Expresion Of IL-1β In Hepatoma On Tumor Growth And Invasiveness And Its Relationship With NK Cell Mediaed Cytolysis

Objective To investigate the role of proinflammatory cytokine IL-1βexpression in hepatoma cells on its growth and invasiveness,as well as the effect on NK cell mediated cytotoxicity,the results will prove with evidence that expression of proinflammatory cytokine by hepatoma cells provided an important mechanism in tumor immunoediting and immune escape from NK cell mediated innate immune surveillance.Methods BALB/c mice at 6-8 weeks was stimulated with 6%starch through abdominal injection for at least 24 to 48 hours,macrophages were collected from the abdominal cavity and treated with LPS(5μg/ml) for 4h,total RNA was extracted cells as guided by the manufacture.Full length murine IL-1βwas amplified through RT-PCR using its specific primers and pfu DNA polymerase.Purified PCR products was then digested with the restriction enzyme EcoRâ… plus Xhoâ… and followed by inserting into the eukaryotic expression vector pIRES2-EGFP catalyzed with T4 ligase at 22℃for 2h in a final volume of 10μl.The products totally was transfected into DH5αrecipients using CaCl₂.The plate was cultured overnight and multiple clones were selected for further assay for the recombination of IL-1βthrough clony PCR,restriction enzyme assay using EcoRâ… and Xhoâ… .The recombinant expression vector with IL-1βwas finally verified by DNA sequencing and named as pIRES2-EGFP-mIL-1β.The vector of pIRES2-EGFP-mIL-1βas then transfected into H22 hepatoma cells(H22/mIL-1β) in a transient transfection system mediated by jetPEI as controlled with pIRES2-EGFP transfectants(H22/mock).Expression of the recombinant vector were observed under fluorescent microscope and confocal microscope 48h after transfection.Expression level of mIL-1βin these cells was analyzed by RT-PCR using specific primers.In vitro proliferation of H22 cells was assessed in a MTT method.Sensitivity of H22 cells to NK cell mediated cytolysis was analyzed in the MTT colorimetric assay using spleenic lymphocytes isolated from wide-type mouse.For in vivo assessment,2×10⁵ H22/mIL-1βand H22/mock cells were inoculated subcutaneously into the right oxter of 6-8 weeks old BALB/c mice respectively in order to establish hepatoma models for further assay.Tumor volume was measured and recorded with digital caliper every two days after tumor injection.Survival time of mouse with different type of tumors was calculated. Tumors was dissected at the 3^rd week,tumor mass and volume,local invasiveness and lung metastasis was verified.Spleenic lymphocytes was isolated to analyzed the cytotoxicity of lymphocytes to H22 cells,in vivo activity of NK cells was reflected through its cytolytic capacity to the NK sensitive target cell known as YAC-1. Expression of NK cell inhibitory receptor NKG2A on lymphocytes was detected through RT-PCR.Results Total RNA isolated from the LPS treated macrophages was used in RT-PCR to prepare the full length murine IL-1β.The result showed we prepared a DNA fragment of 843bp in length as expected.Purified PCR products was inserted immediately into the eukaryotic expression vector pIRES2-EGFP catalyzed by T4 ligase.It was shown in the following repeated clony PCR and restriction enzyme by EcoRâ… and Xhoâ… ,that we got several positive clones composing the expected full length DNA fragment of IL-1β.The clones were sent for further DNA sequence analysis,it was proved that the gene segment inserted in the recombinant expressing vector was identical to those reported murine IL-1βin GenBank(M15131),these results indicated that we successfully prepared a recombinant expression vector with murine IL-1βnamed as pIRES2-EGFP-mIL-1β.Purified pIRES2-EGFP-mIL-1βwas transiently transfected into H22 cells(H22/mIL-1β) by jetPEI using pIRES2-EGFP transfectants as control(H22/mock).48 hours later,bright green fluorescence was seen on the cells surface when observed under a fluorescent microscope or screening by confocal microscopy,indicating that the both vector was successfully introduced into and expressed on H22 cells.Simultaneously the expression level of IL-1βwas markedly elevated in H22/mIL-1βas expected in RT-PCR assay.Expression of IL-1βsignificantly stimulated H22 cell proliferation and in vitro survival compared with H22/mock cells.However,expression of IL-1βmade H22/mIL-1βcells to be more resistant to cytolytic potential of spleenic lymphocytes prepared from wild-type mouse which suggested that natural killer cell cytotoxicity was markedly inhibited.Cytotoxicity of NK cells decreased about 16.8%and 28.1%when the effector to target ratio was 20:1 and 40:1 respectively. For in vivo experiment,48 to 72 hours after transfection,2×10⁵/mouse of H22/mIL-1βand H22/mock cells were inoculated subcutaneously into BALB/c mice respectively,and tumor growth was measured.It was indicated that IL-1βpromoted malignant cell outgrowth and proliferation in vivo,as the tumor volume (590.3±211.6mm³) were significantly enhanced compared with H22/mock cells(210.4±126.8mm³).Expression of IL-1βcould also promote tumor invasiveness and lung metastasis(100%,21.6%) when compared with H22/mock cells(15%,0). We further analyzed in vivo cytotoxicity of NK cells.The result showed that cytolysis capacity of spleenic lymphocytes to H22 hepatoma cells decreased to about 14.9%and 27.9%at effector to target ratio 20:1 and 40:1.Using the NK sensitive YAC-1 cells as targets,we proved that the cytotoxicity of NK cells was significantly down-regulated,its cytolytic capacity decreased about 60.5%and 20%at E:T of 20:1 and 40:1,which provided a major molecular mechanisms for the immune suppression status induced by the proinflammatory cytokine IL-1β.Detection of NKG2A in spleenic lymphocytes suggested that IL-1βcould enhance the transcription of NKG2A in mice with H22/mIL-1β,implying its relationship to the decreased NK cell immune suppression.Conclusionsâ‘ the present research,we successfully prepared a murine IL-1βeukaryotic expression vector pIRES2-EGFP-mIL-1β.â‘¡The recombinant vector was successfully introduced and expressed in H22 cells,expression of IL-1βdirectly contribute to in vitro tumor proliferation and survival by inhibiting of NK cell mediated cytotoxicity.â‘¢We established a very useful tumor mouse model for in vivo analysis of biological activity of proinflammatory cytokine IL-1βon tumor progression and immune reaction.â‘£Our results using mice bearing tumors expressing high level of proinflammatory cytokine IL-1βshowed that chronic inflammation in the tumor microenvironment triggered by IL-1βwas a major molecular mechanism for malignant growth and tumor progression,this factor could directly suppress NK cell mediated innate immunity which led to tumor escape from immune recognition and response.