Secretory mechanisms and intercellular transfer of microRNAs in living cells

Abstract

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.

FIGURE 1.

Characterization of extracellular microRNAs. A, miRNAs were released in proportion to the cellular expression level. HEK293 cells were set up at a density of 2.5 × 10⁵ cells/well in a 24-well plate. The following day, the cells were transfected with 0.5 μg of the indicated primary miRNA expression vectors or pCMV empty vector as a control as described under “Experimental Procedures.” Twenty-four h later, after washing three times by Advanced RPMI containing antibiotic-antimycotic and 2 mm l-glutamine (medium A), culture medium was switched to medium A. After a 24-h incubation, preparation of conditioned medium and isolation of total RNAs were performed as described under “Experimental Procedures.” Expression levels of miRNAs were analyzed using quantitative real-time PCP (QRT-PCR). B, siRNAs targeting luciferase gene (siLuc) were secreted into culture medium. HEK293 cells were set up at a density of 2.5 × 10⁵ cells/well in a 24-well plate. Transfection with psiRNA-LucGL3 vector (+) or control vector (−) and preparation of conditioned medium were conducted as described above. The amount of luciferase siRNAs generated from the expression vector was measured by QRT-PCR with a custom-designed TaqMan small RNA Assay (Applied Biosystems) specific for the luciferase siRNAs. C, time course analysis of extracellular miRNAs. Conditioned medium was collected at the indicated time and applied to quantitative miRNA RT-PCR. A–C, cel-miR-39 and hRNU6 were used as invariant control for conditioned medium and cell, respectively. Each bar is presented as mean S.E. (n = 3).

FIGURE 2.

Purification and characterization of secretory exosomes. A, purified exosomes secreted from HEK293 cells are enriched in CD63 protein. The exosome fractions and whole cell lysate were analyzed by immunoblotting with an anti-CD63 antibody or anti-β-actin antibody. B, total RNAs isolated from conditioned medium, exosome fractions, and their donor HEK293 cells were detected using a Bioanalyzer 2100. The obtained data show the amount and the size distribution of total RNAs. The peak migrating around 25 nucleotides (nt) represents an internal standard. FU, fluorescence units. C, effect of RNase treatment on extracellular miR-21. Conditioned medium from HEK293 cells was exposed to RNase A and RNase T1 at 37 °C followed by the isolation of miRNAs at the indicated times. The amount of miR-21 was determined using quantitative miRNA RT-PCR. The values on the y axis are depicted relative to the amount of miR-21 at 0 min, which is arbitrarily defined as 1. D and E, CD63⁺ exosome fraction is enriched in miR-155 and miR-16 (D) and luciferase siRNA (E). Conditioned medium of HEK293 cells was immunoprecipitated with anti-CD63 antibody or isotype control. The values on the y axis are depicted relative to control, which is arbitrarily defined as 1. C–E, each bar is presented as mean S.E. (n = 3). n.s. represents not significant.

FIGURE 3.

Secretion of miRNA was attenuated by GW4869. A, secretion of miRNAs was suppressed by the treatment with GW4869. HEK293 cells were transfected with pri-miR-146a vector in a 6-well plate. The cells were reseeded and cultured in a 24-well plate for 48 h in the indicated concentrations of GW4869. After the incubation, the medium was subjected to QRT-PCR for miR-16 and -146a. The values on the y axis are depicted relative to the amount of each miRNA at 0 μm GW4869, which is arbitrarily defined as 1. B, after the treatment with the indicated concentrations of GW4869 for 24 h, the total amounts of proteins in the exosomal pellet purified from large scale cultures of stably miR-146a-transduced HEK293 cells were quantified by a BCA assay and are presented as the values per 10 million secreting cells. A and B, each bar is presented as mean S.E. (n = 3). C, purified exosomes secreted by equal numbers of control or GW4869-treated HEK293 cells and equal amounts of total exosomal proteins (quantified by BCA assay) secreted by either control or GW4869-treated HEK293 cells were analyzed by immunoblotting for the presence of CD63 protein. (*, p < 0.05, **, p < 0.005, as compared with untreated cells; Student's t test).

