Research On The Mechanism Of Panaxynol Remold The Phenotype And Function Of Macrophage To Inhibit The Occurrence And Development Of Breast Cancer

ObjectiveMacrophages infiltrated into solid tumors can be differentiated into M1 type macrophage with anti-tumor functions and M2 type macrophage with tumor promoting functions under the different stimulation factors.Based on the functional characteristics of the two subtype of macrophages,this paper intends to explore a new possible target for tumor intervention by inducing the transformation of M2-type into M1-type macrophages.Panaxynol are naturally polyacetylenes,exist extensively,yet a large body of data indicate that it has a significant proliferation inhibition effect on a variety of tumor cells.However,there is no reports on panaxynol in remodeling macrophage phenotype and functions and inhibiting breast cancer cells.In this paper,panaxynol acts on M2-type macrophages to detect the changes in the phenotype and functions and explore the mechanism to observe its impact on the biological function of breast cancer cells.Method(1)The proliferation of 4T1 cells(6,12,24h)treated with panaxynol at different concentrations(0,8,16μg/mL)was detected by CCK-8 kit.Annexin V/7AAD kit was used to detect the rate of tumor cell apoptosis.Meanwhile,the expression of caspase-3,Cleaved caspase-3,Bax,Bcl-2 and other apoptosis-related proteins in 4T1 cells were determined by Western blot.The migration and invasion ability of tumor cells was examined by putting the 4T1 tumor cells and panaxynol at different concentrations(0,8,16μg/mL)in the upper chamber and lower chamber of Transwell for 24 h respectively.(2)The supernatant of 4T1 tumor cells was used to culture RAW264.7 macrophages for 48 h,and then different doses of panaxynol were given for 24 h.The expression level of Arg-1 and iNOS in RAW264.7 was analyzed by Western blot.Meanwhile,qRT-PCR was used to detect Arg-1 and iNOS in the mRNA level.The expression of antigen-presenting related molecules CD80,CD86 and MHCII on the surface of macrophages was probed by Flow Cytometry and the changes of macrophage secretion spectrum were explored by RT-qPCR and ELISA.(3)Macrophages under treatment conditions were co-cultured with 4T1 cells,The growth status of 4T1 cells was observed under the microscope.Transwell assay was performed to detect the effect of macrophages on migration and invasion ability of 4T1 cells.(4)qRT-PCR was used to screen and verify differentially expressed LncRNA in macrophages.Compare the expression of LncRNA before and after panaxynol stimulation.SiRNA was synthesized and transfected with RAW264.7 to analyze the functional changes of macrophages after down-regulation of LncRNA.Results(1)The results of CCK-8 test showed that the proliferation of 4T1 cells treated with different concentrations of panaxynol was inhibited at 6,12 and 24 h,and the proliferation was time-dependent and concentration-dependent.Different doses of panaxynol can induce different degrees of apoptosis in 4T1 tumor cells after 24 h.Western blot results indicated that the expression level of Bax and Cleaved caspase-3 were markly increased,but that of Bcl-2 was decreased.The results of Transwell migration and invasion experiment displayed that,compared with the untreated group,the higher concentration of panaxynol,the more obvious inhibitory effect on the migration and invasion ability of tumor cells.(2)Western blot and qRT-PCR results showed that the expression of Arg-1 in macrophages in the treated group was strikingly declined at the protein level and gene level,while iNOS was rised.FCM indicated that the expression of antigen-presenting related molecules CD80,CD86 and MHCII on the surface of macrophages was also upregulated.qRT-PCR and ELISA results showed increased TNF-α and IL-1β secretion,decreased IL-4,IL-8,and IL-10 secretion.(3)The results of Transwell experiment revealed that macrophages pretreated with panaxynol have an inhibitory effect on the migration and invasion ability of 4T1 cells.(4)The results of qRT-PCR displayed that the phenotypic changes of macrophages in the treated group were accompanied by the decrease of LncRNA039932.Conclusion(1)Panaxynol can directly promote the apoptosis of tumor cells and inhibit their invasion and migration ability.(2)Panaxynol can reverse macrophage phenotype,affect its function and secretion spectrum,and indirectly affect the biological behavior of tumor cells.(3)Changes in macrophage function under treatment were accompanied by low expression of LncRNA039932.