CNOT7 Knockdown Reduces The Effect Of GM-CSF Secretion On The Immune Microenvironment Of Hepatocellular Carcinoma By JAK/STAT Pathway

Objective:The human CCR4-NOT transcriptional complex subunit 7（CNOT7）knockdown reduced the secretion of tumor-derived GM-CSF via the JAK/STAT pathway,and then intervened the role of bone marrow-derived inhibitory cells（MDSCs）in the local tumor immune microenvironment and related mechanisms.Methods:1.ELISA method was used to detect the secretion level of GM-CSF in peripheral blood of liver cancer,hepatitis and healthy volunteers,and the secretion level of GM-CSF in TTCS and NTCS,and compare them;2.Flow cytometry to detect the number of MDSCs in tumor tissue,tumor margin,and non-tumor normal liver tissue;3.Screening appropriate hepatoma cell lines;using CNSS technology,transfecting CNOT7 recombinant plasmid and corresponding control vector plasmid into HepG2cells;4.Western blot analysis was used to detect the protein expression levels of STAT1,STAT3 and GM-CSF during CNOT7 and signal transduction.Results:1.ELISA results show there was no significant difference in peripheral blood GM-CSF levels between liver cancer group,hepatitis group and control group（P>0.05）.2.Compared with tumor group（9.66±2.73pg/ml）and normal liver tissue group（11.61±2.89pg/ml）,tumor margin group（32.16±8.90pg/ml）GM-CSF was statistically different（P<0.05）.3.Flow cytometry results showed that the ratio of CD33⁺CD11b⁺cells in the tumor margin group（11.90±3.96%）was significantly higher than that in the liver cancer tissue group（4.16±2.22%）and normal liver tissue group（6.22±2.29%）,which was statistically significant（P<0.05）.4.After transfection of HepG2 human hepatoma cell line,the CNOT7 protein in HepG2^shCNOT7 group was lower than that in HepG2^EV group and HepG2 group.（P<0.01）,STAT1 protein expression was up-regulated（P<0.01）,STAT3 protein expression was down-regulated（P<0.01）,and GM-CSF protein expression was significantly decreased（P<0.01）.Conclusion:1.Compared with healthy people and hepatitis B patients,there was no significant difference in peripheral blood GM-CSF between liver cancer patients;the distribution of MDSCs in liver cancer was related to the secretion level of GM-CSF and accumulated at the edge of the tumor.2.CNOT7 knockdown can reduce the secretion of tumor-derived GM-CSF,and then intervene in the local immunosuppressive function of MDSCs to achieve the purpose of regulating tumor immune microenvironment and inhibiting tumor cell proliferation and migration.