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Differentiation of Crohn's disease (CD) from intestinal tuberculosis (ITB) is a big challenge to gastroenterologists because of their indistinguishable features and insensitive diagnostic tools. A non-invasive biomarker is urgently required to distinguish ITB/CD patients particularly in India, a TB endemic region, where CD frequency is increasing rapidly due to urbanization. Among the three differentially expressed miRNAs obtained from small RNA transcriptomic profiling of ileocaecal/terminal ileal tissue of ITB/CD patients (n = 3), only two down-regulated miRNAs, miR-31-5p, and miR-215-5p showed comparable data in qRT-PCR. Out of which, only miR-215-5p was detectable in the patient's plasma, but there was no significant difference in expression between ITB/CD. On the other hand, miR-375-3p, the pulmonary TB specific marker was found in higher amount in the plasma of ITB patients than CD while reverse expression was observed in the ileocaecal/terminal ileal tissues of the same patients. Next, using Bioplex pro-human cytokine 48-plex screening panel, only three chemokines, Eotaxin-1/CCL11, SDF-1α/CXCL12, and G-CSF have noted significantly different levels in the serum of ITB/CD patients. ROC analysis has revealed that compared to a single molecule, a combination of miR-375-3p + Eotaxin-1/CCL11 + SDF-1α /CXCL12 + G-CSF showed a better AUC of 0.83, 95% CI (0.69–0.96) with 100% specificity and positive predictive value while sensitivity, negative predictive value, and accuracy were 56%, 69%, and 78% respectively in distinguishing ITB from CD. This study suggests that a combination of plasma markers shows better potential in differentiating ITB from CD than a single marker and this panel of markers may be used for clinical management of ITB/CD patients. [ABSTRACT FROM AUTHOR]

Diagnosis of hepatocellular carcinoma (HCC) remains challenging to clinicians, particularly in a patient with low alpha‐fetoprotein. Here, in silico, ex vivo and in vitro data were combined to identify liver‐specific exosomal miRNAs as an early diagnostic marker for HCC. Transcriptome profiling for mRNA and small RNA in same HCV‐HCC and normal liver tissues followed by cross‐validation of 41 deregulated miRNAs (log2FoldChange > 1.5, Padj <.1) with GEO/TCGA datasets of HCV/HBV related HCC vs normal/adjacent tissue revealed three miRNAs were commonly deregulated (miR‐10b/miR‐21/miR‐182) among all HCC irrespective of viral etiology. Targets of top deregulated miRNAs were identified by TargetScan/miRwalk and validated in mRNA transcriptome data followed by Panther/Gene Ontology enrichment/Cytoscape analysis suggested that targets were mostly from carcinogenesis pathways. Hence, those miRNAs were validated in normal and HCV‐HCC tissues by qRT‐PCR and subsequently in plasma‐derived‐exosomes of both HBV/HCV infected non‐HCC (chronic hepatitis [CH]/liver cirrhosis [LC]) and HCC samples, and in liver‐specific Anti‐Asgr2 immuno‐enriched exosomes. Exosomes were verified using Nanosight/TEM/immune‐blotting with anti‐Alix/anti‐GRP78/anti‐Asgr2. Along with miR‐21‐5p, miR‐10b‐5p/miR‐221‐3p/miR‐223‐3p was found significantly upregulated in the exosome of HCC patients than CH/non‐HCC. The comparable expression pattern was seen in anti‐Asgr2 immuno‐precipitated exosomes. Interestingly, the AFP level was found below 250 ng/mL in about 94% of HCV‐HCC and 62% of HBV‐HCC patients. ROC analysis showed that miR‐10b‐5p + miR‐221‐3p + miR‐223‐3p + miR‐21‐5p could differentiate CH/non‐HCC(CH + LC) from HCC with AUROC: 0.86 (97.5% CI: 0.77‐0.94)/0.80 (97.5% CI: 0.70‐0.89), sensitivity: 74%/58% and specificity: 86%/95% while miR‐10b‐5p + miR‐221‐3p + miR‐223‐3p showed AUROC: 0.84 (97.5% CI: 0.74‐0.94)/0.74 (97.5% CI: 0.63‐0.84), sensitivity: 86%/86% and specificity:66%/53% for low AFP‐HCC vs CH/non‐HCC, respectively, having better sensitivity than the combination of four miRNAs. Multivariate analysis further revealed low Albumin and high miR‐21‐5p as probable independent risk factor for HCC. What's new? Several microRNAs have been found to distinguish hepatocellular carcinoma (HCC) from normal individuals. However, the use of miRNAs as HCC biomarkers remains controversial due to disease heterogeneity and instability of circulating free miRNA. Here, in silico, ex vivo, and in vitro data were combined to identify liver‐specific exosomal miRNAs as an early diagnostic marker for HCC. Altered circulating hepatocyte‐specific exosomal miRNAs were a risk factor for HCC development in both hepatitis B virus‐ and hepatitis C virus‐infected patients. Diagnosis of low‐AFP HCC with a combination of exosomal miRNAs may improve the clinical management of these difficult‐to‐diagnose patients regardless of viral etiology. [ABSTRACT FROM AUTHOR]

