CNOT7 Depletion Reverses IFN-γ Resistance In Hepatocellular Carcinoma Cells

Objective:Hepatocellular carcinoma（HCC）is the most highly malignant diseases and the most prevalent cause of cancer-related deaths in the world.HCC originates on a diffuse and chronic inflammation in liver,where exists unceasing cellular injury and regeneration,finally developing to cirrhosis.Such chronic inflammation in liver is considered to increase the risk of cancer development.There are many reasons to cause the liver inflammation,e.g.hepatotropic virus,medicines,and alcohol.Among them,the HBV infection is generally acknowledged the major risk factor for HCC in the world.Interferon-γ（IFN-γ;also known as type II interferon）is one type of cytokine that is critical in both innate and adaptive immunity in humans.Owing to the anti-virus and anti-tumor properties,Interferons（IFNs）,including type I,II,and III IFNs,have been considered to be valuable therapeutic reagents,and widely used to be against chronic viral hepatitis and HCC.It is also deemed that IFN-γ,neither IFN-α nor IFN-β（also known as type I interferon）,has the ability to control the immunogenicity of tumor cells in human.Increasing the plasma concentration of IFN-γin HCC patients has generally shown a discouraging response.Several studies have demonstrated that IFN-γ exerted growth-inhibitory effects on HCC cells,but other studies shown opposite results.Some authors suggest that the resistance of cancer cells to IFN-γmay be due to a relative deficiency of IFN signaling molecules STAT1 and overexpression of STAT1 may restore the responsiveness of cancer cells to IFN-γ.CNOT7,a subunit of the CCR4-NOT complex,has been identified as the major cytoplasmic mRNA deadenylase.There are some reports that CNOT7 regulated IFN pathways by controlling STAT1 trafficking through interaction with its inactive form and by degrading STAT1-regulated mRNAs through its deadenylase activity.This experiment aimed to explore the role of CNOT7 in hepatocellular carcinoma with HBV-related cirrhosis（HCCBC）;to further understand the functional role of CNOT7 in the IFN-γ resistance of HCC;to elucidate the immune tolerance mechanisms of CNOT7 in HCC.Method:The plasma concentrations of IFN-γ were detected in HCCBC patients with ELISA.The levels of CNOT7,STAT1 and STAT3 expression were determined in the tumor tissuesof HCCBC patients with ICH.We screened four HCC cell lines to detect the susceptibility to IFN-γ with MTT and flow cytometry.To explore the relationship between CNOT7 and STAT1,HepG2 cells were transfected with CNOT7-specific shRNA,followed by transfection with the recombinant plasmids encoding CNOT7.STAT1 expression in HepG2^shCNOT7 and HepG2CNOT7 cells was determined by RT-qPCR and western blot analysis.The viability of HepG2^shCNOT7 and HepG2 cells was evaluated following treatment with IFN-γ using the MTT assay and flow cytometry.To elucidate the mechanisms underlying the action of STAT1,cyclinE1 and caspase3 expression levels were also analyzed by RT-qPCR and western blot.Result:In our study,We detected the levels of plasma IFN-γ in HBC and HCCBC patients.There was no significant difference between these two groups.We found that the levels of plasma IFN-γ in healthy controls were significantly lower than these two patients groups.Therefore,we assumed that a major factor resulting in the poor outcome maybe IFN-γ resistance of HCC cells,rather than the lower plasma IFN-γ concentrations in HCCBC patients.Then,We analyzed the levels of CNOT7,STAT1 and STAT3 expression in HCCBC patients.STAT1 were significantly lower in HCCBC compared to adjacent non-tumor tissues.Supposed that STAT1 is the crucial signaling molecules activated by IFN-γ,STAT1 deficient may be responsible to the IFN-γ insensitivity.This result supports a previous study.Our hypothesis that CNOT7,presenting the opposite result of STAT1,was much higher expression in the HCCBC than adjacent non-tumor tissues.The current experiment was demonstrated as consistent with this hypothesis.However,the expressions of STAT3 were no significant differences in HCCBC and adjacent non-tumor tissues.This result was different from others reports.One possible reason is that high IFN-γconcentration may stimulate HCC