The Role Of RPB5-mediating Protein In Regulating The Progression Of Hepatocellular Carcinoma By Modulating Macrophage Function

Background and ObjectiveMacrophages(Mφ),involved in regulation of host innate immune and adaptive immune,is important for the initiation and progression of tumor.Tumor-associated Macrophages(TAMs),infiltrate in tumors,act as a key regulator of tumor formation,survival and metastasis.Previous study found that TAMs produced interleukin-6(IL-6)and promoted expansion of human hepatocellular carcinoma stem cell.RPB5-mediating protein(RMP),also named Unconventional prefolding RPB5 Interactor(URI),is high expressed in the hepatocellular carcinoma.And RMP could regulate the initiation of hepatocellular carcinoma by regulating the transcription of IL-6.In our previous study,we found that RMP was down-regulated in the activated macrophages;and inhibiting the mRNA or protein expression of RMP could remarkably enhance the activation of macrophages.These results indicate that RMP may be play a pivotal role in regulation the macrophages activation.However,the precise mechanism remains unclear.Here,we are about to explore the mechanisms of RMP in regulating the activation of macrophages in vitro and in vivo.This project intends to in-depth investigate the function of RMP on macrophage activation and the regulatory role of RMP in promoting liver cancer transformation and progression by modulating the macrophage phenotypes.This study will provide new insight into the importance of RMP in inflammation regulation and liver tUmor progression.Experimental methods:1.Conditional RMP knock-out mouse model was conducted by Cre/floxp method.2.In the lethal dose of LPS endotoxin shock mice model,by contrast to the wild type mice,the survival time of RMP myeloid knock-out mice was significantly lower(26.7h vs 32.6h,P=0.041);the mouse lungs obtained from knock-out mice infiltrated with a large number of inflammatory cells and red blood cells;and the concentrations of serum TNF and IL-1 were almost 1.8(p=0.007)and 1.6-time(p=0.024)than those in the control group,respctively.Above results indicate that RMP myeloid knock-out can cause more serious damage.3.Primary culture of macrophages from abdominal and spine.Western blotting was applied to detect the activation of NF-κB and MAPK signaling pathways in macrophages.RT-PCR and ELISA methods were conducted to assess the cytokines under LPS and TNFa treatment.4.Overexpress/knoek-down RPM in macrophage.Then Western blotting was applied to detect the activation of NF-κB and MAPK signaling pathways in macrophages.RT-PCR and ELISA methods were conducted to assess the cytokines under LPS treatment.5.Western blotting and GC-MS were applied to detect the RMP phosphorylation in RAW 264.7 cells under LPS and TNFa treatment.Co-immunoprecipitation was used to assess the interactions between RMP and key members in IKK-NF-κB signaling pathway.6.Hepatocellular carcinoma model induced by DEN and nude mouse were used to verify the role of RMP played in hepatocellular carcinoma.Results:Following are the main results of this research:1.RMPfloxp/floxp mice were successfiully conducted,and myeloid-specific RMP knock-out mice were obtained by crossing with Lyz2-CRE mice.2.In the lethal dose of LPS endotoxin shock mice model,by contrast to the wild type mice,the survival time of RMP myeloid knock-out mice was significantly lower;and the mouse lungs obtained from knock-out mice infiltrated with a large number of inflarmatory cells and red blood cells,indicating a more serious damage.3.In macrophages,knock-out of RMP increased the synthesis and secretion of inflammatory cytokines by activating the NF-κB and MAPK signaling pathways,and enhanced the response to LPS and TNFa treatments.4.Overexpression of RMP significantly inhibited the macrophage activation and the synthesis and secretion of inflammatory cytokines.5.RMP could inhibit macrophages migrating to tumor.6.Macrophage activation is accompanied by RMP phosphorylation,which could by blocked by PKC and IKK inhibitors.7.Protein co-immunoprecipitation assay showed that RMP could interact with IKKβ.8.In the DEN induced HCC mice model,the tumor nunber were about 3.6(p<0.001)than those in the control group.And the survival time of RMP myeloid knock-out mice was significantly lower(35.4w vs 46.7w,p=0.037).Hepatocellular carcinoma had a more rapid growth in the conditional myeloid RMP knock-out mouse than that in wild type mice.These results indicate that knocking-out of RMP in macrophages could promote tumorigenesis.Conclusion:Our study explores the role of myeloid RMP in the initiation and progression of hepatocellular carcinoma.Our conclusions are as below:1.RPM can be phosphorylated by IKKp.2.Myeloid RMP involves in hepatocellular earcinoma by negatively regulating the function of macrophages and changing the tumor microenvironment via NF-κB and MAPK signaling pathways.3.This study of the pathological mechanisms of myeloid RMP may shed a light on the immunotheraphy of hepatocellular carcinoma.