OP03 Identification of Micrornas Regulated by Vitamin D and Fatty Acid Treatment in Immortalised Hepatocytes and Hepatic Stellate Cells Through Microrna Sequencing

The effect of vitamin D supplementation in non-alcoholic fatty liver disease (NAFLD) is not fully elucidated.1 MicroRNAs (miRNAs) are small non-coding RNAs playing a vital role in the progression of metabolic diseases by typically supressing gene expression.2 However, the influence of vitamin D regulated miRNAs in the pathogenesis of NAFLD has been unexplored. With an overall aim of investigating the role of vitamin D in NAFLD pathogenesis, the aims of these experiments were to measure miRNA expression in immortalised hepatocytes and hepatic stellate cells treated by vitamin D, fatty acid or both through miRNA sequencing. Immortalised hepatocytes (HepG2) and hepatic stellate cells (LX-2) were cultured for 16 hours in charcoal-stripped, serum-containing medium before 24-hour (for HepG2) and 6-hour (for LX-2) treatment with either 100 nM calcitriol (1- α-dihydroxyvitamin D3) or fatty acid (500 μM, 1:1 oleic acid: palmitic acid) or both. A total of 24 total RNA samples from 3 independent experiments for both cell lines were isolated using ReliaPrepTM RNA Cell Miniprep System (Promega, Z6012). Next Generation Sequencing was done by the Leeds Teaching Hospitals NHS Trust Service. Quality control, read alignment and read count obtain were finished in Linux. Differential expression was examined by log2 relative fold change between control and three different treatment groups individually by DESeq2 in R. The results were compared with a published literature review from our group [1]. Significant level was set to 0.05. For HepG2 and LX-2 cells, 659 and 507 mature miRNAs were detected, respectively. In which, 55 (8.34%, for HepG2) and 63 (12.42%, for LX-2) mature miRNAs were significantly up or down-regulated (figure 1). Comparing to miRNAs identified through our published systematic review [1], 9 miRNAs were identified in 2 cell lines among those regulated miRNAs (hsa-let-7b-5p, hsa-miR-125b-1–3p, hsa-miR-17–3p, hsa-miR-34a-5p, hsa-miR-181a-2–3p, hsa-miR-132–5p, hsa-miR-197–5p, hsa-miR-10b-5p, hsa-miR-200c-3p). In summary, miRNA sequencing identified a total of 55 and 63 mature miRNAs significantly regulated by vitamin D and/or fatty acid treatment in immortalised hepatocytes and hepatic stellate cells. A total of 9 miRNAs overlapped with the systematic review. Ongoing work includes examining potential functional and mechanistic effects using bioinformatics analysis, candidate miRNA validation by qPCR and examining the consequent response to lipid loading with and without vitamin D in miRNA knockdown and overexpression cell models in both cell lines. Reference Zhang Z, Moon R, Thorne JL, Moore JB. NAFLD and vitamin d: Evidence for intersection of microRNA-regulated pathways. Nutrition Research Reviews 2021;1–20. Wang X, He Y, Mackowiak B, Gao B. MicroRNAs as regulators, biomarkers and therapeutic targets in liver diseases. Gut 2021;70(4):784–795.