| Object:Hepatocellular carcinoma (HCC) is one of the common malignant tumors of the world, the annual deaths of HCC in China accounted for more than half of the world, and HCC has become a major killer to threat the health and lives of our people. Although the means for the treatment of HCC, including surgery, radiotherapy, chemotherapy, Chinese medicine treatment, immunotherapy and radiofrequency ablation therapy, but the effect is not ideal with poor prognosis of patients because tumor relapse and metastasis easily. Therefore, it is urgent to find new methods. With the development of molecular biology and immunology, and with the knowledge about the tumorigenesis molecular mechanisms enrich, people now realize that the tumor is a genetic disease with multi-factor, multi-step and multiple genes involved in, additionally, the rapid development of gene therapy made it possible to overcome malignant tumors by ways of gene therapy together with the traditional treatment. The breakthrough in gene therapy of tumor is to find efficient, specific targets.STAT3is an important member of Signal Transducers and Activators of Transcription (STATs). It is seemed as an information transfer in cytoplasm and play transcriptional activation in nucleus. STAT3is involved in a variety of cytokine signal transduction processes and plays an important role in the regulation of cell growth and survival. After stimulating with growth factors and cytokines, STAT3dimerizated and then entered the nucleus to modulate the expression of target gene by binding to a specific gene promoter sequence. Its target genes, containing the anti-apoptotic gene bcl-xl, of mcl-1and cell cycle control genes of cyclin D1, cyclin E, c-myc and p21, which are closely related with cell proliferation, differentiation, survival, malignant transformation and apoptosis inhibition. STAT3plays an indispensable role during the development of embryonic and differentiation of bone marrow cells. In normal physiological conditions, the activation of STAT3is under strict regulation, and its activation is rapid and transient, lasted only a few minutes to several hours, plays a key role for the maintenance of normal physiological functions of the cells. More and more researches show that the sustained activation of STAT3can lead to abnormal cell proliferation and apoptosis suppression to promote tumor formation and development. There has been abnormal expression or sustained activation about STAT3in many cancer, including lymphoma, multiple myeloma, prostate cancer, breast cancer, lung cancer, pancreatic cancer, ovarian cancer tumor tissues and cell lines, suggesting that STAT3is associated with the progression of tumor development. In addition, it is a main reason for the repression of DC and CTL activation because of STAT3abnormal activation. Therefore, as an oncogene, STAT3has been considered as a new target for cancer treatment.MicroRNA (miRNA), small non-coding RNA, is the main regulator to regulate gene expression at post-transcriptional level, is a focus of the field of life science research in recent years. In mammalian cells, miRNA inhibit mRNA translation by sequence-dependent "mis-match". The study found that compared with normal tissues, miRNA expression as well as the type both significantly changed in human tumor tissues; additionally, many miRNA changes had emerged before the formation of tumors, not just occur after tumor formation. miRNAs abnormal or dysfunctional is closely related to liver cell proliferation, differentiation, and primary liver cancer. Therefore, miRNA, as a new target for tumor diagnosis and treatment, has opened up a new idea for conquering malignant.This research focus on hepatoma cell line HepG2, H7402, PLC/PRF/5, observe its impact on the immune function of NK cells by studying the abnormal activation of STAT3signaling pathway in HCC to investigate the molecules mechanism about immunosuppression of innate immune induced by HCC, and to provide more detailed theoretical basis for STAT3as a cancer therapeutic target; meanwhile, looking for the key miRNA molecules associated with STAT3in HCC, studying its interaction with STAT3, exploring the effect of key miRNA associated with STAT3on natural immune function, so that provide a new thinking for the treatment of liver cancer.MethodsLiver cancer cell lines were transfected STAT3decoy ODN with LipofectamineTM2000and transfection specificity was determined by luciferase reporter gene assay. Then, we found that:the