The Role of Gut Microbiome-Derived Short-Chain Fatty Acid Butyrate in Hepatobiliary Diseases.

The short-chain fatty acid butyrate, produced from fermentable carbohydrates by gut microbiota in the colon, has multiple beneficial effects on human health. At the intestinal level, butyrate regulates metabolism, helps in the transepithelial transport of fluids, inhibits inflammation, and induces the epithelial defense barrier. The liver receives a large amount of short-chain fatty acids via the blood flowing from the gut via the portal vein. Butyrate helps prevent nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, inflammation, cancer, and liver injuries. It ameliorates metabolic diseases, including insulin resistance and obesity, and plays a direct role in preventing fatty liver diseases. Butyrate has different mechanisms of action, including strong regulatory effects on the expression of many genes by inhibiting the histone deacetylases and modulating cellular metabolism. The present review highlights the wide range of beneficial therapeutic and unfavorable adverse effects of butyrate, with a high potential for clinically important uses in several liver diseases.

DDX3-mediated miR-34 expression inhibits autophagy and HBV replication in hepatic cells.

HBV entry to the host cells and its successful infection depends on its ability to modulate the host restriction factors. DEAD box RNA helicase, DDX3, is shown to inhibit HBV replication. However, the exact mechanism of inhibition still remains unclear. DDX3 is involved in multitude or RNA metabolism processes including biogenesis of miRNAs. In this study, we sought to determine the mechanism involved in DDX3-mediated HBV inhibition. First, we observed that HBx protein of HBV downregulated DDX3 expression in HBV-infected cells. Overexpression of DDX3 inhibited HBx, HBsAg and total viral load, while its knockdown reversed the result in Hep G2.2.15 cells. Expression of miR-34 was downregulated in HBV-infected cells. Overexpression of pHBV1.3 further confirmed that HBV downregulates miR-34 expression. Consistent with the previous finding that DDX3 is involved in miRNA biogenesis, we observed that expression of miR-34 positively corelated with DDX3 expression. miRNA target prediction tools showed that miR-34 can target autophagy pathway which is hijacked by HBV for the benefit of its own replication. Indeed, transfection with miR-34 oligos downregulated the expression of autophagy marker proteins in HBV-expressing cells. Overexpression of DDX3 in HBV-expressing cells, downregulated expression of autophagy proteins while silencing of DDX3 reversed the results. These results led us to conclude that DDX3 upregulates miR-34 expression and thus inhibits autophagy in HBV-expressing cells while HBx helps HBV evade DDX3-mediated inhibition by downregulating DDX3 expression in HBV-infected cells.

Hepatitis B Virus X Protein Inhibits the Expression of Barrier To Autointegration factor1 via Upregulating miR-203 Expression in Hepatic Cells.

Hepatitis B virus (HBV) infection targets host restriction factors that inhibit its replication and survival. Previous studies have shown that barriers to autointegration factor1 (BANF1) inhibited the replication of herpes simplex virus and vaccinia virus by binding to phosphate backbone of dsDNA. To date, no reports are available for the interplay between BANF1 and HBV. In this study, we elucidated the mechanisms by which HBV inhibit BANF1. First, the effect of HBV on BANF1 was observed in Huh-7, Hep G2, and Hep G2.2.15 cells. Huh-7 cells were transfected with pHBV1.3 or HBx plasmids. The results showed that there was a decreased expression of BANF1 in Hep G2.2.15 cells (P ≤ 0.005) or in HBV/HBx expressing Huh-7 cells (P ≤ 0.005), whereas BANF1 overexpression decreased viral replication (P ≤ 0.05). To study whether phosphorylation/dephosphorylation of BANF1 was responsible for antiviral activity, mutants were created, and it was found that inhibition due to mutants was less significant compared to BANF1 wild type. Previous studies have shown that HBV, at least in part, could regulate the expression of host miRNAs via HBx. It was found that miR-203 expression was high in Hep G2.2.15 cells (P ≤ 0.005) compared to Hep G2 cells. Next, the effect of HBx on miR-203 expression was studied and result showed that HBx upregulated miR-203 expression (P ≤ 0.005). Overexpression of miR-203 downregulated BANF1 expression (P ≤ 0.05) and viral titer was upregulated (P ≤ 0.05), while inhibition of miR-203, reversed these changes. In conclusion, BANF1 downregulated HBV, whereas HBV inhibited BANF1, at least in part, via HBx-mediated miR-203 upregulation in hepatic cells. IMPORTANCE In this study, for the first time, we found that BANF1 inhibited HBV replication and restricted the viral load. However, as previously reported for other viruses, the results in this study showed that BAF1 phosphorylation/dephosphorylation is not involved in its antiviral activity against HBV. HBV infection inhibited the intracellular expression of BANF1, via HBx-mediated upregulation of miR-203 expression. Overexpression of miR-203 downregulated BANF1 and increased the viral titer, while inhibition of miR-203 reversed these changes. This study helped us to understand the molecular mechanisms by which HBV survives and replicates in the host cells.

