miR-34a and miR-223 as diagnostic biomarkers for the severity of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to non-alcoholic steatohepatitis (NASH) and fibrosis, yet current non-invasive tools lack accuracy in distinguishing these stages. This study aimed to evaluate the circulating miR-34a and miR-223 as biomarkers for NAFLD staging. In this cross-sectional study, 200 participants were equally divided into healthy individuals and patients with steatosis, NASH and fibrosis groups. Serum miRNA expression was measured by stem-loop RT-qPCR and miR-16 was used for normalization, while clinical parameters were measured to validate miRNAs. ANOVA and Kruskal-Wallis tests were used for group comparisons, while ROC analysis and multivariate regression were used to assess the diagnostic performance. Both miRNAs showed significant changes in mean expression across disease stages ( p < 0.001). miR-34a increased from controls (1.42-fold) to NASH (41.28-fold), followed by a relative decline in fibrosis (12.84-fold), while miR-223 remained low in steatosis before rising sharply in NASH (42.17-fold). Both miRNAs correlated strongly with ALT, AST, platelet count, and lipid parameters (|r| > 0.6, p < 0.01). The combined miRNA panel accurately differentiated steatosis from NASH (AUC = 0.978; 96% sensitivity; 94% specificity). Both were independently associated with advanced disease (miR-34a: OR = 1.32, 95% CI: 1.18–1.48; miR-223: OR = 1.28, 95% CI: 1.15–1.42; p < 0.001), and patients in the highest quartiles had a 116-fold higher risk. The addition of both miRNAs to clinical models improved AUC from 0.812 to 0.912 (NRI = 0.267, p < 0.001). The expression patterns of miR-34a and miR-223 offered a practical non-invasive approach for staging of NAFLD.