sensitivity of decoy ODN-treated HCC cells to NK cell lysis was increased after MTT assay; the expression levels of ULBPs, especially ULBP3, were enhanced after blocking STAT3using flow cytometric analysis; the cytolytic activity was enhanced when NK cells were incubated with the supernatant from HCC cells treated with STAT3decoy ODN, with the increase of molecules related to cytolysis by qPCR, ELISA and flow cytometric analysis; at the same time, after blocking STAT3in HCC cells, the mRNAs levels of inflammatory cytokines, including IL-6, IL-8, IL-18, IL-17and IL-23, as well as the immunosuppressive factor TGF-β were downregulated, whereas the mRNAs levels of IFN-a, IFN-β and IFN-y were upregulated; then the regulatory effects of STAT3on the transcriptional activities of the TGF-β1and IL-10promoters were identified by dual-luciferase assay; and the cytolytic activity of NK-92cells incubated with HepG2cell supernatant was increased in the presence of the neutralizing TGF-β1; Additionally, p-STATl levels and transcriptional activity of STAT1in HepG2cells was increased by STAT3decoy ODN, and the cytolytic activity of NK cells was restored by the addition of type I IFN added to the supernatant of HCC cells. These results demonstrated that both TGF-β1reduction and type I IFN production were essential for blocking STAT3-induced NK cell activation.Next, by qPCR assay, we found blocking STAT3still have the potential to modulate the expressions of miRNAs, such as miR-146a downregulation and miR-155upregulation. Then, we examined the changes of HCC biological characteristics and immunological parameters after transfected with miR-146a or miR-155mimics or inhibitors using MTT, qPCR and flow cytometry analysis. Additionally, we confirmed that antitumor therapeutic effects could be attained by miR-146a suppression or miR-155overexpression in vivo. The result showed that not only weight of tumor decreased but also lymphocytes cytolysis activities and molecules related to cytolysis in lymphocytes of liver, spleen, tumor tissue, armpit and inguinal lymph node (LN) were upregulated after miR-146a suppression or miR-155overexpression. Meanwhile, using chromatin co-immunoprecipitation (ChIP) and luciferase reporter gene assays, we confirmed that STAT3can directly bind miR-146a or miR-155promoter. However, the expression of miR-155was significantly upregulated after5-Aza deoxycytidine (5-Aza methyl transferase inhibitors) treatment which inhibited DNA methylation, and therefore we explored the effects of methylation, DNA methyltransferase (DNMT1) and STAT3on the expression of miR-155in HCC by the methylated PCR (MS-PCR) and co-immunoprecipitation precipitation (IP).ResultsPart1:Targeting blockage of STAT3in HCC cells augments NK cell functions via reverse HCC-induced immune suppression1Blocking STAT3in HCC augmented the susceptibility of HCC to NK cell cytolysisHuman NK cell lines NK-92and NKL, as well as human PBMCs were used as effector cells, respectively. The sensitivity of decoy ODN-treated HCC cells to NK cell lysis was increased significantly. In contrast, scramble ODN treatment did not influence the sensitivity of HCC to NK cell lysis.2The NKG2D ligands expressed on HCC cells were upregulated by blocking STAT3Blocking STAT3caused significant upregulation in the expression levels of ULBPs, especially ULBP3, which were contributed to the sensitivity of STAT3-blocked HCC cells to NK cell cytotoxicity.3Blocking STAT3reversed HCC-induced immune suppression on NK cells Blocking STAT3in HCC reversed HCC-mediated immune suppression and promoted NK cell-mediated antitumor effects.4NK cells were activated by the products secreted by STAT3-blocked HCC cellsCompared to the control groups, we found that both mRNA and the protein levels of molecules related to cytolysis, including NKG2D, IFN-y, FasL, perforin, granzyme B and TNF-a, were increased obviously in NK-92cells incubated with the supernatant from STAT3blocked HCC cells. However, the protein level of NKG2A was decreased in NK-92cells by treatment with the supernatant from STAT3decoy ODN-treated HCC cells. Furthermore, similar results were observed when NK-92cells were replaced with human primary CD3-CD56+PBMCs.5The cytokine profile of HCC cells was changed by blocking STAT3In STAT3blocked HCC cells, the mRNAs levels of inflammatory cytokines, including IL-6, IL-8, IL-18, IL-17and IL-23, as well as the immunosuppressive factor TGF-β were downregulated, whereas the levels of immune stimulating