Free fatty acid-induced miR-181a-5p stimulates apoptosis by targeting XIAP and Bcl2 in hepatic cells.

AIMS: Non-alcoholic fatty liver disease is one of the major health concerns in the World. The dietary free fatty acids (FFAs) affect the metabolic status of the hepatocytes by modulating cellular pathways. In this study, we showed that free fatty acids stimulate apoptosis by upregulating miR-181a-5p expression, which in turn targets XIAP and Bcl2. METHODS: Huh7 cells were incubated with FFAs for 72 h and the expression of XIAP, Bcl2, bax, pAkt, Akt, PTEN and ß-actin were determined by Western blots, and miR-181a-5p expression was determined using real-time RT-PCR. The Huh7 cells were transfected with either miR-181a-5p pre-miRs or anti-miR-181a-5p and the regulation of apoptosis and proliferation was studied. Three groups of C57BL/6 mice (n = 6 per group) were fed with standard diet, CSAA or CDAA diet for 6, 18, 32 and 54 weeks. Total protein and RNA were isolated from the liver tissues and used for Western blots and real-time RT-PCR respectively. KEY FINDINGS: FFAs inhibited Akt phosphorylation, expression of XIAP and Bcl2, while upregulating the expression of PTEN, bax, and miR-181a-5p in Huh7 cells. Similar results were observed when the Huh7 cells were transfected with miR-181a-5p premiRs, while these changes were reversed in anti-miR-181a-5p-transfected, FFA-treated Huh7 cells. The CDAA-fed mice showed a significant inhibition of Akt phosphorylation, XIAP and Bcl2, whereas PTEN and bax expression were upregulated. The expression of miR-181a-5p was also significantly higher in CDAA-fed mice. SIGNIFICANCE: These findings showed that free fatty acids induced apoptosis via upregulating miR-181a-5p in hepatic cells.

Biocidal (bacterial and cancer cells) activities of chitosan/CuO nanomaterial, synthesized via a green process.

Biopolymer-based nanomaterials have been developed as antimicrobial and anticancer agents due to their advanced physical, chemical and biomedical characteristics. Herein, chitosan-copper oxide nanomaterial was, successfully synthesized by a green method. In this process, copper salt was nucleated with Psidium guajava leaves extract in order to form the nanomaterial in the chitosan network. Attenuated total reflection-fourier transform, infrared spectroscopy, X-ray diffraction, Dynamic light scattering, Transmission electron microscope, Field emission scanning electron microscopy/Energy dispersive X-ray analysis, X-ray photoelectron spectroscopy and Photoluminescence spectroscopy techniques were, employed to characterize the synthesized nanomaterial. The average size of the nanomaterial was identified to be ∼52.49 nm with XRD. The antibacterial study of CCuO NM showed higher activity than the commercial amoxicillin against gram-positive (G + ve) (Staphylococcus aureus, Bacillus subtilis) and gram-negative (G-ve) bacteria (Klebsiella pneumonia, Escherichia coli). CCuO NM showed in-vitro anticancer potential against human cervical cancer cells (Hela) with an IC50 concentration of 34.69 µg/mL. Photoluminescence spectrum of CCuO NM showed a green emission (oxygen vacancies) observed at ∼516 nm, which is attributed to the generation of reactive oxygen species (ROS) by the nanomaterial, which is believed, to be responsible for the biocidal (cell death) effects. These results suggested that CCuO is a promising nanomaterial that could be suitable for advanced applications in the healthcare industries.

