Zhaohua Hou

Ectopic expression of microRNA-155 enhances innate antiviral immunity against HBV infection in human hepatoma cells

BackgroundHost innate antiviral immunity is the first line of defense against viral infection, and is precisely regulated by thousands of genes at various stages, including microRNAs. MicroRNA-155 (miR-155) was found to be up-regualted during viral infection, and influence the host immune response. Besides, the expression of miR-155, or its functional orthologs, may also contribute to viral oncogenesis. HBV is known to cause hepatocellular carcinoma, and there is evidence that attenuated intracellular immune response is the main reason for HBV latency. Thus, we assume miR-155 may affect the immune response during HBV infection in human hepatoma cells.ResultsWe found that ectopic expression of miR-155 upregulated the expression of several IFN-inducible antiviral genes in human hepatoma cells. And over-expression of miR-155 suppressed suppressor of cytokine signaling 1 (SOCS1) expression and subsequently enhanced signal transducers and activators of transcription1 (STAT1) and signal transducers and activators of transcription3 (STAT3) phosphorylation. We further demonstrate that ectopic expression of miR-155 inhibits HBV X gene expression to some extent in vitro.ConclusionMiR-155 enhances innate antiviral immunity through promoting JAK/STAT signaling pathway by targeting SOCS1, and mildly inhibits HBV infection in human hepatoma cells.

Histone deacetylase inhibitor SAHA epigenetically regulates miR-17-92 cluster and MCM7 to upregulate MICA expression in hepatoma.

Background:Epigenetic therapy using histone deacetylase inhibitors (HDACi) has shown promise in clinical trials for the treatment of human malignancies. In addition to the immediate effects on the tumour cell growth, HDACi upregulates the expression of MHC class I-related chain molecules A and B (MICA and MICB), resulting in an enhanced susceptibility of tumour cells to natural killer cell-mediated lysis. The molecular mechanism underlying is still unclear.Methods:The transcriptional regulation mechanism underlying suberoylanilide hydroxamic acid (SAHA)-mediated regulation of MICA and related miRNA expression was investigated using promoter acetylation assays, bioinformatics analysis and chromatin immunoprecipitation assay.Results:SAHA upregulates the transcription of MICA/B by promoting MICA-associated histone acetylation while suppressing the MICA/B-targeting miRNAs miR-20a, miR-93 and miR-106b. The mechanism by which SAHA repressed miRNAs transcription involved repression of their host genes (miR-17-92 cluster and MCM7). SAHA downregulated the miR-17-92 cluster by abolishing tyrosine phosphorylation of STAT3 and decreased MCM7 transcription through localised histone deacetylation.Conclusions:The HDACi SAHA epigenetically upregulates MICA expression through regulating the expression of miR-17-92 cluster and MCM7 in hepatoma, thus enhancing the sensitivity of HCC to natural killer cell-mediated lysis. This novel mechanism of action provides promise for HDACi in therapy of HCC.

miR-146a is directly regulated by STAT3 in human hepatocellular carcinoma cells and involved in anti-tumor immune suppression

MicroRNAs (miRNAs) play an important role in tumorigenesis, but their role in tumor-induced immune suppression is largely unknown. STAT3 signaling, a key pathway mediating immune suppression in the tumor microenvironment, is responsible for the transcription of several important miRNAs. In this study, we observed that miR-146a, a known important regulator of immune responses, was downregulated by blocking activated STAT3 in hepatocellular carcinoma (HCC) cells. Furthermore, miR-146a inhibition in HCC cells not only altered the STAT3 activation–associated cytokine profile but also reversed HCC-induced NK cell dysfunction in vitro and improved the anti-tumor effect of lymphocytes in vivo. Importantly, ChIP and luciferase reporter assays confirmed that STAT3 directly bound to the miR-146a promoter and induced miR-146a expression. These findings indicated that miR-146a expression was regulated by aberrantly activated STAT3 in HCC cells and exerted negative effects on anti-tumor immune response, which resulted in the upregulation of cytokines such as TGF-β, IL-17, VEGF and downregulation of type I IFN to create an immunosuppressive microenvironment. This further insight into understanding the mechanism responsible for tumor-induced immune suppression highlights the potential application of miR-146a as a novel immunotherapeutic target for HCC.

miR-146a negatively regulates NK cell functions via STAT1 signaling.