Imbalance in lipid metabolism induces steatosis in liver during Chronic hepatitis C (CHC). Contribution of microRNAs in regulating lipid homoeostasis and liver disease progression is well established using small RNA-transcriptome data. Owing to the complexity in the development of liver diseases, the existence and functional importance of yet undiscovered regulatory miRNAs in disease pathogenesis was explored in this study using the unmapped sequences of the transcriptome data of HCV-HCC liver tissues following miRDeep2.pl pipeline. MicroRNA-c12 derived from the first intron of LGR5 of chromosome 12 was identified as one of the miRNA like sequences retrieved in this analysis that showed human specific origin. Northern blot hybridization has proved its existence in the hepatic cell line. Enrichment of premiR-c12 in dicer-deficient cells and miR-c12 in Ago2-RISC complex clearly suggested that it followed canonical miRNA biogenesis pathway and accomplished its regulatory function. Expression of this miRNA was quite low in CHC tissues than normal liver implying HCV-proteins might be regulating its biogenesis. Promoter scanning and ChIP analysis further revealed that under expression of p53 and hyper-methylation of STAT3 binding site upon HCV infection restricted its expression in CHC tissues. Centrosomal protein 350 (CEP350), which sequestered PPARα, was identified as one of the targets of miR-c12 using Miranda and validated by luciferase assay/western blot analysis. Furthermore, reduced triglyceride accumulation and enhanced PPARα mediated transcription of β-oxidation genes upon restoration of miR-c12 in liver cells suggested its role in lipid catabolism. Thus this study is reporting miR-c12 for the first time and showed its' protective role during chronic HCV infection. [ABSTRACT FROM AUTHOR]

Controversies about the origin of circulating miRNAs have encouraged us to identify organ specific circulating miRNAs as disease biomarkers. To identify liver-specific miRNAs for hepatocellular carcinoma (HCC), global expression profiling of miRNAs in liver tissue of HBV-HCC and HBV-control with no or mild fibrosis was evaluated. A total of 40 differentially expressed miRNAs were identified in HCC. Among ten highly altered miRNAs, six miRNAs were successfully validated in tissues, whereas only two miRNAs, miR-126 and miR-142-3p showed increased expression in plasma of HBV-HCC compared to HBV-non-HCC patients. Subsequently, ROC curve analysis revealed that neither miR-126 nor miR-142-3p performed better than AFP in discriminating HCC from non-HCC while combination of each with AFP showed significantly higher efficiency rather than AFP alone (AUC: 0.922, 0.908 vs. 0.88; sensitivity: 0.84, 0.86 vs. 0.82 and specificity: 0.92, 0.94 vs. 0.86 respectively). Interestingly, triple combination of markers (miR-1261miR-142-3p1AFP) showed no additive effect on efficiency (AUC: 0.925) over the dual combination. Again, the expression of only miR-126 was noticed significantly higher in HBV-HCC patients with low-AFP [<250 ng/ml] compared to either non-HCC or liver cirrhosis (AUC: 0.77, 0.64, respectively). Furthermore, no alteration in expression of mir-126 in HCV-HCC or non-viral-HCC revealed that miR-1261AFP might be specific to HBV-HCC. To understand the physiological role of these two miRNAs in hepato-carcinogenesis, target genes related to cancer pathways (APAF, APC2, CDKN2A, IRS1, CRKL, LIFR, EGR2) were verified. Thus, combination of circulating miR-1261AFP is a promising noninvasive diagnostic biomarker for HBV-HCC and may be useful in the management of HCC patients. [ABSTRACT FROM AUTHOR]