Cloning of human ABCB11 gene in E. coli required the removal of an intragenic Pribnow-Schaller Box before it's Insertion into genomic safe harbor AAVS1 site using CRISPR-Cas9.

Background: Genomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn't cause any foreseeable risk to the host. The AAVS1 site is the genetic location which is disrupted upon integration of adeno associated virus (AAV) and is considered a 'safe-harbor' in human genome because about one-third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man.  Therefore, we chose to target the AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. Methods: We used the CRISPR Cas9 a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Results: We found that human ABCB11 sequence has a "Pribnow- Schaller Box" which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli; the removal of this was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool.  We also found that the ABCB11 protein has similarity with E. coli endotoxin (lipid A) transporter MsbA. Conclusions: We inserted ABCB11 at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful in vivo.

Glibenclamide, ATP and metformin increases the expression of human bile salt export pump ABCB11.

Background: Bile salt export pump (BSEP/ABCB11) is important in the maintenance of the enterohepatic circulation of bile acids and drugs. Drugs such as rifampicin and glibenclamide inhibit BSEP. Progressive familial intrahepatic cholestasis type-2, a lethal pediatric disease, some forms of intrahepatic cholestasis of pregnancy, and drug-induced cholestasis are associated with BSEP dysfunction.  Methods: We started with a bioinformatic approach to identify the relationship between ABCB11 and other proteins, microRNAs, and drugs. A microarray data set of the liver samples from ABCB11 knockout mice was analyzed using GEO2R. Differentially expressed gene pathway enrichment analysis was conducted using ClueGo. A protein-protein interaction network was constructed using STRING application in Cytoscape. Networks were analyzed using Cytoscape. CyTargetLinker was used to screen the transcription factors, microRNAs and drugs. Predicted drugs were validated on human liver cell line, HepG2. BSEP expression was quantified by real-time PCR and western blotting. Results:ABCB11 knockout in mice was associated with a predominant upregulation and downregulation of genes associated with cellular component movement and sterol metabolism, respectively. We further identified the hub genes in the network. Genes related to immune activity, cell signaling, and fatty acid metabolism were dysregulated.  We further identified drugs (glibenclamide and ATP) and a total of 14 microRNAs targeting the gene. Western blot and real-time PCR analysis confirmed the upregulation of BSEP on the treatment of HepG2 cells with glibenclamide, ATP, and metformin. Conclusions: The differential expression of cell signaling genes and those related to immune activity in ABCB11 KO animals may be secondary to cell injury. We have found glibenclamide, ATP, and metformin upregulates BSEP. The mechanisms involved and the clinical relevance of these findings need to be investigated.

Augmenter of liver regeneration enhances cell proliferation through the microRNA-26a/Akt/cyclin D1 pathway in hepatic cells.

