关键词: MiRNA-550a, 肺癌, 巨噬细胞, 外泌体

Abstract: Objective Investigating the cellular communication between Lewis lung adenocarcinoma cells (LLC) and macrophages, and exploring the possible regulatory mechanism of LLC on macrophage phenotypic transformation. Methods The normal alveolar epithelial cells (nAECs) and LLCs were co-culturing with macrophages by Transwell technique. The experiment was divided into two groups: control group and cancer cell stimulation group, in which the macrophages in lower ventricular were stimulated by LPS. After 7 days of co-culture, immunofluorescence assay was used to detect the changes of macrophage surface markers and phenotypic transformation; ELISA was used to detect the expression of inflammatory related factors IL-1β、TGF-α、IL-10 and TGF-β in the supernatant of the macrophages in two groups; Exosomes were identified by transmission electron microscopy (TEM) and Western blotting; The expression of microRNA was detected by RT-PCR in two groups. The LLC was transfected with siRNA-550a and siRNA-182 by RNAi, and then inflammatory factors expression was detected by ELISA kit. Results In the co-culture system, the expression of CD68 was high and CD163 was low in the control group (P<0.05), macrophages were M1; While in the cancer stimulation group CD68 was low and CD163 was high (P<0.05), it proved phenotype of macrophages was transformed into M2. Compared with the control group, the expression of pro-inflammatory factors IL-1β and TGF-α were high, and the expression of anti-inflammatory factors IL-10 and TGF-β were low in the cancer stimulation group (P<0.05); The results of TEM, particle size analysis and Western blotting showed there were exosomes in the supernatant of the LLC, and PKH26 staining result showed that macrophages exerted obvious phagocytic effect on exosomes. Compared with nAECs, the contents of miR-550a and miR-182 in exosomes of LLC were significantly higher (P<0.05). After transfected with siRNA-550a, the LLC exosomes significantly promoted IL-1β and TGF-α secretion of macrophages, while the levels of IL-10 and TGFβ were lower compared to siRNA-182 group (P<0.05). Conclusion LLC can transform phenotype of macrophages in tumor microenvironment into cancer supporting cells’ through secreting exosomes. The mechanism may be that MiRNA-550a in exosomes can regulate macrophages phenotype transformation.

Key words: MiRNA-550a, lung cancer, macrophages, exosomes