Authors:

Conwy Zheng

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About Paper:

Neutrophils are the most abundant white blood cells that form the first line of defense against infections through mechanisms like ROS, Phagocytosis, NETosis and recruitment of other immune cells. However, excessive recruitment of neutrophils or overstaying at a site of infection can have negative impacts like inflammation or additional tissue damage. Therefore, proper regulation of neutrophil activity is important to maintain a healthy immune system. This project focuses on the plasma membrane calcium ATPase gene ATP2b1, one of many target genes of MiR-375, as a regulator of neutrophil motility in both zebrafish and human leukemia (HL-60) cells. Discovery of potential regulator genes of neutrophil migration was achieved through RNA sequencing of MiR-375 overexpressing zebrafish, where ATP2b1 was verified to have a significant effect on neutrophil motility using our CRISPR/Cas9 system of Tissue-Specific-Knockout (TSKO). Human relevance was further determined through developing ATP2b1 knockdown cell lines through lentiviral transduction of shRNA expressing plasmids. In both systems, ATP2b1 knockdown cells showed significant decrease in their recruitment and migration when compared to control. Furthermore, this phenotype was rescued when full-length ATP2b1 was reintroduced into both TSKO zebrafish and knockdown HL-60 cells, giving strength to our hypothesis that proper calcium channeling via ATP2b1 is crucial for proper neutrophil activity. Currently, the project is gearing towards exploring whether ATP2b1 regulates other neutrophils functions while also looking into other target genes of MiR-375 as potential regulators of neutrophil motility. Keywords: Neutrophil; microRNA; Zebrafish; CRISPR/Cas9

Source:

Purdue University / 2024

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Co-authors:

Conwy Zheng