Yingjun Guo

A Long Noncoding RNA Activated by TGF-β Promotes the Invasion-Metastasis Cascade in Hepatocellular Carcinoma

The role of TGF-β-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-β signaling is still unknown. In this study, we observed that the lncRNA-activated by TGF-β (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB upregulated ZEB1 and ZEB2 by competitively binding the miR-200 family and then induced EMT and invasion. In addition, lncRNA-ATB promoted organ colonization of disseminated tumor cells by binding IL-11 mRNA, autocrine induction of IL-11, and triggering STAT3 signaling. Globally, lncRNA-ATB promotes the invasion-metastasis cascade. Thus, these findings suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose HCC patients to metastases and may serve as a potential target for antimetastatic therapies.

Plasma MicroRNA-122 as a Biomarker for Viral-, Alcohol-, and Chemical-Related Hepatic Diseases

BACKGROUND The liver is frequently subject to insult because of viral infection, alcohol abuse, or toxic chemical exposure. Extensive research has been conducted to identify blood markers that can better discern liver damage, but little progress has been achieved in clinical practice. Recently, circulating microRNAs (miRNAs) have been reported as potential biomarkers for the noninvasive diagnosis of cancer. In this study, we investigated whether plasma miRNAs have diagnostic utility in identifying liver disease. METHODS The study was divided into 2 phases: marker selection by real-time quantitative PCR analysis of a small set of plasma samples, and marker validation with a large set of plasma samples from 83 patients with chronic hepatitis B viral infections, 15 patients with skeletal muscle disease, and 40 healthy controls. Two mouse model systems, d-galactosamine- and alcohol-induced liver injury, were also developed to evaluate whether differences in miRNA concentration were associated with various liver diseases. RESULTS Among the miRNA candidates identified, miR-122 presented a disease severity-dependent change in plasma concentration in the patients and animal models. Compared with an increase in aminotransferase activity in the blood, the change in miR-122 concentration appeared earlier. Furthermore, this change was more specific for liver injury than for other organ damage and was more reliable, because the change was correlated with liver histologic stage. CONCLUSIONS Our findings suggest that circulating miR-122 has potential as a novel, predictive, and reliable blood marker for viral-, alcohol-, and chemical-induced liver injury.

Down-regulated microRNA-152 induces aberrant DNA methylation in hepatitis B virus–related hepatocellular carcinoma by targeting DNA methyltransferase 1†

The hepatitis B virus (HBV) X protein has been implicated as a potential trigger of the epigenetic modifications of some genes during hepatocarcinogenesis, but the underlying mechanisms remain unknown. MicroRNAs (miRNAs), which are noncoding RNAs that regulate gene expression, are involved in diverse biological functions and in carcinogenesis. In this study, we investigated whether some miRNAs are aberrantly expressed and involved in the regulation of the abnormal DNA methylation status in HBV‐related hepatocellular carcinoma (HCC). Our results showed that the expression of microRNA‐152 (miR‐152) was frequently down‐regulated in HBV‐related HCC tissues in comparison with adjacent noncancerous hepatic tissues and was inversely correlated to DNA methyltransferase 1 (DNMT1) messenger RNA (mRNA) expression in HBV‐related HCCs. The forced expression of miR‐152 in liver cell lines resulted in a marked reduction of the expression of DNMT1 at both the mRNA and protein levels by directly targeting the 3′ untranslated regions of DNMT1. This in turn led to a decrease in global DNA methylation, whereas inhibition of miR‐152 caused global DNA hypermethylation and increased the methylation levels of two tumor suppressor genes, glutathione S‐transferase pi 1 (GSTP1) and E‐cadherin 1 (CDH1). Conclusion: Our findings suggest that miR‐152 is frequently down‐regulated and regulates DNMT1 in HBV‐related HCC. These findings support a tumor‐suppressive role of miR‐152 in the epigenetic aberration of HBV‐related HCC and the potential development of miRNA‐based targeted approaches for the treatment of HBV‐related HCC. HEPATOLOGY 2010

Hepatitis B virus X protein (HBx)‐related long noncoding RNA (lncRNA) down‐regulated expression by HBx (Dreh) inhibits hepatocellular carcinoma metastasis by targeting the intermediate filament protein vimentin

