Investigating functional effects of microRNAs contained in extracellular vesicles from Natural Killer cells

Abstract

Natural Killer (NK) cells are lymphocytes that secrete cytotoxic extracellular vesicles (EVs), which are being investigated for their anti-cancer properties and therapeutic potential. Cancer remains a significant global health concern, emphasizing the importance of continued research in cancer therapy. Understanding how NK-EVs target and eliminate cancer cells is crucial to exploit their anti-cancer properties. We hypothesize that microRNAs (miRNAs) mediate some of the NK-EV-induced cell death, which is why this thesis focuses on measuring the apoptotic-inducing effect of miRNAs previously shown by our group to be enriched in cytotoxic NK-EVs.

Cell viability of the colon cancer cell line HCT 116 was evaluated following treatment with miRNA mimics by using both a caspase-3/7 based flow cytometric assay and an adenosine triphosphate (ATP)-based assay. Gene expression analysis of genes involved in cell cycle, cellular proliferation, and apoptosis was conducted following treatment of HCT 116 cells with miRNAs, measured using quantitative polymerase chain reaction (qPCR). Specific miRNAs were inhibited in the NK cell line NK-92, and changes in the EVs’ cytotoxicity on HCT 116 cells were assessed using the caspase-3/7 based flow cytometric assay.

The main aim of the thesis was to investigate the functional effects of miRNAs contained in NK-EVs. Apoptotic effects on HCT 116 cancer cells were observed in miR-15b-5p, miR-16- 5p, miR-19b-3p, miR-20a-5p, miR-22-3p, miR-29b-3p, miR-142-3p, and miR-181b-5p. MiR16-5p appeared to downregulate mRNA for the anti-apoptotic protein Bcl2, while miR-19b3p seemed to upregulate RICTOR mRNA, a regulator of cell growth. Inhibiting miR-29b-3p in NK-92 cells appeared to enhance NK-EVs cytotoxicity against HCT 116 cells. However, the observations lacked statistical significance, rendering them inconclusive.

In summary, this thesis indicates that miRNAs enriched in cytotoxic NK-EVs may indeed have a functional effect on the cancer cell line HCT 116. These findings provide a foundation for further research into these miRNAs to conclude their functional effects.