IFN? Up-regulates The Expression Of CMPK2 In Liver Cancer Cells And Activates Macrophages

Liver cancer greatly threatens human health,whose mortality rate is ranked second in the world.As liver cancer can escape immune elimination by the loss of antigenicity and immunogenicity and orchestrating an immunosuppressive microenvironment,anti-tumor immunotherapy has received increased attention.Subcutaneous injection of interferon a(IFNa),as a kind of simple,convenient and cheap immunotherapy,has been widely applied in the treatment of liver cancer.Through analyzing the cDNA microarray data of the hepatocellular carcinoma(HCC)cell line Huh7 with and without IFNa treatment,we found that CMPK2 significantly upregulated in Huh7 cells 6 hours after IFNa treatment.CMPK2 is a nucleoside monophosphate kinase located in the mitochondrion and is involved in the nucleotide salvage pathway.CMPK2 may play an important role in the activation of macrophages.Expression levels of CMPK2 and inflammatory factors rise significantly in macrophages after the simulation of IFNa,LPS or Poly(I:C).This study intends to understand the impact of CMPK2 on HCC cells,and to explore weather CMPK2 can promote antitumor immunity.We treated the Huh7,Hela and 293T with IFNa,IFN? and LPS separately,and found that the transcription level of CMPK2 elevated most significantly in Huh7 after the IFNa treatment.When the treatment concentration of IFNa is between 1×106 U/L and 5×106 U/L,the transcription level of CMPK2 raised most significantly in Huh7.The CMPK2 transcription level increased one hour after the IFNa treatment,reached its peak around the 6th hour,and significantly declined at the 24th hour.The results of Western blot showed that the protein level of CMPK2 and the phosphorylation level of STAT1 were significantly elevated 6 hours after the IFNa treatment.Next,we established the CMPK2 overexpressing Huh7 cells by stably infecting with leentivirus.Real-time PCR analysis of the transcription levels of the cyclins suggested that CMPK2 did not have a significant influence on the proliferation of Huh7 cells.Immunofluorescent staining of the superoxide in the mitochondria by MitoSOX Red showed that there was no significant difference between the state of oxidative stress in CMPK2 overexpressing Huh7 cells and the control group.As CMPK2 can transform nucleoside monophosphates into nucleoside diphosphates,the increase of CMPK2 level will lead to a rise in the nucleoside diphosphate level and promote the production of nucleoside triphosphates.We measured the ATP levels and found that the ATP level in the cells and supernatant of CMPK2 overexpressing Huh7 cells is significantly higher than the control group.ATP can combine with purinergic receptors on the surface of macrophages,activates macrophages,and thus promotes the expression of cytokines,such as IL-1?.We simulated macrophages with the supernatant of CMPK2 overexpressing Huh7 cells and found that the transcriptional levels of IL-1?,IL-6,and CCL5 in the macrophages were much higher than the control group.It is suggested that CMPK2 may activate the expression of cytokines by increasing the ATP level in Huh7 cells.In summary,this study found that the transcription and protein levels of CMPK2 were significantly enhanced after the treatment of IFNa for 6 hours.CMPK2 increased the ATP level in the cells and supernatant of Huh7 cells.The supernatant of CMPK2 overexpressing Huh7 cells activated the expression of IL-1?,IL-6,and CCL5 in macrophages.It suggests that the immune-mediated antitumor effect of IFNa may is related to the significant elevation of CMPK2 expression,which increases the ATP level in Huh7 cells and promotes the activation of macrophages,but the specific mechanisms need further experiments to verify.