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. 2010 Mar 30;285(23):17442–17452. doi: 10.1074/jbc.M110.107821

FIGURE 6.

FIGURE 6.

Secretory small RNAs can be transferred to recipient cells. A, treatment with conditioned medium enriched in luciferase siRNA down-regulated luciferase activity. HEK293 cells stably transduced with firefly luciferase were incubated with the indicated medium for 3 days. The conditioned medium was prepared from HEK293 cells transfected with psiRNA-LucGL3 or empty vector. The luciferase siRNA was not detected in RPMI medium and control CM as well as miR-146a CM using QRT-PCR, whereas luciferase siRNA (siLuc) CM contained 200 pm luciferase siRNA as calculated under “Experimental Procedures.” The concentration of miR-146a in miR-146a CM was 123 pm. The cells were harvested and applied to luciferase reporter assay. The values on the y axis are depicted relative to firefly luciferase activity of RPMI medium-treated cells, which is defined as 100%. B, the diagram shows a reporter assay for testing the direct regulation of target gene expression by extracellular miRNAs. C, extracellular miR-146a derived from COS-7 and HEK293 cells suppressed luciferase activity of the sensor vector. COS-7 cells transfected with miR-146a sensor vector were used as recipient cells. The recipient cells were incubated in the conditioned medium containing extracellular miRNAs of the indicated concentrations. After a 2-day incubation, luciferase reporter assay was performed as described under “Experimental Procedures.” D, miR-146a did not reduce luciferase activity from the mutated sensor vector. COS-7 cells transfected with the mutated miR-146a sensor vector were used as recipient cells. The recipient cells were transfected with synthetic miRNAs (left graph) or incubated in the conditioned medium containing extracellular miRNAs at the indicated concentrations (right graph). Luciferase assay was carried out as described above. C and D, the values on the y axis are depicted relative to normalized Renilla luciferase activity of control cells, which is defined as 100%. A, C, and D, each bar is presented as mean S.E. (n = 3). (*, p < 0.05, **, p < 0.005, as compared with control recipient cells; Student's t test); n.s. represents not significant.