It is known that natural killer (NK) cell function is downregulated in chronic hepatitis B (CHB)-infected patients and in hepatic carcinoma (HCC) patients, but the mechanisms underlying this functional downregulation are largely unclear. In this study, microRNA (miR)-146a expression increased in NK cells from CHB and HCC patients compared with NK cells from healthy donors, and miR-146a levels were negatively correlated to NK cell functions. Overexpression of miR-146a reduced NK cell-mediated cytotoxicity and the production of interferon (IFN)-γ and tumor necrosis factor-α, which were reversed upon inhibition of miR-146a. In NK cells, miR-146a expression was induced by interleukin (IL)-10 and transforming growth factor-β, but reduced after treatment with interleukin-12, IFN-α and IFN-β. We further revealed that miR-146a regulated NK cell functions by targeting STAT1. Taken together, upregulated miR-146a expression, at least partially, attributes to NK cell dysfunction in CHB and HCC patients. Therefore, miR-146a may become a therapeutic target with great potential to ameliorate NK cell functions in liver disease.

Poly(I:C) exhibits an anti-cancer effect in human gastric adenocarcinoma cells which is dependent on RLRs.

Poly(I:C), an agonist of TLR3 and RLRs, has been used as an immune adjuvant in clinical trials for many years. Although it has been found to trigger apoptosis in a variety of cancers, its mechanisms in human gastric adenocarcinoma is unclear. The purpose of this study was to assess the effect of poly(I:C) on human gastric adenocarcinoma cells. Our observations showed that intracellular delivery of poly(I:C) by liposomes had a pro-apoptotic effect in vitro, and significantly inhibited xenograft growth of human gastric adenocarcinoma in nude mice. Further investigations indicated that RLRs, as intrinsic RNA sensors, contributed to the poly(I:C)-triggered apoptotic effect through upregulation of RIG-I, MDA-5, and most significantly, LGP2, accompanied by increased expression of Bcl-2 family members. Conversely, this apoptotic effect was greatly reduced by silencing RIG-I, MDA-5, or LGP2. Although LGP2 is considered an innate immune negative regulator of RIG-I and MDA-5, it exhibited a positive regulatory effect on poly(I:C)-induced apoptosis in human gastric adenocarcinoma cells. These findings suggested that poly(I:C) may be a promising chemotherapeutic agent against human gastric adenocarcinoma.

Hepatitis B virus inhibits intrinsic RIG-I and RIG-G immune signaling via inducing miR146a

Previous studies showed that hepatitis B virus (HBV), as a latency invader, attenuated host anti-viral immune responses. miRNAs were shown to be involved in HBV infection and HBV-related diseases, however, the precise role of miRNAs in HBV-mediated immunosuppression remains unclear. Here, we observed that down-regulated RIG-I like receptors might be one critical mechanism of HBV-induced suppression of type I IFN transcription in both HBV+ hepatoma cell lines and liver cancer tissues. Then, miR146a was demonstrated to negatively regulate the expression of RIG-I-like receptors by directly targeting both RIG-I and RIG-G. Further investigation showed that antagonizing miR146a by anti-sense inhibitors or sponge approach accelerated HBV clearance and reduced HBV load both in vitro and in a HBV-carrying mouse model. Therefore, our findings indicated that HBV-induced miR146a attenuates cell-intrinsic anti-viral innate immunity through targeting RIG-I and RIG-G, and silencing miR146a might be an effective target to reverse HBV-induced immune suppression.

The anti-HBV effect mediated by a novel recombinant eukaryotic expression vector for IFN-α

BackgroundChronic hepatitis B is a primary cause of liver-related death. Interferon alpha (IFN-α) is able to inhibit the replication of hepadnavirus, and the sustained and stable expression of IFN-α at appropriate level may be beneficial to HBV clearance. With the development of molecular cloning technology, gene therapy plays a more and more important role in clinical practice. In light of the findings, an attempt to investigate the anti-HBV effects mediated by a eukaryotic expression plasmid (pSecTagB-IFN-α) in vitro was carried out.MethodsHBV positive cell line HepG2.2.15 and its parental cell HepG2 were transfected with pSecTagB-IFN-α or empty plasmid by using Lipofectamine™ 2000 reagent. The expression levels of IFN-α were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA methods. The effects of pSecTagB-IFN-α on HBV mRNA, DNA and antigens were analyzed by real-time fluorescence quantitative PCR (qRT-PCR) and ELISA assays. RT-PCR, qRT-PCR and western blot were employed to investigate the influence of pSecTagB-IFN-α on IFN-α-induced signal pathway. Furthermore, through qRT-PCR and ELISA assays, the suppressive effects of endogenously expressed IFN-α and the combination with lamivudine on HBV were also examined.ResultspSecTagB-IFN-α could express efficiently in hepatoma cells, and then inhibited HBV replication, characterized by the decrease of HBV S gene (HBs) and HBV C gene (HBc) mRNA, the reduction of HBV DNA load, and the low contents of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Mechanism research showed that the activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal pathway, the up-regulation of IFN-α-induced antiviral effectors and double-stranded (ds) RNA sensing receptors by delivering pSecTagB-IFN-α, could be responsible for these phenomena. Furthermore, pSecTagB-IFN-α vector revealed effectively anti-HBV effect than exogenously added IFN-α. Moreover, lamivudine combined with endogenously expressed IFN-α exhibited stronger anti-HBV effect than with exogenous IFN-α.ConclusionOur results showed that endogenously expressed IFN-α can effectively and persistently inhibit HBV replication in HBV infected cells. These observations opened a promising way to design new antiviral genetic engineering drugs based on IFN-α.

