| Objective: Ovarian cancer is one of the most common female malignant tumors.The prognosis of ovarian cancer patients is poor which the mortality ranks first in gynecologic malignant tumors.At present,ovarian cancer rebulking operation combined with chemotherapy is the first-line treatment for ovarian cancer.As we all known,chemoresistance is one of the important factors which leading to the recurrence and death of ovarian cancer patients.In recent years,studies have shown that tumor microenvironment provides an environment which is good for the drug resistance and immune escape of cancer cells.Therefore,increasing researches focused on the treatment of cancer by targeting tumor immune microenvironment.The aim of the study is to investigate the effects of macrophages and macrophages treated with mi R-21 on the chemoresistance of ovarian cancer cells,and the possible molecular mechanisms,that is,whether mi R-21 can regulate the polarization of macrophages,and further regulate the PI3K/AKT signaling pathway in the ovarian cancer cells that co-cultured with macrophages.Methods: Firstly,according to the data in GEO database,the expression patterns of 22 kinds of immune cells in the tumor microenvironment of ovarian cancer tissue were analyzed by CIBERSORT algorithm.Through the clinicopathological data of ovarian cancer in TCGA,the correlation between the expression levels of the clinicopathological data with different phenotypes of macrophages were analyzed,and the prognosis of ovarian cancer patients was analyzed by the Kalpan-meier methods.According to the data of GEO database,we analyzed the relationship between the expression level of mi R-21 and chemoresistance of ovarian cancer cells,as well as macrophages of different phenotypes.The expression level of mi R-21 in human ovarian cancer tissues was detected by real-time PCR,and the correlation between the expression level of mi R-21 and clinicopathological parameters of ovarian cancer was analyzed by TCGA database.The human monocyte cell line THP-1,the human ovarian cancer cell lines OVCAR3 and HO-8910 were cultured for the experiments.PMA was used to transform THP-1 cells into M0 macrophages.LPS and IFN-? was used to induce M0 macrophages into M1 macrophages,and IL-4 and IL-13 was used to induce M0 macrophages into M2 macrophages.The expressions of macrophages markers were detected by real-time PCR and flow cytometry.M0,M1 and M2 macrophages were co-cultured with ovarian cancer cells to detect the drug resistance.Real-time PCR was used to detect the expression levels of mi R-21 in ovarian cancer cells and macrophages.The expression of mi R-21 was detected in ovarian cancer cells co-cultured with M0,M1 and M2 macrophages.The M2 macrophages transfected with mi R-21 mimic and mi R-21 inhibitor were co-cultured with ovarian cancer cells,and IC50 of ovarian cancer cells were detected by CCK8.Western blot was used to detect the chemoresistance and apoptosis related proteins,including cleaved caspase 3,BCL-2,ABCG2 and MDR1.What's more,the apoptosis ability and the cell cycle were detected by flow cytometry.The expressions of macrophage related markers in M2 macrophages transfected with mi R-21 mimic and mi R-21 inhibitor were detected by real-time PCR.GSEA software was used to analyze the related signaling pathways of M2 macrophages on drug resistance of ovarian cancer cells.The expression levels of PI3K?AKT?p-AKT were detected by western blot.LY294002,worked as the selective inhibitor of PI3 K,was used in the rescue experiment of PI3K/AKT signaling pathway in the downstream passage.Results: According to the analysis of CIBERSORT algorithm,the expressions of immune cells in chemosensitivity ovarian cancer tissues and chemoresistance ovarian cancer tissues were significantly different,in which the relative expression of macropahges was higher in ovarian cancer tissues,and the expression of M2 macrophages was higher in chemoresistance ovarian cancer tissues.In addition,according to the data of TCGA,ovarian cancer patients with higher expression of M1 macrophages have longer overall survival time,while patients with higher expression of M2 macrophages showed shorter survival time.The results of bioinformatics analysis showed that mi R-21 expression in chemoresistance ovarian cancer cells was significantly higher than that in chemosensitivity ovarian cancer cells,and the expreesion of mi R-21 in M2 macrophages was higher than that in M0 and M1 macrophages.According to the analysis of tissues samples we collected,the expression of mi R-21 in ovarian cancer tissue was significant up-regulated.Due to the analysis of TCGA database,the expression of mi R-21 is closely related to the histological grade and lymph node invasion and metastasis of ovarian cancer.Real-time PCR results showed that mi R-21 expreesion in M2 macrophages was significantly higher than that in ovarian cancer cells,M0 and M1 macrophages,and co-cultured of ovarian cancer cells with M2 macrophages increased the expression of mi R-21 in ovarian cancer cells.CCK8 and western blot results showed that ovarian cancer cells co-cultured with M2 macrophages showed stronger chemoresistance.Transfection mi R-21 mimic into macrophages and co-cultured with ovarian cancer cells could enhance the resistance of ovarian cancer cells,inhibit the apoptosis of ovarian cancer cells,and regulate the cell cycle.However,transfection mi R-21 inhibitor showed the opposite biological behaviors.In addition,transfection of mi R-21 mimic in macrophages promoted the transformation of the macrophages into M2 macrophages,whereas transfection of mi R-21 inhibitor promoted the transformation into M1 macrophages.The GSEA software was used to analyze of effect of PI3K/AKT signaling pathways involved in the effect of M2 macrophages worked on ovarian cancer cells.Western blot analysis showed that macrophages transfected with mi R-21 could further regulate the expression of downstream PI3 K,p-AKT and AKT proteins to regulate the chemoresistance of ovarian cancer cells.After pre-treated LY294002 with ovarian cancer cells for 1h,CCK8 and western blot results indicated that M2 macrophages treated by mi R-21 could affect the chemoresistance of ovarian cancer cells through PI3K/AKT signaling pathway.Conclusion: 1.The expression levels of immune cells in chemoresistance ovarian cancer tissues and chemosensitivity ovarian cancer tissues were significantly different,among which the expression of M2 macrophages in chemoresistance ovarian cancer tissues was increased.2.Patients with higher expression of M1 macrophages have a better prognosis;while patients with higher expression of M2 macrophages have a poor prognosis.3.Compared to the benign ovarian tissues,mi R-21 is highly expressed in ovarian cancer tissues,and is closely related to histological grade and lymph node invasion and metastasis.4.M2 macrophages co-cultured with ovarian cancer cells can promote chemoresistance.Transfected mi R-21 mimic into M2 macrophages and then co-cultured with ovarian cancer cells could promote ovarian cancer cells chemoresistance,inhibit ovarian cancer cells apoptosis,and promote S phase arrest of cell cycle,while transfected the mi R-21 inhibitor showed the opposite biological behaviors.5.Transfection of mi R-21 mimic in macrophages can promote its transformation into M2 macrophages,while transfection of mi R-21 inhibitor can promote its transformation into M1 macrophages.6.Ovarian cancer cells co-cultured with M2 macrophages and M2 macrophages treated with mi R-21 could regulate the chemoresistance of ovarian cancer cells by regulating the PI3K/AKT signaling pathway. |
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