The hepatitis B virus X protein (HBx) has been implicated as an oncogene in both epigenetic modifications and genetic regulation during hepatocarcinogenesis, but the underlying mechanisms are not entirely clear. Long noncoding RNAs (lncRNAs), which regulate gene expression with little or no protein‐coding capacity, are involved in diverse biological processes and in carcinogenesis. We asked whether HBx could promote hepatocellular carcinoma (HCC) by regulating the expression of lncRNAs. In this study we investigated the alteration in expression of lncRNAs induced by HBx using microarrays and real‐time quantitative polymerase chain reaction (PCR). Our results indicate that HBx transgenic mice have a specific profile of liver lncRNAs compared with wildtype mice. We identified an lncRNA, down‐regulated expression by HBx (termed lncRNA‐Dreh), which can inhibit HCC growth and metastasis in vitro and in vivo, act as a tumor suppressor in the development of hepatitis B virus (HBV)‐HCC. LncRNA‐Dreh could combine with the intermediate filament protein vimentin and repress its expression, and thus further change the normal cytoskeleton structure to inhibit tumor metastasis. We also identified a human ortholog RNA of Dreh (hDREH) and found that its expression level was frequently down‐regulated in HBV‐related HCC tissues in comparison with the adjacent noncancerous hepatic tissues, and its decrement significantly correlated with poor survival of HCC patients. Conclusion: These findings support a role of lncRNA‐Dreh in tumor suppression and survival prediction in HCC patients. This discovery contributes to a better understanding of the importance of the deregulated lncRNAs by HBx in HCC and provides a rationale for the potential development of lncRNA‐based targeted approaches for the treatment of HBV‐related HCC. (HEPATOLOGY 2013)

Immune response in mice inoculated with plasmid DNAs containing multiple-epitopes of foot-and-mouth disease virus

This paper focuses on the development of candidate DNA vaccine encoding antigenic epitopes of type O foot-and-mouth disease virus (FMDV). A series of plasmids encoding different combinations of B cell epitopes and a T cell epitope were constructed and characterized by inoculating BALB/c mice. The specific antibodies were only detectable in the mice inoculated with plasmids encoding the T cell epitope and B cell epitopes from sites 5 and 1, within which site 5 includes residues 135-167 of VP1 and site 1 includes 141-160 region (G-H loop) and carboxyl terminus of VP1. Stronger cellular immune responses were also observed in these mice using T cell proliferation assay.

Enhanced Immunogenicity of Modified Hepatitis B Virus Core Particle Fused with Multiepitopes of Foot-and-Mouth Disease Virus

Abstract Hepatitis B virus core (HBc) particles, self‐assemble into capsid particles and are extremely immunogenic, hold promise as an immune‐enhancing vaccine carrier for heterologous antigens. However, formation of virus‐like particles (VLP) can be restricted by size and structure of heterlogous antigens. In the study, we investigated formation of VLP by modified HBc fused with specified foot‐and‐mouth disease virus (FMDV) multiepitopes and evaluated their immune effects. Firstly, three HBc display vectors (pHBc1, pHBc2 and pHBc3) were constructed by deletions of different lengths within the HBc c/e1 region: 75–78 amino acid (aa), 75–80 aa and 75–82 aa respectively. Secondly, we inserted different compositions of FMDV multiepitopes, BT [VP1(141–160)–VP4(21–40)] and BTB [VP1(141–160)–VP4(21–40)–VP1(141–160)], into modified regions. As a result, only plasmid pHBc3‐BTB of six recombinant vectors was expressed as soluble protein, which resulted in the formation of complete VLP confirmed by electron microscopy. Recombinant VLP could be taken up by cells and presented in vitro and in vivo. Furthermore, the modified VLP displayed a significantly stronger immunogenicity than other five recombinant proteins and GST‐BTB with a higher titer of peptide‐specific and virus‐specific antibody, elevated IFN‐γ and interleukin‐4 production, especially enhanced lymphocyte proliferation. The results encourage further work towards the development of FMDV vaccines using hepatitis B virus core particles fused with FMDV epitopes.

Transient inhibition of foot-and-mouth disease virus replication by siRNAs silencing VP1 protein coding region.

Abstract Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, a severe, clinically acute, vesicular disease of cloven-hoofed animals. RNA interference (RNAi) is a mechanism for silencing gene expression post-transcriptionally that is being exploited as a rapid antiviral strategy. To identify efficacious small interfering RNAs (siRNAs) to inhibit the replication of FMDV, candidate siRNAs corresponding to FMDV VP1 gene were designed and synthesized in vitro using T7 RNA polymerase. In reporter assays, five siRNAs showed significant sequence-specific silencing effects on the expression of VP1-EGFP fusion protein from plasmid pVP1-EGFP-N1, which was cotransfected with siRNA into 293T cells. Furthermore, using RT-qPCR, viral titration and viability assay, we identified VP1-siRNA517, VP1-siRNA113 and VP1-siRNA519 that transiently acted as potent inhibitors of FMDV replication when BHK-21 cells were infected with FMDV. In addition, variations within multiple regions of the quasispecies of FMDV were retrospectively revealed by sequencing of FMDV genes, and a single nucleotide substitution was identified as the main factor in resistance to RNAi. Our data demonstrated that the three siRNA molecules synthesized with T7 RNA polymerase could have transient inhibitory effects on the replication of FMDV.

