M icroRNAs (miRNAs) are a large family of small (ϳ21-nucleotide [nt]) noncoding RNAs that interact with complementary target sites in their target mRNAs to induce translational repression, deadenylation, and degradation (1). However, the reciprocal effect of target mRNA on miRNA activity is largely unknown. is the most abundant liverspecific miRNA, accounting for approximately 70% of the total miRNA population in the adult liver (2). It has been found to play key roles in liver development and hepatic function (3, 4), hepatocyte growth, neoplastic transformation and tumorigenicity (5-8), lipid metabolism (9, 10), and regulation of hepatitis B virus (HBV) and hepatitis C virus (HCV) replication (11-13).HBV is a small (ϳ3.2 kb), enveloped, partially doublestranded DNA virus. The HBV genome contains four overlapping open reading frames (ORFs). The RNA transcripts are polyadenylated; capped; 3.5, 2.4, 2.1, and 0.7 kb in length; and named the pre-C/C or pregenomic RNA (pgRNA), pre-S, S, and X mRNAs, respectively. These mRNAs encode several overlapping viral proteins, including the polymerase, core, HBe, pre-S1, S2, S, and X proteins (14). There are approximately 350 million chronic HBV carriers worldwide, and chronic HBV infection is the major etiological factor in hepatocellular carcinoma (HCC) (15, 16). The relative risk for the development of HCC in chronic hepatitis B (CHB) patients is estimated to be 25 to 100 times higher than that in those without infection (15,17,18).Several possible pathways and molecular mechanisms have been reported for the involvement of HBV infection in malignant transformation of liver cells, including both direct and indirect mechanisms that likely act synergistically. Direct effects by viral factors include HBV DNA integration into the hepatocyte genome (which acts via cis-or trans-activation of nearby genes or enhances host chromosomal instability), the antiapoptotic and procarcinogenic functions of the HBx and truncated pre-S2/S viral proteins, and HBV mutants and genotypes (14,15,19,20). The indirect effects of chronic viral infection on malignant transformation include persistent inflammation and liver cirrhosis (which may significantly contribute to the transformation of hepatocytes and promote hepatocarcinogenesis through an integrated multistep process [21,22]), aberrant DNA methylation of specific cellular genes (23), and host susceptibility (24). However, the molecular mechanisms underlying HBV-induced carcinogenesis remain elusive and await further investigation (14, 15).Our previous study showed that loss of miR-122 induced by HBV infection enhances HBV replication through cyclin G1-modulated p53 activity, thereby possibly contributing to viral persistence (25). Moreover, miR-122 repression is only found in HCC arising in HBV-infected livers but not in HCV-infected liv-

ABSTRACT

Persistent inflammation in chronic hepatitis plays a major role in the development of hepatocellular carcinoma (HCC). In this study, the major inflammatory cytokines expressed in chronic hepatitis, IL-6 and TNF-α, induced a marked decrease in microRNA-122 (miR-122) levels, and miR-122 expression was downregulated in the livers of chronic hepatitis B (CHB) patients. The decrease of miR-122 caused upregulation of the proinflammatory chemokine CCL2. IL-6 and TNF-α suppressed miR-122 both by directly downregulating the transcription factor C/EBPα and indirectly upregulating c-myc, which blocks C/EBPα-mediated miR-122 transcription. In addition, IL-6 and TNF-α levels were elevated and miR-122 levels were decreased in mouse and rat models of diethylnitrosamine (DEN)-induced HCC. Restoration of miR-122 levels through delivery of agomir-122 suppressed DEN-induced hepatocarcinogenesis in mice. Our results show that inflammation-induced miR-122 downregulation in hepatitis contributes to carcinogenesis and suggest that increasing miR-122 may be an effective strategy for preventing HCC development in CHB patients.

