and non-alcoholic fatty liver disease (NAFLD). Acquired resistance to endocrine therapies (ET) develops in approximately 20% of BCa patients with estrogen receptor α positive (ER+) tumors following treatment. The mechanisms by which tumor cells evade ET are not completely understood. Using a cell line model, we investigated the role of an m6A reader protein, HNRNPA2B1 (A2B1) that is upregulated in ET-resistant ER+ BCa cells. Stable overexpression of A2B1 in ET-sensitive MCF-7 cells (MCF-7-A2B1), results in ET resistance, whereas knockdown of A2B1 in ET-resistant cells restored ET-sensitivity. microRNAs (miRNAs) downregulated by transient overexpression of A2B1 were identified to target two key enzymes (PSAT1 and PHGDH) in the serine biosynthetic pathway (SSP) which is upregulated in ET-resistant BCa cells and in tumors from patients with ET-resistant disease. Using luciferase assays, PSAT1 and PHGDH were validated as bona fide targets of miRNAs downregulated by A2B1 (miR-145-5p and miR-424-5p targeting PSAT1, miR-34b-5p and miR-876-5p targeting PHGDH). Exogenous overexpression of the validated miRNAs decreased endogenous PSAT1 and PHGDH in ET-resistant BCa cells, resulting in increased sensitivity to ET in vitro. In the second model, alterations in the m6A epitranscriptome were identified in the livers of male C57Bl/6Jmice after a single, oral exposure to polychlorinated biphenyls (PCB), a class of persistent organic pollutants, in combination with 12 weeks on a high fat diet (HFD). Our results demonstrated that exposure to PCBs in combination with a HFD resulted in major changes to the mRNA and miRNA transcriptomes, and m6A epitranscriptome. Pathway analysis of the genes in which m6A peaks were altered identified pathways involved in the progression from steatosis to steatohepatitis in NAFLD. PCB exposures also resulted in changes to alternative splicing (AS) mechanisms and events, suggesting that PCB-induced m6A changes contribute to altered isoforms expression in NAFLD. Taken together, the results in this dissertation demonstrate the significant role of altered m6A in two common human diseases