Sponging of five tumour suppressor miRNAs by lncRNA-KCNQ1OT1 activates BMPR1A/BMPR1B-ACVR2A/ACVR2B signalling and promotes chemoresistance in hepatocellular carcinoma.

Diverse mechanisms have been established to understand the chemoresistance of hepatocellular carcinoma (HCC), but the contribution of non-coding RNAs is not surveyed well. Here, we aimed to explore the lncRNA-miRNA axis in Hepatitis C and B virus (HCV and HBV) infected HCC to investigate the molecular mechanism of chemoresistance and to identify a potential therapeutic target for HCC. The small RNA transcriptome analysis followed by qRT-PCR validation with the liver tissues of both HCV and HBV infected HCC patients revealed that miR-424-5p, miR-136-3p, miR-139-5p, miR-223-3p, and miR-375-3p were the most downregulated miRNAs in HCC compared to normal (log2 fold change ≤-1.5, Padj ≤ 0.05). In silico pathway analysis with the validated targets of each miRNA revealed that the signalling pathway regulating pluripotency of stem cells is commonly targeted by these five miRNAs. Subsequent validation by 3'UTR-luciferase assay and western blot analysis unveiled that these five miRNAs impeded either same or diverse genes, but all linked to BMP signalling pathway such as BMPR1A/BMPR1B by miR-139-5p, miR-136-3p, and miR-375-3p, and ACVR2A/ACVR2B by miR-424-5p and miR-223-3p. Furthermore, restoration of each miRNA in Huh7/SNU449 cells inhibited phosphorylation of downstream SMAD1/5 and ERK1/2, and attenuated Epithelial-mesenchymal transition, stemness, spheroid formation, chemoresistance, invasion and migration of cells. To investigate the mechanism of suppression of these miRNAs, "DIANA" tool was employed and lncRNA-KCNQ1OT1 was retrieved as interacting partner of all the five miRNAs. In vitro RNA pull-down assay revealed that lncRNA-KCNQ1OT1 physically interacted and sequestered these five miRNAs in the cytoplasm. Hence, KCNQ1OT1 was suppressed in Huh7/SNU449 cells using CRISPR technology and observed regression of oncogenic properties with enhanced chemosensitivity and reduced metastasis in cancer cells. Shrinkage of tumour size and volume in NOD-SCID mice injected with KCNQ1OT1-sgRNA cells further strengthened our observations. Thus, lncRNA-KCNQ1OT1 is the main regulator, which reduces the level of beneficiary miRNAs in the tumour milieu and modulates BMP signalling pathway to promote chemoresistance in HCC, suggesting lncRNA-KCNQ1OT1 might have robust potential to be a therapeutic target in HCC.

Influence of polymorphisms in TNF-α and IL1ß on susceptibility to alcohol induced liver diseases and therapeutic potential of miR-124-3p impeding TNF-α/IL1ß mediated multi-cellular signaling in liver microenvironment.

Background and aims: Alcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p. Materials and methods: Bio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required. Results: The combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1ß/IL8/MCP1/IL6/TGFß) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1ß were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1ß was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1ß-31CT+TT than TNF-α-238GG/IL1ß-31CC. The TNF-α/IL1ß promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1ß over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFß and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1ß were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFßR2/MCP1, and ICAM1 respectively. Conclusion: Thus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1ß gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1ß and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients.

Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis.