AIM: Hepatocytes can proliferate and regenerate when injured by toxins, viral infections, and so on. Augmenter of liver regeneration (ALR) is a key regulator of liver regeneration, but the mechanism is unknown. The role of ALR in other cell types is not known. In the present study, we investigated the relationship between microRNA (miRNA)-26a and ALR in the Huh7 cell line and adipose tissue-derived mesenchymal cells from chronic liver disease patients and healthy individuals. METHODS: Huh7 cells were transfected independently with ALR and miRNA-26a expression vectors, and their effects on cell proliferation, the expression of miRNA-26a, and activation of the phosphatase and tensin homolog and Akt signaling pathways were determined. The experiments were repeated on mesenchymal stem cells derived from healthy individuals and chronic liver disease patients to see whether the observations can be replicated in primary cells. RESULTS: Overexpression of ALR or miRNA-26a resulted in an increase of the phosphorylation of Akt and cyclin D1 expression, whereas it resulted in decreased levels of p-GSK-3ß and phosphatase and tensin homolog in Huh7 cells. The inhibition of ALR expression by ALR siRNA or anti-miR-26a decreased the Akt/cyclin D1 signaling pathway, leading to decreased proliferation. Mesenchymal stem cells isolated from the chronic liver disease patients had a higher ALR expression, while the mesenchymal stem cells isolated from healthy volunteers responded to the growth factor treatments for increased ALR expression. It was found that there was a significant increase in miRNA-26a expression and proliferation. CONCLUSIONS: These data clearly showed that ALR induced the expression of miRNA-26a, which downregulated phosphatase and tensin homolog, resulting in an increased p-Akt/cyclin D1 pathway and enhanced proliferation in hepatic cells.

TGF-ß induces liver fibrosis via miRNA-181a-mediated down regulation of augmenter of liver regeneration in hepatic stellate cells.

OBJECTIVE: To study the role of miRNA-181a and augmenter of liver regeneration in TGF-ß-induced fibrosis in hepatic stellate cells. METHODS: LX2 cells were treated with 20 ng/ml TGF-ß for 24 h. miRNA-181a, ALR plasmid and empty vectors were transfected using siPORT NeoFx reagent. Cells were harvested after 48 h or 72 h of transfection for protein or RNA analysis. Western blotting was performed for ALR, TGF-ß receptor II (TGFß-RII), collagen 1A1 (COLL1A1), alpha-smooth muscle cell actin (α-SMA), rac1, E-cadherin and ß-actin. Quantitative RT-PCR was performed for ALR, GAPDH, miRNA-181a or 5S rRNA. RESULTS: TGF-ß induced the expression of miRNA-181a, which in turn down-regulated ALR thereby induced the fibrosis markers, such as COLL1A1, α-SMA and rac1 in hepatic stellate cells. Over-expression of miRNA-181a down-regulated expression of ALR and up-regulated expression of fibrosis markers. On the other hand, ALR over-expression resulted in a decrease in miRNA-181a expression and fibrosis markers. Over-expression of ALR also inhibited the expression of TGFß-RII and increased expression E-cadherin. CONCLUSION: TGF-ß induced miRNA-181a, which in turn induced fibrosis, at least in part, by inhibiting ALR. ALR inhibited TGF-ß action by decreasing the expression of TGFß-RII, thereby inhibiting miRNA-181a expression and fibrosis markers. ALR could serve as a potential molecule to inhibit liver fibrosis.

Butyrate inhibits HBV replication and HBV-induced hepatoma cell proliferation via modulating SIRT-1/Ac-p53 regulatory axis.

Butyrate, a histone deacetylase inhibitor, has several therapeutic applications, including cancer. However, the effect of butyrate in HBV replication is not known so far. It was hypothesized that butyrate might inhibit HBV replication and host cell proliferation via SIRT-1. It was found that the increased expression of SIRT-1 in Hep G2.2.15 cells (HBV expressing cells) than Hep G2 cells. Next the expression of SIRT-1 and Acetylated p53 (Ac-p53) were measured in the liver biopsy samples of chronic hepatitis B (CHB) patients with high viral load and compared to CHB patients with low viral load and found that there was a high SIRT-1 expression and a low Ac-p53 levels in CHB patients with high viral load compared to CHB patients with low viral load. Incubation of butyrate inhibited SIRT-1 expression and cell proliferation. Inhibition of SIRT-1 by butyrate or SIRT-1 siRNA increased the levels of Ac-p53. The elevated Ac-p53 decreased p-akt, cyclin D1, and thereby inhibited cell proliferation. Incubation of butyrate with Hep G2.2.15 cells also inhibited HBx protein expression, HBV-DNA and hepatitis B surface antigen (HBsAg). Taken together, the data showed that butyrate inhibited HBV replication and cell proliferation by inhibiting SIRT-1 expression in hepatoma cells.

