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CircSPI1 functions as an oncogene through antagonizing SPI1 and interacting with microRNAs in acute myeloid leukemia

ABSTRACT: Background: Strict control of PU.1, encoded by SPI1, is importmant for myeloid development, and inhibition of PU.1 expression and activity, even moderate, can lead to acute myeloid leukemia (AML). Much effort has focused on regulatory elements at the SPI1 locus on controlling PU.1. Discovery of circular RNAs (circRNAs), a novel class of noncoding regulators, has raised additional questions regarding their impacts on PU.1 and the development of AML. Methods: We used the pan-cancer circRNA compendium MiOncoCirc to investigate the existence and expression pattern of circSPI1s. We designed short hairpin RNAs (shRNAs) targeting the back-spliced site to silence circSPI1 and then performed CCK8 proliferation assay, flow cytometry analysis and Wright-Giemsa stain to explore the function of circSPI1. We performed RNA-sequencing with and without circSPI1 knockdown, western bloting and luciferase assay to explore the potential mechanisms. We utilized machine learning techniques and integrated bioinformatic analysis to evaluate the prognostic value of circSPI1 for AML, based on TCGA and BeatAML cohorts. Results: We found that circSPI1, the circular RNA derived from the SPI1 gene (encoding PU.1), was highly expressed in AML than that in normal counterparts. Functional experiements showed circSPI1 acted as an oncogene, as evidenced by the observation that circSPI1 knockdown induced myeloid differentiation and apoptosis of AML cells. This was different from the parental gene SPI1 that is generally low expressed in AML and functions as a tumor suppressor. Mechanistic investigations revealed that circSPI1 exerted multiple regulatory roles in AML progression. On one hand, circSPI1 contributed to the orchestrated differentiation of AML cells by antagonizing SPI1 expression. On the other hand, circSPI1 was involved in proliferation and apoptosis by interacting with microRNAs miR-1307-3p, miR-382-5p and miR-767-5p, which was uncoupled with SPI1. Finally, circSPI1-regulated genes were associated with the clinical outcome of AML patients, implicating the clinical significance of circSPI1 in AML. Conclusions: We demonstrate that highly expressed circSPI1 acts as an oncogene in AML, through both antognizing PU.1 expression and binding to microRNAs that is independent to PU.1. Our data provide new insight into complex SPI1 gene regulation now involving cicrSPI1 as well as the independent role of cicrRNA in AML.

ORGANISM(S): Homo sapiens

PROVIDER: GSE159931 | GEO | 2021/03/24

REPOSITORIES: GEO

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Project description:More than forty loci contribute to genetic risk for Alzheimer’s disease (AD). These risk alleles are enriched in myeloid cell enhancers suggesting that microglia, the brain-resident macrophages, contribute to AD risk. We have previously identified SPI1/PU.1, a master regulator of myeloid cell development in the brain and periphery, as a genetic risk factor for AD. Higher expression of SPI1 is associated with increased risk for AD, while lower expression is protective. To investigate the molecular and cellular phenotypes associated with higher and lower expression of PU.1 in microglia, we used stable overexpression and knock-down of PU.1 in BV2, an immortalized mouse microglial cell line. Transcriptome analysis suggests that reduced PU.1 expression suppresses expression of homeostatic genes similar to the disease-associated microglia response to amyloid plaques in mouse models of AD. Moreover, PU.1 knock-down resulted in activation of protein translation, antioxidant action and cholesterol/lipid metabolism pathways with a concomitant decrease of pro-inflammatory gene expression. PU.1 overexpression upregulated and knock-down downregulated phagocytic uptake in BV2 cells independent of the nature of the engulfed material. However, cells with reduced PU.1 expression retained their ability to internalize myelin similar to control albeit with a delay, which aligns with their anti-inflammatory profile. Here we identified several microglial responses that are modulated by PU.1 expression levels and propose that risk association of PU.1 to AD is driven by increased pro-inflammatory response due to increased viability of cells under cytotoxic conditions. In contrast, low expression of PU.1 leads to increased cell death under cytotoxic conditions accompanied by reduced pro-inflammatory signaling that decreased A1 reactive astrocytes signature supporting the protective effect of SPI1 genotype in AD. These findings inform future in vivo validation studies and design of small molecule screens for therapeutic discovery in AD.