factor, such as IFN-a, IFN-β and IFN-y were upregulated.6TGF-β1reduction and type I IFN production by STAT3blocked HCC cells were critical for NK cell activationThe regulatory effects of STAT3on the transcriptional activities of the TGF-β1and IL-10promoters were identified by dual-luciferase assay. The cytolytic activity of NK-92cells incubated with HepG2cell supernatant was increased in the presence of the neutralizing TGF-β1. Additionally, p-STAT1levels and transcriptional activity of STAT1in HepG2cells was increased by STAT3decoy ODN. Investigation of the role of interferons by antibody neutralization showed that anti-IFNAR mAb suppressed the cytolytic activity of NK cells induced by the supernatant from STAT3-blocked HCC cells. These results demonstrated that both TGF-β1reduction and type I IFN production were essential for blocking STAT3-induced NK cell activation.Part2:STAT3contributes to HCC-induced the suppression on NK cell antitumor immune responses via epigenetical control of miR-155expression1miR-155was upregulated after blocking STAT3in human HCC cellsThe level of miR-155was upregulated by blocking STAT3.2Suppression of miR-155promoted the expression of inflammatory cytokines associated with STAT3activation in HCC cells and resulted in HCC-induced NK cell dysfunctionMR-155suppression inhibited the expression of inflammatory cytokines associated with STAT3activation, such as TGF-β, IL-6and IL-8, as well as IFN-a mRNA reduction. It was indicated that miR-155might be involved in HCC-promoting inflammation to generate an inflammatory microenvironment that further supports tumor progression.After incubated with the supernatant from HepG2cells treated with miR-155mimics, the cytolysis and molecules related to cytolysis were both enhanced when compared to that of NK cells incubated with the supernatant from negative control RNA (NC)-treated HepG2cells. It was suggested that miR-155could contribute to anti-tumor function of NK cell through altering the inflammatory condition and strengthen expression of miR-155promote anti-tumor effect of NK cells in vitro.3The overexpression of miR-155promoted anti-tumor immune response in vivoAfter transfected with miR-155inhibitor,2×106these Hepa1-6cells were injected s.c. into the right posterior flank of C57/B6mice. The results showed that the liver and spleen lymphocytes from mice-bearing miR-155mimics-treated tumor cell exhibited enhanced cytolysis activities against Hepa1-6cells. Meanwhile, the tumor weight was lighter and spleen weight was heavier in mice-bearing miR-155mimics-treated Hepa1-6cells comparede to that in mice-bearing control RNA treated-tumor cell. Moreover, the expressions of CD69, NKG2D and FasL in CD3-NK1.1+and CD3+NK1.1-lymphocytes from iver, spleen, tumor tissue, armpit and inguinal lymph node (LN) were all upregulated in mice-bearing miR-155 mimics-treated tumor cells. These results suggested that the expressions of miR-155modulated by STAT3both in human HCC and in murine HCC exerted an important role in anti-tumor immune response in vivo.4STAT3directly bound the promoter of miR-155Using Chromatin immunoprecipitation (ChIP) assay we veified that STAT3mediated regulation of miR-155dependent on binding to their promoters directly.55-Aza treatment enhanced the expression of miR-155significantlyThe expression levels of miR-155were remarkably restored after5-Aza treatment. These result indicated that the repression of miR-155in liver cancer may be partly ascribed to DNA methylation.6DNA methylation inhibites miR-155transcriptional activityUsing Chromatin immunoprecipitation (ChIP) and Luciferase assay we veified that DNA methylation contributed to miR-155expression except for STAT3which lead to low level of miR-155in HCC cells.7DNMT1was crucial for miR-155methylation in human HCC The result showed that aberrant activation of STAT3in HCC cells promoted the methylation of miR-155promoter via recruiting DNMT1.Part2:STAT3aggrevates HCC-induced immune suppression by modulating miR-146a directly1Blocking STAT3inhibited the expression of miR-146a and increased the expressions of miR-146a target genes in human HCC cellsBlocking STAT3in HCC cells not only suppressed the expression of miR-146a, but also increased the expression of miR-146a target genes, including the transcriptions of IFIT3, STAT1and TRAF6enhancement.2miR-146a didn’t influence the proliferation of HCC cells in vitroThere was no significant change in the proliferation of cells treated with miR-146a mimics or