Augmenter of liver regeneration: A key protein in liver regeneration and pathophysiology.

Liver is constantly exposed to pathogens, viruses, chemicals, and toxins, and several of them cause injury, leading to the loss of liver mass and sometimes resulting in cirrhosis and cancer. Under physiological conditions, liver can regenerate if the loss of cells is less than the proliferation of hepatocytes. If the loss is more than the proliferation, the radical treatment available is liver transplantation. Due to this reason, the search for an alternative therapeutic agent has been the focus of liver research. Liver regeneration is regulated by several growth factors; one of the key factors is augmenter of liver regeneration (ALR). Involvement of ALR has been reported in crucial processes such as oxidative phosphorylation, maintenance of mitochondria and mitochondrial biogenesis, and regulation of autophagy and cell proliferation. Augmenter of liver regeneration has been observed to be involved in liver regeneration by not only overcoming cell cycle inhibition but by maintaining the stem cell pool as well. These observations have created curiosity regarding the possible role of ALR in maintenance of liver health. Thus, this review brings a concise presentation of the work done in areas exploring the role of ALR in normal liver physiology and in liver health maintenance by fighting liver diseases, such as liver failure, non-alcoholic fatty liver disease/non-alcoholic steatohepatitis, viral infections, cirrhosis, and hepatocellular carcinoma.

Triiodothyronine stimulates VEGF expression and secretion via steroids and HIF-1α in murine Leydig cells.

Leydig cells are the principal steroidogenic cells of the testis. Leydig cells also secrete a number of growth factors including vascular endothelial growth factor (VEGF) which has been shown to regulate both testicular steroidogenesis and spermatogenesis. The thyroid hormone, T3, is known to stimulate steroidogenesis in Leydig cells. T3 has also been shown to stimulate VEGF production in a variety of cell lines. However, studies regarding the effect of T3 on VEGF synthesis and secretion by the Leydig cells were lacking. Therefore, we investigated the effect of T3 on VEGF synthesis and secretion in a mouse Leydig tumour cell line, MLTC-1. The effect of T3 was compared with that of LH/cAMP and hypoxia, two known stimulators of Leydig cell functions. The cells were treated with T3, 8-Br-cAMP (a cAMP analogue), or CoCl2 (a hypoxia mimetic) and VEGF secreted in the cell supernatant was measured using ELISA. The mRNA levels of VEGF were measured by quantitative RT-PCR. In the MLTC-1 cells, T3, 8-Br-cAMP, and CoCl2 stimulated VEGF mRNA levels and the protein secretion. T3 also increased steroid secretion as well as HIF-1α protein levels, two well-established upstream regulators of VEGF. Inhibitors of steroidogenesis as well as HIF-1α resulted in inhibition of T3-stimulated VEGF secretion by the MLTC-1 cells. This suggested a mediatory role of steroids and HIF-1α protein in T3-stimulated VEGF secretion by MLTC-1 cells. The mediation by steroids and HIF-1α were independent of each other. ABBREVIATIONS: 8-Br-cAMP: 8-bromo - 3', 5' cyclic adenosine monophosphate; CoCl2: cobalt chloride; HIF-1α: hypoxia inducible factor -1α; LH: luteinizing hormone; T3: 3, 5, 3'-L-triiodothyronine; VEGF: vascular endothelial growth factor.

Global microRNA expression profiling in the liver biopsies of hepatitis B virus-infected patients suggests specific microRNA signatures for viral persistence and hepatocellular injury.