cells constantly,and then inhibit the STAT3 expression of HCCBC patients relatively.In order to further validate our hypothesis,we screened four HCC cell lines to detect the susceptibility to IFN-γ.We finally found that the levels of CNOT7 and STAT1 expression were correlated with IFN-γ resistance.Considering the results described above,HepG2 cells appeared to be the most suitablecell line for further evaluating the mechanism.To explore the relationship between CNOT7 and STAT1,HepG2 cells were transfected with CNOT7-specific shRNA,followed by transfection with the recombinant plasmids encoding CNOT7.The level of STAT1 protein was greater in HepG2^shCNOT7 cells than in HepG2 cells.In parallel,the level of STAT1 protein wasdown-regulated in HepG2CNOT7 cells compared to that in HepG2 cells.However,there was no significant difference in STAT1 mRNA expression between HepG2^shCNOT7 and HepG2cells and between HepG2CNOT7.These results prompted us to investigate a possible physical interaction between CNOT7 and STAT1 proteins.The Co-IP assay revealed a clear interaction between STAT1 and CNOT7 in HepG2 cells.Therefore our results indicate that CNOT7 may be a negative regulator of the IFN-γ pathways by controlling STAT1 trafficking through interaction with its latent form.To further understand the functional role of CNOT7 in the IFN-γ resistance of HepG2 cells,the viability of HepG2^shCNOT7 and HepG2 cells was evaluated following treatment with IFN-γ using the MTT assay.IFN-γsignificantly inhibited the proliferation of HepG2 sh CNOT7 cells but not HepG2 cells.Furthermore,flow cytometry with PI staining showed that following IFN-γ treatment,the percentage of HepG2^shCNOT7 cells in the G0/G1-phase increased significantly,suggesting that IFN-γ induced G0/G1-phase cell cycle arrest;Evaluation of apoptosis with flow cytometry and AnnexinV-APC/7-AAD staining showed that following IFN-γ treatment,the early apoptosis rates of HepG2^shCNOT7 cells increased significantly,suggesting that IFN-γsignificantly induced HepG2^shCNOT7 apoptosis.These results suggest that knocking down CNOT7 protein expression could reverse IFN-γ resistance in HepG2 cells.Finally,to determine whether IFN-γ signaling is redirected through feedback from alterations in STAT1 protein.STAT1 expression increased in both HepG2^shCNOT7 and HepG2 cells after IFN-γ treatment in a time-dependent manner,although the expression level of STAT1 protein was consistently greater in HepG2^shCNOT7 cells.However,STAT3 expression was significantly down-regulated in HepG2^shCNOT7 compared to HepG2 cells after treatment with IFN-γ.To elucidate the mechanisms underlying the action of STAT1,cyclinE1 and caspase3 expression levels were also analyzed by RT-qPCR and western blot.There was no significant difference in the levels of cyclinE1 and caspase3 between HepG2^shCNOT7 and HepG2 cells.However,after IFN-γ stimulation,the expression level of cyclinE1 in HepG2^shCNOT7 cells was significantly inhibited,whereas caspase3 expression was significantly enhanced,with no changes observed in HepG2 cells.On the basis of our results,we tried to prove the possible mechanism by which STAT1 and STAT3 may show mutual competitive inhibition after IFN-γ treatment.The effects of IFN-γ on tumors appear to be determined by the ratio of STAT1 to STAT3.STAT1 deficiency redirects IFN signaling through feedback activation of STAT3;if STAT1 is overexpressed,the opposite occurs.Conclusion:In summary,our results demonstrate that the occurrence of HCCBC partially attribute to IFN-γ resistance rather than IFN-γ deficiency.IFN-γ resistance is correlated with CNOT7 overexpression and STAT1 deficiency.Given that CNOT7 is a key factor in IFN-negative regulation,the combination of CNOT7 depletion and IFN-γ appears to be highly effective for the inhibition of HCC cell proliferation in vitro,which may contribute to the development of new therapeutic approaches against liver cancer.CNOT7 knockdown had little or no effect on primary tumor cell,indicating that pharmaceutical suppression CNOT7 may not be unacceptably toxic in the clinic.Furthermore,due to its enhancing immunologic function,CNOT7 depletion is considered to be a superior adjuvant therapy in immunological therapy for HCC.