inhibitors. And this result was confirmed by the cell cycle assay.3miR-146a promoted the expression of inflammatory cytokines associated with STAT3activation in HCC cellsMiR-146a overexpression increased the expression of inflammatory cytokines associated with STAT3activation, such as TGF-p, IL-6and IL-8, as well as IFN-a mRNA reduction. It was indicated that miR-146a might be involved in HCC-promoting inflammation to generate an inflammatory microenvironment that further supports tumor progression.4The expression of miR-146a was contributed to human HCC-induced NK cell dysfunctionAfter incubated with the supernatant from HepG2cells treated with miR-146a inhibitors, both the molecules related to NK cytolysis and the cytolysis were enhanced when compared to that of NK cells incubated with the supernatant from negative control RNA (NC)-treated HepG2cells. It was suggested that miR-146a inhibition could contribute to antitumor function of NK cell through altering the inflammatory condition.5STAT3directly bound the promoter of miR-146a and regulated the expression of miR-146aUsing Chromatin immunoprecipitation (ChIP) and Luciferase assay we veified that STAT3can directly bind the promoter of miR-146a and promote its expression.6The inhibition of miR-146a promoted anti-tumor immune response in vivoAfter transfected with miR-146a inhibitor,2×106these Hepa1-6cells were injected s.c. into the right posterior flank of C57/B6mice. The results showed that the liver and spleen lymphocytes from mice-bearing miR-146a inhibitors-treated tumor cell exhibited enhanced cytolysis activities against Hepa1-6cells. Meanwhile, the tumor weight was lighter and spleen weight was heavier in mice-bearing miR-146a inhibitors-treated Hepa1-6cells comparede to that in mice-bearing control RNA treated-tumor cell. Moreover, the expressions of CD69, NKG2D and FasL in CD3-NK1.1+and CD3+NK1.1-lymphocytes from iver, spleen, tumor tissue, armpit and inguinal lymph node (LN) were all upregulated in mice-bearing miR-146a inhibitor-treated tumor cells. These results suggested that the expressions of miR-146a modulated by STAT3both in human HCC and in murine HCC exerted an important role in anti-tumor immune response in vivo.ConclusionIn this study, we found that blocking STAT3in HCC cells enhanced NK cell antitumor function. On one hand, after blocking STAT3HCC cells, NKG2D ligands were upregulated, which promoted recognition by NK cells. On the other hand, the inflammatory and immunosuppressive cytokines of HCC cells was altered, in particular, TGF-β and IL-10expression was reduced, and type ⅠIFN was induced. The cytokines changes lead to a series of cascading effects, containing changing the tumor microenvironment, reversal of immune tolerance, facilitating NK cell activation with a concomitant elevation of molecules associated with NK cytolysis, and cytotoxicity restoration of NK cell.Further studying found that constitutive activation of STAT3in HCC still have the potential to modulate the expressions of miRNAs. MiR-146a downregulation and miR-155upregulation were detected after STAT3blockage in HCC. Then, we found that miR-146a and miR-155were both involved in HCC-promoting inflammation to generate an inflammatory microenvironment that further supports tumor progression after transfected with miR-146a or miR-155mimics or inhibitors. Furthermore, we confirmed that antitumor therapeutic effects could be attained by miR-146a suppression or miR-155overexpression in vivo. Meanwhile, we verified that STAT3can directly bind miR-146a or miR-155promoter through ChIP and luciferase reporter assay. However, the expression of miR-155was significantly upregulated after5-Aza treatment which inhibited DNA methylation, and we demonstrated that miR-155expression is controlled by promoter methylation and verified miR-155was repressed by STAT3through recruitment of DNMT1. These findings indicate that miR-155may act as a tumor suppressor in HCC and reveal critical mechanisms for STAT3-driven miRNA suppression and rational therapeutic targets of epigenetic modifications in HCC. These results showed that the dysregulations of miR-146a and miR-155were both involved in HCC-induced immune suppression, and corrected microRNA expressions could augments NK cell function. These findings not only provided important insight into the molecular mechanism of hepatocarcinogenesis, but also shed new light on the targeting strategy for HCC therapy. |
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