SAT-109 tiRNA-5 Reflects the Severity of Mitochondrial Oxidative Stress in Nonalcoholic Fatty Liver Disease

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disease with complicated mechanisms among which disturbance of mitochondria function weighs a lot. Overexpression of CypD, the initial factor in mitochondrial permeability transition pore, led to mitochondrial stress triggered hepatic TG accumulation. tRNA fragments (tRFs) are non-coding RNAs produced by precise cleavage of tRNA and divided into subtypes including tiRNA-5, tiRNA-3, tRF-5, tRF-3 and tRF-1. It is reported that tRFs, especially tiRNA-5 associated with stress. However, whether tRFs connect with NAFLD is unclear. To solve the problem, CypD overexpression models were constructed by injecting mouse AdPPIF virus to 8-weeks male C57BL/6 mice as study group (CH, n=6). Control group (WT, n=6) was established by injecting empty vector virus (AdEGFP). Then tRFs in liver were detected by high-throughput sequencing. Proportion of tRFs in CH was 40.8% higher vs WT. A connection might exist between CypD and tRFs. To validate this speculation, CypD knockdown mice were established as study group and 8-weeks male C57BL/6 as control. Then both groups were fed with 60 kcal% fat diet for 4 weeks or 8 weeks and named as CKO4, CWT4, CKO8 or CWT8 (n=6). Sequencing showed that proportion of tRFs in CKO4 and CKO8 was 42.7% and 31.2% lower vs control respectively, consistent with the speculation. Therefore, bioinformatics analysis was used to explore further. Pie plot based on proportion of each tRFs subtype showed that over 90% tRFs were tRF-5 and tiRNA-5. tiRNA-5 was 38.5% higher in CH vs WT, 27.4% lower in CKO4 vs CWT4 and 36.0% lower in CKO8 vs CWT8, indicating a connection between tiRNA-5 and CypD. Integration of pathological sections, metabolic index and more analysis were done for futher study. PCA and Venn diagram described the difference on the whole while absolute value of Pearson correlation coefficient (CH vs WT=0.84, CKO4 vs CWT4=0.93, CKO8 vs CWT8=0.73) and Scatter plot (CH vs WT =0.69, CKO4 vs CWT4=0.85, CKO8 vs CWT8=0.66) reflected the similarity between study and control group. These data indicated a connection between tRF and the severity of NAFLD assessed by pathology and metabolism index. Finally, a cutoff of fold change ≥ 1.5 was employed to identify the differentially expressed tRFs and most of those were tiRNA-5 as speculated. qRT-PCR was conducted for validation, among which fold change of AS-tDR-000666 (GlyGCC derived tiRNA-5) was significative (CH vs WT=4.29, CKO4 vs CWT4=0.22, CKO8 vs CWT8=0.05). It might be a potential biomarker related with severity of NAFLD. Because the CypD-change-induced mitochondrial oxidative stress was the direct mechanism triggering NAFLD in mouse models above, we could conclude that mitochondrial oxidative stress in NAFLD changed the profile of tRFs and tiRNA-5 had the potential to reflect the severity of mitochondrial oxidative stress in NAFLD. This study provided a new understanding of NAFLD.