Hepatitis B virus (HBV) can manipulate the microRNA (miRNA) regulatory networks in infected cells to create a permissive environment for viral replication, cellular injury, disease onset, and its progression. The aim of the present study was to understand the miRNA networks and their target genes in the liver of hepatitis B patients involved in HBV replication, liver injury, and liver fibrosis. We investigated differentially expressed miRNAs by microarray in liver biopsy samples from different stages of HBV infection and liver disease (immune-tolerant [n = 8], acute viral hepatitis [n = 8], no fibrosis [n = 16], early [F1+F2, n = 19] or late [F3+F4, n = 14] fibrosis, and healthy controls [n = 7]). miRNA expression levels were analyzed by unsupervised principal component analysis and hierarchical clustering. Analysis of miRNA-mRNA regulatory networks identified 17 miRNAs and 18 target gene interactions with four distinct nodes, each representing a stage-specific gene regulation during disease progression. The immune-tolerant group showed elevated miR-199a-5p, miR-221-3p, and Let-7a-3p levels, which could target genes involved in innate immune response and viral replication. In the acute viral hepatitis group, miR-125b-5p and miR-3613-3p were up, whereas miR-940 was down, which might affect cell proliferation through the signal transducer and activator of transcription 3 pathway. In early fibrosis, miR-34b-3p, miR-1224-3p, and miR-1227-3p were up, while miR-499a-5p was down, which together possibly mediate chronic inflammation. In advanced fibrosis, miR-1, miR-10b-5p, miR-96-5p, miR-133b, and miR-671-5p were up, while miR-20b-5p and miR-455-3p were down, possibly allowing chronic disease progression. Interestingly, only 8 of 17 liver-specific miRNAs exhibited a similar expression pattern in patient sera. CONCLUSION: miRNA signatures identified in this study corroborate previous findings and provide fresh insight into the understanding of HBV-associated liver diseases which may be helpful in developing early-stage disease diagnostics and targeted therapeutics. (Hepatology 2018;67:1695-1709).

Oxidative stress plays a key role in butyrate-mediated autophagy via Akt/mTOR pathway in hepatoma cells.

Hepatocellular Carcinoma (HCC) is one of the most aggressive forms of cancer, responsible for a number of deaths in humans. Butyrate, one of the short chain fatty acids produced by the gut microbiota during anaerobic fermentation, was shown to be beneficial for inhibiting cancer growth. In this study, we showed that sodium butyrate induced autophagy via reactive oxygen species (ROS) in hepatoma cells. Butyrate (0-6 mM) incubation significantly increased intracellular ROS levels (45.2% compared to control), which in turn inhibited phosphorylation of akt and mTOR, leading to the upregulation of autophagic proteins, such as beclin 1, ATG 5, LC3-II, followed by the increased autophagosome formation (34.4% compared to control cells). Addition of a known antioxidant, N-acetyl cysteine (NAC), reversed these butyrate-induced ROS and autophagy. It was also found that butyrate-induced ROS enhanced MAPK activation, which was inhibited by NAC. In conclusion, our data showed that butyrate induced ROS, which in turn induced autophagy via inhibition of akt/mTOR pathway. Hence, butyrate could be considered as a potential candidate for HCC treatment.

Butyrate induces ROS-mediated apoptosis by modulating miR-22/SIRT-1 pathway in hepatic cancer cells.