FIGURE 4.

miRNAs were secreted through nSMase2-dependent pathway. A and B, siRNA-mediated knockdown of nSMase2. HEK293 cells were transfected with pri-miR-146a vector in a 6-well plate. One day later, these cells were reseeded in a 24-well plate and transfected with either negative control or nSMase2 siRNA. The following day, conditioned medium and cell extract were applied to QRT-PCR for miR-16 as well as miR-146a (B). In parallel, cell extract was applied to immunoblot for nSMase2 and β-actin proteins (A). The values on the y axis are depicted relative to the amount of miRNAs of control, which is arbitrarily defined as 1. C and D, augmentation of miRNA secretion by overexpressed human nSMase2. HEK293 cells were transfected with human nSMase2 expression vector or control vector, along with pri-miR-146a vector. After a 24-h incubation, conditioned medium and cell extract were applied to QRT-PCR (D), and cell extract was applied to immunoblot (C). The values on the y axis are depicted relative to the amount of miRNAs of nSMase2−, which is arbitrarily defined as 1. B and D, each bar is presented as mean S.E. (n = 3). (*, p < 0.05, **, p < 0.005, as compared with control cells; Student's t test); n.s. represents not significant.

FIGURE 5.

The secretion of miRNAs does not require ESCRT system. A, knockdown of human Alix protein. HEK293 cells transfected with negative control siRNA or Alix siRNA were harvested and applied to immunoblot for Alix and β-actin proteins. B, the depletion of Alix impaired miRNA activity. Upper schematic, our sensor vector possesses Renilla luciferase and firefly luciferase for assessing the miRNA activity and for normalizing transfection efficiency, respectively. Lower graph, HEK293 cells were transfected with 0.1 μg of miR-146a sensor vector and the indicated siRNAs together with 200 or 0 pm pre-miR-146a in a 96-well plate. The following day, the cells were applied to a Dual luciferase reporter assay. The values on the y axis are normalized Renilla luciferase activity. C and D, cellular and extracellular miR-146a were not affected by Alix siRNA. MiR-146a stably overexpressing HEK293 cells were transfected with the indicated siRNAs. After the medium switch, the cells were cultured for another 24 h, and the cell lysate (C) and conditioned medium (D) were then applied to miRNA QRT-PCR analysis. The values on the y axis are depicted relative to control siRNA, which is arbitrarily defined as 1. B–D, each bar is presented as mean S.E. (n = 3). (*, p < 0.05, **, p < 0.005, as compared with control siRNAs; Student's t test); n.s. represents not significant.

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.

FIGURE 7.

Secretory tumor-suppressive miRNAs attenuated PC-3M cell proliferation. A, schematic representation of a cell proliferation assay. B, cell growth inhibition by synthetic miR-146a and extracellular miR-146a. PC-3M-luc cells were transfected with synthetic miRNAs (left graph) or incubated in the conditioned medium containing extracellular miRNAs at the indicated concentrations (right graph) followed by cell growth assay as described under “Experimental Procedures.” C, miR-146a-mediated ROCK1 suppression in PC-3M-luc cells. The final concentrations of synthetic and extracellular miR-146a are 10 and 21 pm, respectively. D, the treatment with GW4869 to donor cells restored the reduced cell growth by the exosomal miR-146a. Donor COS-7 cells transfected with pri-miR-146a expression vector were incubated in the presence (lane 2) or absence of 10 μm GW4869 for 3 days. The conditioned medium from COS-7 cells transfected with empty vector was used as a control (lane 1). GW4869-untreated conditioned medium enriched in miR-146a was divided into two aliquots, one of which was treated with 10 μm GW4869 (lane 3) and the other of which was not treated (lane 4) before the transfer to recipient cells. The following assay was conducted as described above. B and D, the values on the y axis are depicted relative to normalized Renilla luciferase activity of control cells, which is defined as 100%. Each bar is presented as mean S.E. (n = 3). (*, p < 0.05, **, p < 0.005, as compared with untreated PC-3M-luc cells; Student's t test); n.s. represents not significant.