Rowan Abdelbary ^1,2 Manon Ragheb ^1,2 Shereen Ahmed El Sobky ² Nagwa El-Badri ³ Nourhan Aboud ² Asmaa Ibrahim ⁴ Ahmed Tawheed ⁵ Mona Zidan ⁶ Ramy Karam Aziz ⁶ Abd Elrahman Abouzid ³ Radwa Ayman Salah ³ Mohamed El-Kassas ⁵ Imam Waked ⁴ Ahmed Moustafa ^1,7 Injie Omar Fawzy ² Nada El-Ekiaby ² Ahmed Ihab Abdelaziz ^2*

• ¹ Biotechnology Graduate Program, American University in Cairo, New Cairo, Cairo, Egypt
• ² School of Medicine, Newgiza University, Giza, Beni Suef, Egypt
• ³ Center of Excellence for Stem Cells and Regenerative Medicine, Zewail City of Science and Technology, Giza Governorate, Egypt
• ⁴ Department of Hepatology and Gastroenterology, National Liver Institute, Menoufia University, Shebin El-Kom, Monufia, Egypt
• ⁵ Endemic Medicine Department, Faculty of Medicine, Helwan University, Cairo, Egypt
• ⁶ Microbiology and Immunology Research Program, Children’s Cancer Hospital Egypt 57357, Cairo, Egypt
• ⁷ Department of Biology, American University in Cairo, New Cairo, Egypt

The role of miRNAs in regulating variable molecular functions has been sought by scientists for its promising utility in regulating the immune response and, hence, in treating various diseases. In hepatocellular carcinoma specifically, a reduction in the number and efficiency of circulating and intrahepatic natural killer (NK) cells has been reported. Our project aims to delve into understanding the role of miR-216a-3p in the regulation of NK cell cytotoxicity, especially since it plays a tumor suppressor role in the context of HCC. To achieve our aim, we isolated NK cells from the whole blood of 86 patients with HCC and 23 healthy controls. We assessed the expression profile of miR-216a-3p in NK cells of patients and controls. Furthermore, we induced the expression of miR-216a-3p in NK cells isolated from healthy controls, followed by measuring the release of interferon-gamma (IFN-γ), tumor necrosis factoralpha (TNF-α), perforins (PRF) and granzyme B (GrB) using ELISA as well as NK cells cytolytic activity against Huh7 cells using lactate dehydrogenase (LDH) cytotoxicity assay. After that, we performed an in silico analysis to understand the mechanistic regulation imposed by miR-216a-3p on NK cells to study its impact on one of its potential downstream targets. Our results have indicated that miR-216a-3p has higher expression in NK cells of patients with HCC, and simulating this elevated expression pattern via forcing miR-216a-3p expression in normal NK cells has negatively impacted the release of TNF-α, IFN-γ, GrB, and PRF. Consequently, a decrease in cell cytolysis was observed. Our in silico analysis revealed that the predicted downstream targets of miR-216a-3p are enriched in the FOXO-signaling pathway. Among those targets is FOXO-1, which has been reported to play a role in NK cell maturation. Thus, we evaluated FOXO-1 expression upon mimicking miR-216a-3p in control NK cells that showed significant downregulation of FOXO-1 on both RNA and protein levels. In conclusion, we report miR-216-3p as a negative regulator of NK cell cytotoxicity.Keywords: microRNAs (miRNAs), miR-216a-3p, hepatocellular carcinoma (HCC), natural killer (NK) cells, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), granzymes (GrB), perforins (PRF)