BACKGROUND: The complement system functions primarily as a first-line host defense against invading microbes, including viruses. However, the interaction of Hepatitis B virus (HBV) with the complement-components during chronic HBV infection remains largely unknown. We investigated the mechanism by which HBV inhibits the formation of cytolytic complement membrane-attack complex (MAC) and studied its impact on MAC-mediated microbicidal activity and disease pathogenesis. METHODS: Blood/liver tissues were collected from chronically HBV-infected patients and controls. HepG2hNTCP cells were infected with HBV particles and Huh7 cells were transfected with full-length linear HBV-monomer or plasmids containing different HBV-ORFs and expression of complement components or other host genes were evaluated. Additionally, ELISA, Real-time PCR, Western blot, bioinformatics analysis, gene overexpression/knock-down, mutagenesis, chromatin immunoprecipitation, epigenetic studies, immunofluorescence, and quantification of serum HBV-DNA, bacterial-DNA and endotoxin were performed. RESULTS: Among the MAC components (C5b-C9), significant reduction was noted in the expression of C9, the major constituent of MAC, in HBV-infected HepG2hNTCP cells and in Huh7 cells transfected with full-length HBV as well as HBX. C9 level was also marked low in sera/liver of chronic hepatitis B (CHB) and Immune-tolerant (IT) patients than inactive carriers and healthy controls. HBX strongly repressed C9-promoter activity in Huh7 cells but CpG-island was not detected in C9-promoter. We identified USF-1 as the key transcription factor that drives C9 expression and demonstrated that HBX-induced hypermethylation of USF-1-promoter is the leading cause of USF-1 downregulation that in turn diminished C9 transcription. Reduced MAC formation and impaired lysis of HBV-transfected Huh7 and bacterial cells were observed following incubation of these cells with C9-deficient CHB sera but was reversed upon C9 supplementation. Significant inverse correlation was noted between C9 concentration and HBV-DNA, bacterial-DNA and endotoxin content in HBV-infected patients. One-year Tenofovir therapy resulted in improvement in C9 level and decline in viral/bacterial/endotoxin load in CHB patients. CONCLUSION: Collectively, HBX suppressed C9 transcription by restricting the availability of USF-1 through hypermethylation of USF-1-promoter and consequently hinder the formation and lytic functions of MAC. Early therapy is needed for both CHB and IT to normalize the aberrant complement profile and contain viral and bacterial infection and limit disease progression.

LncRNA NEAT1 regulates HCV-induced Hepatocellular carcinoma by modulating the miR-9-BGH3 axis.

Chronic hepatitis C virus (HCV) infection is a leading cause of end-stage liver diseases, such as fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Several cellular entities, including paraspeckles and their related components, are involved in viral pathogenesis and cancer progression. NEAT1 lncRNA is a major component of paraspeckles that has been linked to several malignancies. In this study, analysis of the Cancer Genome Atlas (TCGA) database and validation in HCV-induced HCC tissue and serum samples showed significantly high expression of NEAT1 in patients with liver cancer. Moreover, we found that NEAT1 levels increased upon HCV infection. To further understand the mechanism of NEAT1-induced HCC progression, we selected one of its targets, miR-9-5 p, which regulates BGH3 mRNA levels. Interestingly, miR-9-5 p levels were downregulated upon HCV infection, whereas BGH3 levels were upregulated. Additionally, partial NEAT1 knockdown increased miR-9-5 p levels and decreased BGH3 levels, corroborating our initial results. BGH3 levels were also upregulated in HCV-induced HCC and TCGA tissue samples, which could be directly correlated with NEAT1 levels. As a known oncogene, BGH3 is directly linked to HCC progression mediated by NEAT1. We also found that NEAT1 levels remained upregulated in serum samples from patients treated with direct-acting antivirals (DAA), indicating that NEAT1 might be a molecular trigger that promotes HCC development. Collectively, these findings provide molecular insights into HCV-induced HCC progression via the NEAT1-miR-9-BGH3 axis.

A combination of circulating microRNA-375-3p and chemokines CCL11, CXCL12, and G-CSF differentiate Crohn's disease and intestinal tuberculosis.

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.

Persistence of Hepatitis B Virus Infection: A Multi-Faceted Player for Hepatocarcinogenesis.

Hepatitis B virus (HBV) infection has a multi-dimensional effect on the host, which not only alters the dynamics of immune response but also persists in the hepatocytes to predispose oncogenic factors. The virus exists in multiple forms of which the nuclear localized covalently closed circular DNA (cccDNA) is the most stable and the primary reason for viral persistence even after clearance of surface antigen and viral DNA. The second reason is the existence of pregenomic RNA (pgRNA) containing virion particles. On the other hand, the integration of the viral genome in the host chromosome also leads to persistent production of viral proteins along with the chromosomal instabilities. The interferon treatment or administration of nucleot(s)ide analogs leads to reduction in the viral DNA load, but the pgRNA and surface antigen clearance are a slow process and complete loss of serological HBsAg is rare. The prolonged exposure of immune cells to the viral antigens, particularly HBs antigen, in the blood circulation results in T-cell exhaustion, which disrupts immune clearance of the virus and virus-infected cells. In addition, it predisposes immune-tolerant microenvironment, which facilitates the tumor progression. Thus cccDNA, pgRNA, and HBsAg along with the viral DNA could be the therapeutic targets in the early disease stages that may improve the quality of life of chronic hepatitis B patients by impeding the progression of the disease toward hepatocellular carcinoma.