A round 400 million people worldwide are infected with hepatitis B virus (HBV). Chronic hepatitis B (CHB), which is triggered by HBV infection, results in a huge health burden on the global community, as it is correlated with a significantly increased risk for the development of cirrhosis, liver failure, and hepatocellular carcinoma (HCC) (1). Currently, treatment of CHB consists mainly of pegylated alpha interferon (IFN-␣) and nucleoside or nucleotide analogs (e.g., lamivudine, adefovir, and entecavir). IFN-␣ was the first drug licensed to treat HBV infection. As an important first-line treatment option, pegylated IFN-␣ as monotherapy can effectively treat CHB in 25 to 40% of patients, and greater sustained virological responses (SVRs) and hepatitis B virus e antigen (HBeAg) seroconversion rates in HBeAg-positive patients were observed with addition of nucleoside/nucleotide analogue therapies (2, 3). In fact, treatment with pegylated IFN results in the highest rate of off-treatment sustained responses among currently available drugs (4). Moreover, responses to IFNbased therapy are accompanied by the potential for hepatitis B virus surface antigen (HBsAg) loss or seroconversion, and early serum HBsAg loss was recently reported to have predictive value for SVRs to IFN in both HBeAg-positive and -negative CHB patients (5-7).As a member of the type I interferons, IFN-␣ can initiate the activation of Jak/STAT and NF-B signaling, which induces hundreds of IFN-stimulated genes (ISGs) and may play an important role in IFN-mediated anti-HBV activity. Both in vitro and in vivo studies have shown that besides a stimulating effect on cytotoxic T lymphocytes and natural killer cell function, IFN-based therapy (IFN-␣-2b and pegylated IFN-␣-2a or -2b) also has a direct antiviral effect by preventing the formation or accelerating decay of viral capsids and/or inducing antiviral ISGs that inhibit HBV expression and replication (8-13). Inhibition of IFN-␣ signaling by HBV has been suggested to antagonize the IFN response (14).Nevertheless, these studies also strongly suggest that there is significant potential, in principle, to modulate the effectiveness of IFN-mediated anti-HBV activities. Moreover, the antiviral activity of ISGs remains elusive and still awaits further investigation (15). Responses to IFN-␣ therapy vary greatly in CHB patients, but the underlying mechanisms are almost unknown (4-6). Notably, IFN-␣/␤ was recently found to suppress HBV replication in HBV transgenic mice when the viral load was high, whereas it enhanced HBV replication when the viral load was low, indicating its dual function for HBV (16). Taken together, the data show that the precise mechanism of action of IFN-␣ has not been understood fully.MicroRNAs (miRNAs) are a class of small RNAs of approximately 22 nucleotides (nt) which interact with complementary target sites, usually in the 3=-untranslated region (3=-UTR) of target mRNAs, and induce their translational repression, deadenylation, and degradation. MicroRNA-122 (miR-122), a mammalian liv...

BackgroundHepatitis B virus (HBV) infection correlated with the development of cirrhosis, liver failure and hepatocellular carcinoma (HCC), poses a huge health burden on the global community. However, the pathogenesis of chronic hepatitis B (CHB) remains unclear. Apolipoprotein A1 (ApoA1) mainly secreted by hepatocytes, represents the major protein component of high-density lipoprotein. ApoA1 secretion may be disrupted by HBV infection. In this study, we mainly investigated the molecular mechanism of ApoA1 down regulated by HBV for revealing the pathogenesis of CHB.MethodsApoA1 expression in livers of CHB patients as well as healthy controls were performed by Real-time PCR (RT-PCR) and Western blot. The serum ApoA1 levels were measured by Enzymed-linked immunosorbent assay (ELISA). Expression of ApoA1 mRNA and protein levels were performed by RT-PCR and Western blot in human hepatoma HepG2 cells and subline HepG2.2.15 cells. HBV expression construct, pHBV1.3 were transfected into HepG2, the changes of ApoA1 mRNA and protein expression were detected by RT-PCR and Western blot. To further study the mechanism of ApoA1 down regulation by HBV, 11 CpG islands in ApoA1 promotor were tested for DNA methylation status by MSP. HepG2.2.15 cell lines were treated with DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC), then, expression of ApoA1 mRNA and HBV particles in the supernatant, as well as ApoA1 protein levels were detected by RT-PCR and Western blot. Secretion of HBsAg and HBeAg in HepG2 cells cotransfected with pApoA1 and pHBV1.3 constructs was tested by ELISA. Meanwhile, secretion of HBsAg and HBeAg in the supernatant were quantified by ELISA in the HepG2.2.15 cells treated with 5-aza-dC plus ApoA1 siRNA.ResultsExpression of ApoA1 mRNA and protein levels, as well as serum ApoA1 levels in CHB patients were decreased corresponding healthy controls in vivo. In addition, the expression of ApoA1 mRNA and protein levels were down regulated in HepG2.2.15 cells correponding HepG2 cells, 11 CpG islands in ApoA1 promoter were tested for methylation status by MSP in HepG2.2.15 cells compared to HepG2 cells, while two CpG islands were found hypermethylated. Expression of ApoA1 mRNA and protein levels were increased in HepG2.2.15 cells treated with DNA methyltransferase inhibitor 5-aza-dC. Furthermore, overexpression of ApoA1 can enhance HBV expression in HepG2 cells while the inhibitory effect of 5-aza-dC on HBV expression was completely abolished by blocking 5-aza-dC-induced up-regulation of ApoA1 using RNAi.ConclusionsEpigenetic silencing of ApoA1 gene expression by CpG island DNA hypermethylation induced by HBV may contribute to the pathogenesis of CHB.

