CircWWC3 Promotes Breast Cancer Progression Through Affecting Macrophages Polarization And PD-L1 Expression Via IL-4

Part 1 Effect of circWWC3 on the expression of IL-4 in breast cancercells.Objective: To investigate the effect of circWWC3 on the expression of IL-4 in breast cancer cells.Methods:1.After knockdown of circWWC3 in breast cancer cell MDA-MB-231,the key genes affecting cell pathway were screened by high-throughput microarray technology.2.qRT-PCR was used to detect the expression of IL-4 in breast cancer cells after overexpression or knockdown of circWWC3.3.Western blot was used to detect the expression of IL-4 in breast cancer cells after overexpression or knockdown of circWWC3.4.The changes of IL-4 secretion in breast cancer cells after overexpression or knockdown of circWWC3 were detected by ELISA.Results:1.The results of microarray analysis showed that IL-4 was one of the most relevant candidate genes affecting cell pathway after knockdown of circWWC3 in MDA-MB-231 cells.2.The results of qRT-PCR showed that the expression level of IL-4 in MDA-MB-231 was significantly decreased after knockdown of circWWC3(P< 0.01),and the expression level of IL-4 in MDA-MB-453 was significantly increased after overexpression of circWWC3(P < 0.01).3.The results of western blot showed that the expression level of IL-4 in MDA-MB-231 decreased significantly after knockdown of circWWC3(P <0.01),while the expression level of IL-4 in MDA-MB-453 increased significantly after overexpression of circWWC3(P < 0.01).4.The results of ELISA showed that the secretion level of IL-4 in MDA-MB-231 was significantly decreased after knockdown of circWWC3(P< 0.01),and the secretion level of IL-4 in MDA-MB-453 was significantly increased after overexpression of circWWC3(P < 0.01).Summary:Circ WWC3 can promote the expression and secretion of IL-4 in breast cancer cells.Part 2 Circ WWC3 promotes the development of breast cancer byregulating the expression of IL-4 and affecting the polarizationof tumor associated macrophagesObjective: To investigate the effect of circWWC3 on the polarization of tumor associated macrophages by regulating the expression and secretion of IL-4 in breast cancer cells.Methods:1.After IL-4 induced macrophages to polarize towards M2,the expression of macrophage surface markers was detected by qRT-PCR.2.After macrophages were induced to polarize towards M2 by IL-4,the secretion of cytokines and chemokines were detected by ELISA.3.The supernatant of breast cancer cells overexpressing or knockdown circWWC3 was cocultured with macrophages.The expression of macrophage surface markers was detected by qRT-PCR.4.The supernatant of breast cancer cells with overexpression or knockdown of circWWC3 was cocultured with macrophages.The secretion levels of cytokines and chemokines in macrophages were detected by ELISA.Results:1.The results of qRT-PCR showed that the expression levels of IL-10、TGF-β、CCL-8、CCL-17 and CCL-22 were significantly increased after IL-4induced macrophage polarization(P < 0.05).2.The results of ELISA showed that the secretion levels of IL-10、TGF-β、CCl-8、CCL-17 and CCL-22 were significantly increased after IL-4 induced macrophage polarization in vitro(P < 0.05).3.The results of qRT-PCR showed that the expression levels of macrophage surface markers IL-10、TGF-β、CCL-8、CCL-17 and CCL-22 were significantly decreased after coculture of MDA-MB-231 supernatant and macrophages(P < 0.05);after coculture of MDA-MB-453 supernatant and macrophages,the expression levels of IL-10、TGF-β、CCL-8、CCL-17 and CCL-22 were significantly increased(P < 0.01).4.The results of ELISA showed that the secretion of IL-10、TGF-β、CCL-8、CCL-17 and CCL-22 decreased significantly after coculture of MDA-MB-231 supernatant of breast cancer cells with knockdown of circWWC3 and macrophages(P < 0.05).The secretion of IL-10、TGF-β、CCL-8、CCL-17 and CCL-22 increased significantly after coculture of MDA-MB-453 supernatant of breast cancer cells with overexpression of circWWC3 and macrophages(P < 0.01).Summary:IL-4 can induce macrophages to polarize towards M2.Circ WWC3 can promote the development of breast cancer by affecting the expression and secretion of IL-4.Part 3 Circ WWC3 promotes the development of breast cancer byregulating the expression of PD-L1 on breast cancer cells andmacrophages through regulating IL-4.Objective: To investigate the effect of circWWC3 on the expression of PD-L1 on breast cancer cells and tumor associated macrophages by regulating the expression and secretion of IL-4 in breast cancer cells.Methods:1.The expression of PD-L1 on macrophage surface was detected by qRT-PCR after IL-4 induced macrophage polarization towards M2.2.After macrophages were induced to polarize towards M2 by IL-4,the changes of PD-L1 on macrophage surface were detected by immunofluorescence.3.After overexpression or knockdown of circWWC3 in breast cancer cells,the changes of PD-L1 on the surface of breast cancer cells were detected by qRT-PCR.4.After overexpression or knockdown of circWWC3 in breast cancer cells,the changes of PD-L1 on the surface of breast cancer cells were detected by immunofluorescence.5.The supernatant of breast cancer cells overexpressing or knockdown circWWC3 was cocultured with macrophages,and the changes of PD-L1 on macrophages were detected by qRT-PCR.6.The supernatant of breast cancer cells overexpressing or knockdown circWWC3 was cocultured with macrophages,and the changes of PD-L1 on macrophages were detected by immunofluorescence.Results:1.The results of qRT-PCR showed that after IL-4 induced macrophage polarization,the expression level of PD-L1 on macrophage surface was significantly increased(P < 0.05).2.Immunofluorescence results showed that the expression level of PD-L1 on macrophage surface increased significantly after IL-4 induced macrophage polarization.3.The results of qRT-PCR showed that the expression level of PD-L1 in MDA-MB-231 cells with knockdown of circWWC3 was significantly decreased(P < 0.05),while the expression level of PD-L1 in MDA-MB-453 cells with overexpression of circWWC3 was significantly increased(P <0.01).4.Immunofluorescence results showed that the expression of PD-L1 in MDA-MB-231 cells with knockdown of circWWC3 was significantly decreased;PD-L1 expression level in breast cancer with overexpression of circWWC3 was significantly increased.5.The results of qRT-PCR showed that the expression level of PD-L1 on macrophage surface was significantly decreased after coculture with the supernatant of MDA-MB-231 cells with knockdown of circWWC3(P < 0.05);the expression level of PD-L1 on macrophage surface was significantly increased after coculture with the supernatant of MDA-MB-453 cells with overexpression of circWWC3(P < 0.05).6.Immunofluorescence results showed that the expression of PD-L1 on the surface of macrophages was significantly decreased when the supernatant of MDA-MB-231 cells with knockdown of circWWC3 was cocultured with macrophages;When the supernatant of MDA-MB-453 cells overexpressing circWWC3 was cocultured with macrophages,the expression of PD-L1 on macrophages increased significantly.Summary:IL-4 can induce macrophages to polarize to M2 direction,which can increase the expression level of PD-L1 on macrophages.circWWC3 can also increase the expression level of PD-L1 on macrophages by affecting the expression of IL-4 in breast cancer cells.Meanwhile,circWWC3 can also increase the expression level of PD-L1 in tumor cells,which can promote the progress of breast cancer.Conclusion:Our research can draw the following conclusions: in breast cancer cells,circWWC3 can induce macrophage polarization to M2 type by promoting the expression and secretion of IL-4,and induce the expression of PD-L1 on the surface of macrophage and breast cancer cells,so as to promote the progress of breast cancer.