Study On The Mechanism Of Macrophages Regulating Ovarian Cancer Cell Metastasis Induced By TWEAK Via Exosomes

Part one Macrophages inhibit ovarian cancer cell metastasis induced by TWEAK via exosomesObjective: To investigate whether macrophages could inhibit ovarian cancer cell metastasis induced by TWEAK via exosomes.Methods: Macrophages, which were differentiated from THP-1 by PMA, were stimulated by TWEAK for 24 h. Exosomes were isolated from macrophages' conditioned medium by exosome isolation kit and were then detected by transmission electron microscopy. To evaluate whether secreted exosomes could be taken up by recipient cells, the isolated exosomes labeled with PKH67(green fluorescent membrane linker-dye), were incubated with the ovarian cancer cell line SKOV3 for 12 h before the confocal laser scanning microscopy were captured. The ovarian cancer cell lines SKOV3 and HO-8910 PM were separately cocultured with PBS, exosomes secreted by macrophages and exosomes secreted by TWEAK-treated-macrophages for 48 h respectively, and then were detected the ability of migration and invasion by using transwell methods.Results: Exosomes purified from macrophages' conditioned medium exhibited round morphology by electron microscopy and a size range of 30 to 100 nm. According to the results of the confocal microscopy, the labeled exosomes secreted by the macrophages, were located in the cytoplasm of SKOV3 cells, especially encircled the nucleus, and could be taken up by the recipient cells. Compared with the blank group, the ability of migration and invasion of ovarian cancer cells, stimulated by exosomes secreted by macrophages, was significantly increased(P<0.05). However, once treated by TWEAK, the macrophages reduced the ability of migration and invasion of ovarian cancer cells via their secreted exosomes.Conclusions: The ovarian cancer cells could internalize the exosomes secreted by the macrophages, TWEAK could reverse the ability of macrophages to promote ovarian cancer cell metastasis via exosomes.Part two Identify differentially expressed mi RNAs between the exosomes secreted by macrophages and those secreted by TWEAK-treated-macrophagesObjective: To determine the mi RNA profiles and to identify the differentially expressed mi RNAs between the exosomes secreted by macrophages and those secreted by TWEAK-treated-macrophages.Methods: Macrophages, which were differentiated from THP-1 by PMA, were stimulated by TWEAK for 24 h. Exosomes were isolated from macrophages' conditioned medium by exosome isolation kit. The exosomes' RNA were extracted and quantified, and then were assessed using Agilent microarray technologies to identify the differentially expressed mi RNAs.Results: According to the microarray analysis, there were 55 mi RNAs upregulated and 17 mi RNAs downregulated when compared the exosomes secreted by TWEAK-treated-macrophages with those secreted by macrophages. Among the above differentially expressed mi RNAs, 15 upregulated ones including mir-7, mir-148 a, mir-140 and so on, and 4 downregulated ones were viewed as fuctional according to the reported studies.Conclusions: Once treated by TWEAK, the exosomes secreted by macrophages could exhibit a totally different mi RNA profiles, and we choose mir-7 as the targeted mi RNA to further our study.Part three Investigate the mechanism of macrophages inhibiting epithelial ovarian cancer cell metastasis induced by TWEAK via exosomesObjective: To investigate the mechanism of macrophages inhibiting epithelial ovarian cancer cell metastasis induced by TWEAK via exosomes.Methods: Macrophages, which were differentiated from THP-1 by PMA, were stimulated by TWEAK for 24 h. Exosomes were isolated from macrophages' conditioned medium by exosome isolation kit. The ovarian cancer cell lines SKOV3 and HO-8910 PM were separately cocultured with PBS, exosomes secreted by macrophages and exosomes secreted by TWEAK-treated-macrophages. After incubation for 24 h, RNAs were extracted from the ovarian cancer cells and were then detected the levels of mir-7 using Real-time PCR. After incubation for 48 h, proteins were extracted and were then detected the expression of EGFR and the phosphorylation of its downstream AKT and ERK signaling pathways.Results: The results of Real-time PCR indicated that exosomes secreted by TWEAK-treated-macrophages could upregulate the levels of mir-7 in both two ovarian cancer cells. The results of Western Blot also showed that exosomes secreted by TWEAK-treated-macrophages could inhibit the expressions of EGFR and downregulate the Phosphorylated forms of AKT and ERK, while their total forms were kept unchanged.Conclusions: Once treated by TWEAK, the exosomes secreted by macrophages could upregulate the levels of mir-7 and downregulate the expressions of EGFR, causing the inactivation of its downstream AKT and ERK signaling pathways in the ovarian cancer cells.