A novel microRNA boosts hyper-ß-oxidation of fatty acids in liver by impeding CEP350-mediated sequestration of PPARα and thus restricts chronic hepatitis C.

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 ß-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.

The exosome encapsulated microRNAs as circulating diagnostic marker for hepatocellular carcinoma with low alpha-fetoprotein.

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 (log2 FoldChange > 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.

Oncogenic potential of hepatitis B virus subgenotype D1 surpasses D3: significance in the development of hepatocellular carcinoma.

Despite widespread distribution of hepatitis B virus (HBV)-genotype D, the clinical implications of its ten subgenotypes (D1-D10) have not been well documented. Here, we have investigated the impact of two major circulating HBV/D subgenotypes, D1 and D3 in Eastern India towards pathogenesis of liver disease progression to hepatocellular carcinoma (HCC). HBV subgenotypes were determined using full-length genome sequences of HBV isolates from patients with chronic hepatitis B (CHB), liver cirrhosis (LC) and HCC. Impact of D1 and D3 on viral lifecycle and disease progression was assessed by several in vitro assays. Phylogenetic tree analysis revealed that HBV/D1 and HBV/D3 were the two predominating HBV subgenotypes circulating in Eastern India. Interestingly, the frequency of patients infected with HBV/D1 was noticed progressively rising from CHB to HCC through LC while the increasing frequency of HBV/D3 declined suddenly in HCC implicating HBV/D1 might have greater oncogenic potential than HBV/D3. Similar to higher viral load noted in HCC patients infected with HBV/D1 than HBV/D3, the larger amount of intracellular/extracellular viral DNA and secreted HBsAg levels in transfected cell lines also implicated that HBV/D1 might replicate faster than HBV/D3. Again, higher expression of marker genes related to endoplasmic reticulum stress, epithelial-mesenchymal transition, DNA double strand breaks, angiogenesis etc. and faster rate of cellular migration and anchorage independent growth cumulatively suggested that compared to HBV/D3, HBV/D1 generates more liver injuries which eventually culminates into HCC. Therefore, our results highlight the importance of determination of subgenotypes of HBV in CHB patients, so that high-risk individual can be monitor periodically that may help to detect HCC at early stages.

Hepatitis B Virus X Protein Upregulates hELG1/ ATAD5 Expression through E2F1 in Hepatocellular Carcinoma.

The precise mechanism by which HBx protein of hepatitis B virus (HBV) impacts on hepato-carcinogenesis remain largely elusive despite strong evidences for its' involvement in the process. Here, we have investigated the role of HBx on expression of a novel gene hELG1/ATAD5, which is required for genome maintenance and its' importance in hepatocarcinogenesis. This study has for the first time showed that the expression of this gene was significantly higher in human cancer such as HBV-associated hepatocellular carcinoma (HCC) and in different HCC cell lines compared to normal liver. In addition, a significant elevation in ATAD5 expression was also found in HBx transfected HCC cell lines implicating HBx mediated transcriptional regulation on ATAD5. Using different deletion mutant constructs of putative promoter, the active promoter region was first identified here and subsequently the regulatory region of HBx was mapped by promoter-luciferase assay. But ChIP assay with anti-HBx antibody revealed that HBx was not physically present in ATAD5 transcription machinery whereas anti-E2F1 antibody showed the presence of E2F1 in the complex. Luciferase assay with E2F1 binding site mutant had further confirmed it. Moreover, both loss-and gain-of-function studies of ATAD5 showed that ATAD5 could enhance HBV production in transfected cells whereas knock down of ATAD5 increased the sensitivity of HCC cell line to chemotherapeutics 5-fluorouracil. Overall, this data suggests that a positive feedback loop regulation between ATAD5 and HBV contributed to both viral replication and chemo-resistance of HCC cells.