Butyrate is one of the short chain fatty acids, produced by the gut microbiota during anaerobic fermentation of dietary fibres. It has been shown that it can inhibit tumor progression via suppressing histone deacetylase and can induce apoptosis in cancer cells. However, the comprehensive pathway by which butyrate mediates apoptosis and growth arrest in cancer cells still remains unclear. In this study, the role of miR-22 in butyrate-mediated ROS release and induction of apoptosis was determined in hepatic cells. Intracellular expression of miR-22 was increased when the Huh 7 cells were incubated with sodium butyrate. Over-expression of miR-22 or addition of sodium butyrate inhibited SIRT-1 expression and enhanced the ROS production. Incubation of cells with anti-miR-22 reversed the effects of butyrate. Butyrate induced apoptosis via ROS production, cytochrome c release and activation of caspase-3, whereas addition of N-acetyl cysteine or anti-miR-22 reversed these butyrate-induced effects. Furthermore, sodium butyrate inhibited cell growth and proliferation, whereas anti-miR-22 inhibited these butyrate-mediated changes. The expression of PTEN and gsk-3 was found to be increased while p-akt and ß-catenin expression was decreased significantly by butyrate. These data showed that butyrate modulated both apoptosis and proliferation via miR-22 expression in hepatic cells.

Circulating microRNAs: Possible role as non-invasive diagnostic biomarkers in liver disease.

Liver is the central organ for metabolism and the hepatocytes metabolize several drugs, hepatotoxins, alcohol, etc. Continuous exposure of the hepatocytes to these toxins result in various chronic diseases, such as alcoholic liver disease, non-alcoholic fatty liver disease, viral hepatitis and hepatocellular carcinoma. Although several diagnostic methods, such as serum markers, liver biopsy or imaging studies are currently available, most of these are either invasive or detect the disease at advanced stages. Hence, there is a need for new molecular markers that can be used for early detection of the disease. MicroRNAs (miRNAs) are naturally occurring, 20-22 nucleotide long, non-coding RNA molecules that regulate the gene expression at post-transcriptional levels, thereby modulating various biological functions. Their expression is deregulated under pathological conditions, and recent studies showed that they are secreted and can be detected in various body fluids. Since the cellular changes occur at earlier stages of the disease, detecting miRNAs in the body fluids could make them as potential novel biomarkers. Albeit, the difficulties in standardization procedures, cost and availability should be addressed before using them in the clinical arena. This review highlights the possible role of secreted miRNAs to use as early non-invasive diagnostic markers for liver disease.

Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells.

Hepatitis B Virus (HBV) utilizes several mechanisms to survive in the host cells and one of the main pathways being autophagosome formation. Humic acid (HA), one of the major components of Mineral pitch, is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. We hypothesized that HA could induce cell death and inhibit HBV-induced autophagy in hepatic cells. Incubation of Hep G2.2.1.5 cells (HepG2 cells stably expressing HBV) with HA (100 µM) inhibited both cell proliferation and autophagosome formation significantly, while apoptosis induction was enhanced. Western blot results showed that HA incubation resulted in decreased levels of beclin-1, SIRT-1 and c-myc, while caspase-3 and ß-catenin expression were up-regulated. Western blot results showed that HA significantly inhibited the expression of HBx (3-fold with 50 µM and 5-fold with 100 µM) compared to control cells. When HA was incubated with HBx-transfected Hep G2 cells, HBx-induced autophagosome formation and beclin-1 levels were decreased. These data showed that HA induced apoptosis and inhibited HBV-induced autophagosome formation and proliferation in hepatoma cells.

Mineral pitch induces apoptosis and inhibits proliferation via modulating reactive oxygen species in hepatic cancer cells.

BACKGROUND: Mineral Pitch (MP) is a dark brown coloured humic matter originating from high altitude rocks. It is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. METHODS: The Huh-7 cells were treated with different concentrations of MP for 24 h, and both apoptosis and proliferation was determined by the TUNEL and MTT assays respectively. The formation of ROS and nitric oxide was analysed by DCFH-DA and Griess reagent respectively. The expression of miRNA-21 and miRNA-22 were checked by the real time PCR. Effect of miRNA-22 on proliferation and c-myc was studied by over-expressing miRNA-22 premiRs in Huh-7 cells. RESULTS: We found that MP enhanced anti-cancer effects by inducing apoptosis and inhibiting proliferation. MP induced both ROS and NO, upon neutralizing them, there was a partial recovery of apoptosis and proliferation. MP also induced miRNA-22 expression, while miRNA-21 expression was inhibited. Over-expression of miRNA-22 resulted in a significant inhibition of proliferation. miRNA-22 directly targeted c-myc gene, thereby inhibited proliferation. These results clearly show that MP induces its anti-cancer activity by more than one pathway. CONCLUSION: The data clearly indicate that MP induced apoptosis via the production of ROS, and inhibited proliferation by inducing miRNA-22 and inhibiting miRNA-21 in Huh-7 cells.