Human Serum Amyloid P Functions as a Negative Regulator of the Innate and Adaptive Immune Responses to DNA Vaccines

The utility of DNA vaccines has been limited by their failure to elicit sufficiently potent immune responses in many human applications, whereas DNA vaccinations in mice have been very successful. However, the underlying mechanisms remain unknown. We hypothesize that serum amyloid P component (SAP), which has a species-specific, DNA-binding ability, contributes to the differences between human and mice and then limits DNA vaccine’s efficacy in vivo. In our study, DNA vaccine-induced adaptive immune responses were also significantly decreased in the human SAP (hSAP) transgenic mice. Using human promonocytic cell line THP-1–derived macrophages as a cell model, we found that cells incubated with a hSAP–DNA complex showed significant defects in innate immune activations, whereas mouse SAP had similar, albeit very weak, activities. hSAP also significantly inhibited the functions of two identified DNA sentinels, high-mobility group B protein 1 and antimicrobial peptide LL37, and redirected DNA update to FcRs leading to endocytosis and endosomal degradation. We also found that a chemical SAP inhibitor strongly recovered the suppressed innate immune responses to DNA in the presence of human serum and enhanced the immunogenicity of DNA vaccines in vivo. Our data indicated that SAP is a key negative regulator for innate immune responses to DNA and may be partly responsible for the insufficient immune responses after DNA vaccinations in humans. SAP suppression may be a novel strategy for improving efficacy of human DNA vaccines and requires further clinical investigations.

DNA immunization perturbs lipid metabolites and increases risk of atherogenesis.

In addition to conventional vaccination, DNA-mediated immunization has been developed as an alternative approach in the prevention and treatment of different infectious diseases, including hepatitis B. To define sets of serum protein and metabolite biomarkers that could be employed to determine the efficacy and safety of DNA vaccines, an integrated multiple systems biology approach was undertaken on mice immunized with DNA vaccine, recombinant protein, plasmid vector, and phosphate-buffered solution. Their sera were analyzed by two-dimensional electrophoresis and HPLC coupled with time-of-flight mass spectrometry. We detected an increase in phytosphingosine, dihydrosphingosine, palmitoylcarnitine, and ceramide in the sera of DNA-vaccinated mice. Several protein molecules were found to be altered in DNA-vaccinated mice, including apolipoprotein A-I precursor. Taken together, these results indicated that DNA vaccine stimulated hepatic sphingolipid synthesis, which may have altered the structure of circulating lipoproteins and promoted atherogenesis. This study also underscores the power of metabolomics and proteomics in the definition of DNA-vaccine-mediated metabolic phenotypes.

Identification of an HLA-A*0201-restricted CD8(+) T-cell epitope encoded within Leptospiral immunoglobulin-like protein A.

Leptospirosis is an important zoonosis in humans. Immunity against leptospiral infection was thought to be primarily humoral, and limited studies have addressed the role of CD8(+)T cells. Leptospiral immunoglobulin-like protein A (LigA) is an important protective antigen of Leptospira and a potential target for Leptospira-specific cell-mediated immunity. In this study, twenty LigA-derived peptides were tested their binding affinity and stability for the HLA-A*0201 molecule. Peptides with high binding affinity and stability for HLA-A*0201 were then assessed their capacity to elicit specific cytotoxic T-lymphocyte (CTL) responses using cytotoxicity, ELISPOT assays for IFN-gamma and HLA-A*0201-peptide tetramer assays. We identified a HLA-A*0201-restricted epitope, LigA(305-313) KLIVTPAAL in Leptospira LigA. CTLs specific for LigA(305-313) were elicited both in HLA-A2.1/K(b) transgenic mice and in patients with a clinical and/or laboratory diagnosis of leptospirosis. Staining of the HLA-A*0201-LigA(305-313) tetramer revealed the presence of LigA(305-313)-specific CTLs in peripheral blood mononuclear cells (PBMCs) sourced from five patients infected with three different serovars of Leptospira. In conclusion, we report the existence of specific cytotoxic CD8(+)T cells in patients with leptospirosis and we suggest that the newly identified epitope, LigA(305-313), will be helpful in enhancing the understanding of the mechanism of immunity to leptospirosis.