Effects Of STING Signaling Pathway On Oxaliplatin Against Mouse C26 Colon Cancer Cell In Vitro And In Vivo

BackgroundColorectal cancer ranks the third among malignant tumors in China,and it isincreasing year by year.There are various methods for the combined treatment of colorectal cancer,in which the combination of cytokine-mediated anti-tumor therapy and chemotherapeutic drugs has made some progress in scientific research and clinical.Oxaliplatin is a chemotherapy drug for DNA damage that is widely used in the treatment of colorectal cancer in clinical practice.It is often used in combination with other drugs to treat colorectal cancer.Studies have shown that DNA in the cytoplasm can cause activation of the interferon gene stimulating factor STING?Stimulator of Interferon Genes?,and recent studies have shown that STING-mediated signaling pathway plays an important role in inducing anti-tumor immune response.However,the exact role of the STING signaling pathway in the treatment of colon cancer with oxaliplatin remains to be further explored.PurposeTo investigate the effect of STING signaling pathway on the proliferation ofoxaliplatin against colorectal cancer?CRC?C26 cells in vitro and in vivo and its possible mechanisms.Methods1.C26 was cultured,the shmSTING-C26 cell line and the overexpressed omSTING-C26 cell line were cultured in our lab,and the transfection efficiency of the two groups was observed under a fluorescence microscope.2.The proliferation of C26,C26shmSTING and C26omSTING cells under normoxic conditions was detected by CCK8 method.The cell proliferation of C26 and C26shmSTING at different OXA concentrations was detected by CCK8 method under normoxia and hypoxia.3.Western blotting was used to detect the expression of STING,TBK1,IRF3, CyclinD1 and?-actin in C26,C26shmSTING and C26omSTING cells were treated with oxaliplatin?10?g/ml?and oxaliplatin-free 6 groups..4.q-PCR was used to detect the expression of mSTING and mIFN-?in six groups of cells treated with oxaliplatin and without oxaliplatin in C26,C26shmSTING and C26omSTING cells.5.Flow cytometry was used to detect the cell cycle changes of C26, C26shmSTING and C26omSTING under the same OXA concentration in normoxia and hypoxia?1%O₂?condition.6.Flow cytometry was used to detect apoptosis of C26,C26shmSTING and C26omSTING under hypoxic conditions at the same drug concentration.7.Flow cytometry was used to detect the expression of PD-L1 at the same OXA concentration for C26 and C26omSTING,respectively.8.C26,C26-shmSTING and C26-omSTING cells were inoculated into the left axilla of Balb/c mice,respectively,and the growth inhibition effect of the tumorigenicity and oxaliplatin administration?5 mg/kg?on the transplanted tumor was observed.9.Flow cytometry was used to detect intratumoral CD8⁺T cells and CD107a⁺/CD8⁺T in 6 groups of tumor-bearing mice in C26,C26shmSTING and C26omSTING cell control groups and oxaliplatin-treated group.CD45⁺tumor infilitrating lymphocytes were also detected.Results1.Down-regulation of STING stimulating factor in C26 cells did not affect cell proliferation,and up-regulated the growth rate of STING cells in C26 cells was significantly lower than that in wild-type C26 cells?P<0.05?.Under normal and hypoxic conditions,oxaliplatin reduced the inhibition rate of C26-shmSTING cells,especially under hypoxic conditions.2.Compared with the control group,the expression of STING,TBK1 and IRF3 was increased and the expression of CyclinD1 was decreased in C26-omSTING cells.The three groups were given the same concentration of OXA and compared with the experimental group not treated with OXA,STING,Both TBK1 and IRF3 expression levels increased,and CyclinD1 expression decreased.3.When C26 cells knocked down and overexpressed STING were given the same concentration of OXA,the expression levels of mSTING and mIFN-?were up-regulated compared with the corresponding control groups.4.Under normal-oxygen conditions,the resistance of C26 cells to OXA increased with the increase of drug concentration,while under hypoxic conditions,the resistance to OXA decreased with the increase of drug concentration.Under the conditions of normoxia and hypoxia,with the increase of OXA concentration,the resistance of oxaliplatin to C26-shmSTING and C26-omSTING cells increased gradually.5.Under hypoxic conditions,C26 and C26-omSTING were increased in apoptotic rate compared with the experimental group after adding different concentrations of OXA.Compared with the experimental group,C26-shmSTING cells had a large proportion of viable cells compared with the experimental group,and the apoptotic rate increased slightly with the increase of OXA concentration.6.In the normoxic environment,the levels of PD-L1 in the C26 and C26-shmSTING cell groups were increased after the addition of OXA,but the C26-shmSTING cell group significantly reduced the increase of PD-L1 under the action of OXA.7.Inhibition rate of tumor formation in mice with Balb/c in C26 plus OXA treatment group was 69.98%in mice,and tumor formation and C26-shmSTING mice in C26-shmSTING plus OXA-administered mice.The inhibition rate of tumor formation in vivo was 27.47%;the inhibition rate of tumor formation in the C26-omSTING plus OXA-administered group of Balb/c mice and the C26-omSTING group was 74.86%,and the C26-omSTING plus OXA treatment group The tumor formation inhibition rate in mice and the C26-omSTING group was 33.67%.8.C26,C26-shmSTING and C26-omSTING three groups of tumor-bearing mice were added to the OXA-administered group.Compared with the non-OXA-administered group,the level of CD8+T cells decreased while CD107a+/CD8+.The level of T cells increased,and the levels of TIL in the tumor-bearing CD45+in the tumor-bearing mice of the C26 and C26-shmSTING groups were increased in the OXA-administered group compared with the non-OXA-administered group,in the omSTING group.The level of TIL of CD45+inside the tumor was increased in mice after administration of OXA.Conclusion1.The sting signaling pathway is highly correlated with the proliferation of C26cells and the sensitivity of oxaliplatin;2.Knockdown expression of STING has a significant effect on the proliferation of C26 cells,but reduces the inhibitory effect of oxaliplatin on the proliferation of C26 in vitro and in vivo,and reduces the sensitivity of C26 to oxaliplatin;3.Overexpression of STING can significantly delay the proliferation of C26 cells in vitro and in vivo,which may be related to down-regulation of CyclinD1 and increased type I interferon signaling pathway.4.Knockdown/overexpression of sting could reduce the apoptosis-inducing effect of oxaliplatin on C26 cells in vitro,and the specific mechanism needs further study.