Overexpression of microRNA-30a inhibits hepatitis B virus X protein-induced autophagosome formation in hepatic cells.

Hepatitis B virus (HBV) enters the host and survives by using several mechanisms. One of the ways that HBV survives and replicates in the host cells is by inducing autophagy. Previous reports have shown that microRNA (miRNA)-30a inhibits autophagosome formation in cancer cells. Hence, we hypothesized that overexpression of miRNA-30a could inhibit HBV-induced autophagosome formation in hepatic cells. To study this, both HepG2 cells and HepG2.2.1.5 cells (HBV-expressing stable cell line) were transfected with miRNA-30a, and the cells were collected either for RNA isolation or protein isolation after 72 h of transfection. Beclin-1 expression was significantly higher in untransfected HepG2.2.1.5 cells than in HepG2 cells. Western blots showed that miRNA-30a overexpression resulted in a significant decrease in beclin-1 expression (eight-fold and four-fold in HepG2 and HepG2.2.1.5 cells, respectively) and c-myc expression, whereas the numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells were increased. In contrast, overexpression of HBV X protein (HBx) in HepG2 cells resulted in the enhancement of beclin-1 (six-fold increase as compared with the empty vector-transfected cells) and c-myc expression, whereas the numbers of TUNEL-positive cells were reduced. To confirm these findings, HBx and miRNA-30a were coexpressed in HepG2 cells, and the results showed significant inhibition of autophagosome formation and beclin-1 and c-myc expression, whereas apoptosis increased. These data demonstrate that HBx induces autophagosome formation via beclin-1 expression, whereas miRNA-30a overexpression could successfully inhibit the beclin-1 expression induced by HBx, thereby modulating autophagosome formation in hepatic cells.

Hepatitis B virus induces cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma by targeting programmed cell death protein4 (PDCD4) and phosphatase and tensin homologue (PTEN).

Hepatitis B viral infection-induced hepatocellular carcinoma is one of the major problems in the developing countries. One of the HBV proteins, HBx, modulates the host cell machinery via several mechanisms. In this study we hypothesized that HBV enhances cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma. HBx gene was over-expressed, and miRNA-21 expression and cell proliferation were measured in Huh 7 and Hep G2 cells. miRNA-21 was over-expressed in these cells, cell proliferation and the target proteins were analyzed. To confirm the role of miRNA-21 in HBx-induced proliferation, Hep G 2.2.1.5 cells (a cell line that expresses HBV stably) were used for miRNA-21 inhibition studies. HBx over-expression enhanced proliferation (3.7- and 4.5-fold increase; n = 3; p<0.01) and miRNA-21 expression (24- and 36-fold increase, normalized with 5S rRNA; p<0.001) in Huh 7 and Hep G2 cells respectively. HBx also resulted in the inhibition of miRNA-21 target proteins, PDCD4 and PTEN. miRNA-21 resulted in a significant increase in proliferation (2- and 2.3-fold increase over control cells; p<0.05 in Huh 7 and Hep G2 cells respectively) and decreased target proteins, PDCD4 and PTEN expression. Anti-miR-21 resulted in a significant decrease in proliferation (p<0.05) and increased miRNA-21 target protein expression. We conclude that HBV infection enhances cell proliferation, at least in part, via HBx-induced miRNA-21 expression during hepatocellular carcinoma progression.