During virus infection, viral RNAs and mRNAs function as blueprints for viral protein synthesis and possibly as pathogen-associated molecular patterns (PAMPs) in innate immunity. Here, considering recent research progress in microRNAs (miRNAs) and competitive endogenous RNAs (ceRNAs), we speculate that viral RNAs act as sponges and can sequester endogenous miRNAs within infected cells, thus cross-regulating the stability and translational efficiency of host mRNAs with shared miRNA response elements. This cross-talk and these reciprocal interactions between viral RNAs and host mRNAs are termed “competitive viral and host RNAs” (cvhRNAs). We further provide recent experimental evidence for the existence of cvhRNAs networks in hepatitis B virus (HBV), as well as Herpesvirus saimiri (HVS), lytic murine cytomegalovirus (MCMV) and human cytomegalovirus (HCMV) infections. In addition, the cvhRNA hypothesis also predicts possible cross-regulation between host and other viruses, such as hepatitis C virus (HCV), HIV, influenza virus, human papillomaviruses (HPV). Since the interaction between miRNAs and viral RNAs also inevitably leads to repression of viral RNA function, we speculate that virus may evolve either to employ cvhRNA networks or to avoid miRNA targeting for optimal fitness within the host. CvhRNA networks may therefore play a fundamental role in the regulation of viral replication, infection establishment, and viral pathogenesis.

No abstract

Stroke is one of the major causes of death and long-term disability, of which acute ischemic stroke (AIS) is the most common type. Although circular RNA (circRNA) expression profiles of AIS patients have been reported to be significantly altered in blood and peripheral blood mononuclear cells, the role of exosome-containing circRNAs after AIS is still unknown. Plasma exosomes from 10 AIS patients and 10 controls were isolated, and through microarray and bioinformatics analysis, the profile and putative function of circRNAs in the plasma exosomes were studied. A total of 198 circRNAs were differentially quantified (|log2 fold change| ≥ 1.00, p < 0.05) between AIS patients and controls. The levels of 12 candidate circRNAs were verified by qRT-PCR, and the quantities of 10 of these circRNAs were consistent with the data of microarray. The functions of host genes of differentially quantified circRNAs, including RNA and protein process, focal adhesion, and leukocyte transendothelial migration, were associated with the development of AIS. As a miRNA sponge, differentially quantified circRNAs had the potential to regulate pathways related to AIS, like PI3K-Akt, AMPK, and chemokine pathways. Of 198 differentially quantified circRNAs, 96 circRNAs possessing a strong translational ability could affect cellular structure and activity, like focal adhesion, tight junction, and endocytosis. Most differentially quantified circRNAs were predicted to bind to EIF4A3 and AGO2—two RNA-binding proteins (RBPs)—and to play a role in AIS. Moreover, four of ten circRNAs with verified levels by qRT-PCR (hsa_circ_0112036, hsa_circ_0066867, hsa_circ_0093708, and hsa_circ_0041685) were predicted to participate in processes of AIS, including PI3K-Akt, AMPK, and chemokine pathways as well as endocytosis, and to be potentially useful as diagnostic biomarkers for AIS. In conclusion, plasma exosome-derived circRNAs were significantly differentially quantified between AIS patients and controls and participated in the occurrence and progression of AIS by sponging miRNA/RBPs or translating into proteins, indicating that circRNAs from plasma exosomes could be crucial molecules in the pathogenesis of AIS and promising candidates as diagnostic biomarkers and therapeutic targets for the condition.