Fig. 6.

Ionizing radiation stimulates extracellular vesicle miR-603 export. (a) Western blotting detection of EV markers in EVs secreted by BT-83 cells with or without IR treatment (6 Gy). The EV markers CD9, CD81, TSG101, and HSC70 were used to identify the presence of EVs, ApoA1 was served as an EV negative marker, and α-tubulin was used as an intracellular marker not incorporated into EVs. EV pellets were run with equivalent total numbers of EV particles, as determined by NTA. Cell pellets were run with equivalent total protein input amounts, as determined by α-tubulin levels. (b) IR lowered the number of released EVs in BT-83 and LN340 cells. Culture medium of BT-83 cells (top panel) and LN340 cells (bottom panel) with or without IR treatment was collected and subjected to EV isolation and EV quantification. *p < 0.05 versus unirradiated control group (Student's t-test). (c) and (d) IR increased extracellular secretion of EV-miR-603. miR-603 copy number per EV (c) and total miR-603 copy number (d) were compared between unirradiated control group and irradiated group. *p < 0.05, **p < 0.01 versus unirradiated control group (Student's t-test). (e) EVs secreted by irradiated or unirradiated BT-83 cells were isolated using ExoQuick-TC ULTRA EV isolation kit for tissue culture media and incubated with the indicated treatments before isolating RNA and measuring the levels of miR-603. The EV pellets were incubated with RNase A (0.5 µg/µl) alone or were incubated with proteinase K (0.4 µg/µl) prior to RNase treatment in the presence or absence of 2% Triton X-100. ***p < 0.001, ^n.s.p > 0.05 between indicated groups (one-way ANOVA). (f) Representative fluorescence microscopy images for PKH67-labelled EVs uptake by human microglia HMC3 cells after 24 h incubation. Green: PKH67; red: Alexa Fluor 594 phalloidin; blue: DAPI. Scale bar: 25 µm. (g) Pharmaceutical blocking of EV secretion by GW4869 repressed IR-induced miR-603 reduction in GBM cells. BT-83 cells (left panel) and LN340 cells (right panel) were pretreated with GW4860 (10 µM, 24 h) or DMSO before receiving IR treatment. Cells were collected at indicated time points and subjected to miR-603 quantification using qPCR. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to DMSO group (Student's t-test). (h) Repression of EV secretion through Rab27a knockdown rescued IR-induced cellular miR-603 decrease. BT-83 cells (left panel) and LN340 cells (right panel) were transfected with siRNA targeting Rab27a (siRab27a) or negative control siRNA (siNT) before receiving IR treatment. Cells were collected at indicated time points and the level of miR-603 was measured by qPCR. *p < 0.05 versus siNT-transfected group (Student's t-test).