Signature of Circulating microRNA (miR) Detected by NextSeq Differentiates Non-alcoholic Fatty Liver Disease (NAFLD) Patients With Higher Percentage Hepatic Collagen: A Potential Role for a Prognostic Biomarker

Introduction: Stage of hepatic fibrosis caused by collagen deposition is the only independent histologic predictor of mortality. Emerging evidence suggests a role for miR dysregulation in the pathogenesis of NAFLD. In fact, specific miRs profiles are associated with NAFLD. Our aim was to use sera from NAFLD patients to assess differential miR signature expression associated with fibrosis assessed by percentage (%) collagen deposition. Methods: We included 66 patients with biopsy-proven NAFLD [64% NASH, 61% fibrosis]. Each liver biopsy was stained with Sirius red and % collagen was quantified using Computer Assisted Morphometry (CAM). HTG EdgeSeq sequencing technology was used to sequence for (˜2200 miR) on Next-Seq®500 system. Differential expression (DE) miR defined as those whose log2 fold changes (FC) > log2(2), with false discovery rate (FDR) adjusted p-value < 0.01were tested. Ingenuity Pathway Analysis tool (IPA) was used to generate a network of genes- DE miR interactions. We calculated circulating miR signature score associated with %hepatic collagen using Random Forests (RF) R-package. Circulating miR independently associated with % hepatic collagen were assessed using generalized linear model. Results: Combination of serum miR-1245a, miR-3124-3p, miR-4760-3p, miR-3613-5p miR-4488 could discriminate NAFLD with high % hepatic collagen from NAFLD with low % hepatic collagen with an area under the curve (AUR)= 0.9335, p < 0.0001. In addition, multivariate analysis revealed that miR-3169 (β =0.54395, SE=0.23057, p=0.02) and miR-4792(β=-1.9838, SE=0.34902, p < 0.0001) were independently assoicated with higher % hepatic collagen depition in NAFLD. Interestingly, IPA showed that miR-3124-3p and miR-4488 are regulators for genes of hypoxia-inducible factor 1 alpha (HIF1α) signaling pathway which regulates stellate cell activation and fibrosis progression. Furthermore, miR-4792 is the direct regulator of Forkhead box (FOXC1) pathway of epitheilal to mesencymal transition (EMT) which is a crucial process in determining the outcomes of hepatic fibrosis. Conclusion: We have identified biologicaly relevant miR signature that can segregate NAFLD according to the presence of high and low hepatic collagen deposition. These results indicate involvement of miRs in the pathogeneis of NAFLD-related fibrosis and their potential role as biomarkers to predict high hepatic collagen deposition (significant fibrosis), the most imporrant prognostic factor in NAFLD.