Irradiated microglia exhibited M1-type polarization and hampered A549 cells MET transition
To explore the impact of irradiation on microglial phenotype, CHME5 cells were treated with 0, 2, 3, 4 Gy of irradiation. Then the mRNA levels of iNOS and IL10 as the markers for M1 and M2 types, respectively, were measured. There was a noticeable up-regulation in iNOS expression and down-regulation of IL 10 expression in irradiated microglia, particularly in the group treated with 3 Gy (Fig. 1A). Western Blot analysis revealed up-regulation of iNOS expression and down-regulation of Arg1 expression after treatment with 3 Gy irradiation (Fig. 1B). Further analysis showed that the effects of irradiation were extremely obvious at 48 h using qRT-PCR and Western Blot (Fig. 1C and 1D). Immunofluorescence staining results were consistent with these observations (Fig. 1E).
Next, we plan to explore the role of irradiated M1 microglia on the phenotypical modulation of NSCLC cells. The expression of E-cadherin and Vimentin in A549 cells was analyzed by Western Blot, both of which vital markers of epithelial/mesenchymal phenotype. In the group with control culture media, the mesenchymal phenotype A549 cells quickly reversed into epithelial type with high E-cadherin and low Vimentin expression. In contrast, in the group treated with irradiated CHME5 supernatant, A549 cells retained their mesenchymal phenotype with low E-cadherin and high Vimentin expression. Introduction to these groups of CHME5 supernatant without irradiation or U87 supernatant did not affect the observed phenotype changes (Fig. 1F).
Irradiation-induced M1 microglia increased both intra and extra-cellular miR-9 level
Several recent studies indicated microRNAs played crucial roles in regulation of EMT/MET process[26, 27]. Thus, we analyzed microRNA expression in LPS-treated group and control groups. miR-9 levels increased dramatically in LPS-induced M1 microglia (Fig. 2A). We confirmed that the level of miR-9 was significantly elevated in irradiated CHME5 and HMO6 cells (3 Gy and 3 Gy*2f), when compared with non-irradiated microglia, both intracellularly (Fig. 2B and 2D) and extracellularly (Fig. 2C and 2E). These results showed that miR-9 expression was up-regulated in irradiated microglia and it was secreted into the extracellular space, suggesting that miR-9 secreted by irradiated microglia played an important role in inducing a mesenchymal phenotype in metastatic A549 cells.
Irradiated M1-type microglia elevated intracellular level of miR-9 in A549 cell lines
To confirm the effects of miR-9 produced by irradiated M1 microglia on the MET process, we modulated the expression of miR-9 in A549 or brain metastatic A549-F3 cells, using an miR-9 mimic and inhibition of plasmid transduction (Fig. 3A and 3B). Levels of intracellular miR-9 were detected via qRT-PCR in each of three groups: negative control, over-expression, and inhibition group. Significantly elevated/decreased miR-9 levels were detected in the overexpression/inhibition group respectively, confirming the success of in vitro miR-9 expression modulation in A549 and A549-F3 cells (Fig. 3C and 3D).
We added irradiated or non-irradiated microglia culturing supernatant into A549 and A549-F3 cells transfected with control/miR-9 mimic/miR-9 inhibition plasmid, respectively, observing the highest level of miR-9 in miR-9 mimic group treated with irradiated conditioned medium. In contrast, the lowest miR-9 level were detected in the miR-9 inhibition group, where the non-irradiated microglia supernatant was added (Fig. 3E and 3F). When the miR-9 inhibition A549/A549-F3 group was treated with irradiated microglia supernatant, their intracellular levels of miR-9 were significantly higher than those in the miR-9 inhibition group treated with non-irradiated microglia supernatant. This result suggests that irradiated M1 microglia increased their production and secretion of miR-9; and so, the uptake of miR-9 by A549 and A549-F3 was increased. This absorbed miR-9 might play a role in reducing NSCLC brain metastasis.
Irradiated M1 type microglia inhibited the MET via miR-9/CDH1
To explore the influence of miR-9 levels on phenotypic conversions of A549 and A549-F3 cells we conducted Western blot analysis. In Fig. 4A, groups with controlled culture media and negative plasmid the mesenchymal phenotype A549 cells quickly reversed into epithelial-type with high E-cadherin and low Vimentin expression. In contrast, groups with up-regulated miR-9 retained their mesenchymal phenotype (low E-cadherin and high Vimentin expression). Among groups with up-regulated miR-9, those treated with irradiated microglia supernatant retained the most typical mesenchymal phenotype. Meanwhile, miR-9 downregulation could promote E-cadherin expression. Furthermore, A549 cells with down-regulated miR-9 treated with non-irradiated microglia supernatant transformed into the most typical epithelial phenotype (the highest E-cadherin and the lowest Vimentin expression). Consistent with the above, A549-F3 cells transfected with down-regulated miR-9 plasmid were able to develop an epithelial phenotype, while irradiated microglia supernatant could inhibit the MET process to keep the A549-F3 cells in a mesenchymal state. Meanwhile, A549-F3 cells with up-regulated miR-9 treated with irradiated CHME5 supernatant maintained the most typical mesenchymal phenotype (Fig. 4B).
Given that, miRNAs could inhibit gene expression by binding to the 3’UTR of respective RNAs , WT and MUT of CDH1 3’UTR-driven luciferase vectors were respectively cotransfected with NC or miR-9-5p mimics into 293T cells (Fig. 4C). Results indicated cotransfection with miR-9-5p mimics and WT CDH1 3’-UTR caused inhibition of luciferase activity. Moreover, cotransfection of miR-9-5p mimics and MUT CDH1 3’-UTR had no effects on luciferase activity Fig. 4D). These findings point to CDH1 being the target of miR-9.
Low dose irradiation reduced brain metastases of A549-F3 cells in brain mice model
To confirm the effects of irradiation on NSCLC-BM in vivo, we selected 7 days after irradiation to inject tumor cells for the higher level of miR-9 (Fig. S1). We use bioluminescence imaging (BLI) to assess the incidence rate of BM in a mouse model (Fig. 5A and 5B). BM incidence was reduced in irradiated mice (40%, n = 10), when compared to the control group (70%, n = 10) (Fig. 5C). There are significant differences between the two groups by analysis of the photon flux (Fig. 5D). Furthermore, irradiation increased miR-9 levels in mice brain microenvironment (Fig. 5E). These findings suggest that, in a mouse model, low dose irradiation reduces A549-F3 cells-mediated brain metastases, plausibly by elevating miR-9 expression levels, which inhibit tumor cells MET in brain microenvironment.