Intracellular Poly(I:C) Initiated Gastric Adenocarcinoma Cell Apoptosis and Subsequently Ameliorated NK Cell Functions

Natural killer (NK) cells are granular lymphocytic cells that exert essential functions in viral infection defense and tumor immune surveillance. However, the functions of NK cells were impaired in cancer patients. Polycytidylic acid [poly(I:C)] has been used as an immune adjuvant to improve innate and adaptive immune responses. In this study, intracellular poly(I:C) could trigger gastric adenocarcinoma cells apoptosis quickly. Meanwhile, the sensitivity of poly(I:C)-treated gastric adenocarcinoma cells to NK cell cytolysis was increased, concomitant with the elevated expression of MICA/B and Fas. Furthermore, the cytolytic activity of NK cells against tumor cells was augmented significantly by the supernatant from poly(I:C)-transfected tumor cells compared with NK cells treated by the supernatant from untreated tumor cells, as well as the proliferation and migration abilities of NK cells. In this process, the activating receptors and cytolysis-associated molecules of NK cells were up-regulated. Further investigation showed that type I interferon (IFN) produced by poly(I:C)-transfected gastric adenocarcinoma cells played an important role in this process. Our findings demonstrated that intracellular poly(I:C) not only triggered gastric adenocarcinoma cell apoptosis, but also enhanced NK responses via inducing type I IFN production by gastric adenocarcinoma cells. These functions make poly(I:C) a promising therapeutic medicine for gastric adenocarcinoma.

HBV suppresses expression of MICA/B on hepatoma cells through up-regulation of transcription factors GATA2 and GATA3 to escape from NK cell surveillance

Decreased expression of NKG2D ligands on HBV-infected human hepatoma cells impairs NK cells lysis. However, which components of HBV exert this effect and the precise mechanisms need to be further investigated. In the present study, we observed that the HBx and HBc genes significantly down-regulated MICA expression. Through analysis with the chromatin immunoprecipitation assay, we found that HBV infection promotes the expression of transcription factors GATA-2 and GATA-3, which specifically suppressed MICA/B expression by directly binding to the promoter region of MICA/B. HBx protein, acting as a co-regulator, forms a tripolymer with GATA2 and GATA3, thus promotes the GATA-2 or GATA-3-mediated of MICA/B suppression. HBc protein inhibits MICA/B expression via directly binding to the CpG island in the MICA/B promoter. Thus, our study identified the novel role of transcription factors GATA-2 and GATA-3 in suppressing MICA/B expression and clarified the mechanisms of HBx and HBc in downregulation of MICA/B expression. These findings provide novel mechanisms for the contribution of HBV to hepatoma cells escape from NK cell surveillance.

Perspectives on RNA Interference in Immunopharmacology and Immunotherapy

RNA interference (RNAi), mediated by short interfering RNA (siRNA), vector-derived short hairpin RNA (shRNA) and microRNA (miRNA), brings about revolutionary fea‐ tures to basic biomedical research and clinical application. New drugs based on RNAi have been developed for therapeutic applications. The family of RNAi molecules are effi‐ cient agents to modulate mammalian immune system, and many studies reported that these molecules could manipulate immune defence, surveillance and homeostasis. Both perfect match of siRNA/shRNA and non-perfect match of miRNA could be beneficial for designing RNAi-based drugs for treatment of tumour and viral infection. This chapter provides a view to control or utilize the immune regulation of various small RNAs that should help researchers to understand the successful clinical application of RNAi.