Functional microRNA is transferred between glioma cells

Abstract

MicroRNAs (miRNA) are single-stranded 17- to 27-nucleotide RNA molecules that regulate gene expression by posttranscriptional silencing of target mRNAs. Here, we transformed rat 9L gliosarcoma cells to express cel-miR-67, a miRNA that lacks homology in rat. Coculture of these cells with cells that expressed a luciferase reporter that contained a complementary sequence to cel-miR-67 resulted in significant suppression of luciferase expression. This effect was also observed in the U87-MG human glioma cell line. Moreover, luciferase suppression was inhibited by the addition of carbenoxolone to cocultures, suggesting that gap junction communication regulates intercellular transfer of miRNA. Finally, in situ hybridization revealed the presence of cel-miR-67 in cel-miR-67-null 9L cells after coculture with cel-miR-67-expressing cells. Our data show that miRNA transcribed in glioma cells can be transferred to adjacent cells and induces targeted inhibition of protein expression in the acceptor cells. These findings reveal a novel mechanism of targeted intercellular protein regulation between brain tumor cells.

Figure 1

Cel-miR-67 expressing 9L gliosarcoma cells suppress luciferase expression in, and transfer cel-miR-67 to, neighboring cel-miR-67-negative cells by a gap junction-dependent mechanism. A, Luciferase detected in 9L-Luc cells co-cultured with 9L cells transfected with an empty expression vector (9L-mirNeg), or one encoding for cel-miR-67 (9L-mir67) or cel-miR-239 (9L-mir239). n = 4, ANOVA, P = 0.004. B, Luciferase detected in 9L-Luc67 cells co-cultured with 9L cells stably expressing either cel-miR-67 (9L-mir67s), cel-miR-239 (9L-mir239s) or the empty expression vector (9L-mirNegs). n = 4, ANOVA, P = 0.006. C, Luciferase detected in 9L-Luc67 cells co-cultured with 9L-mir67s, 9L-mir239s, or 9L-mirNegs, in the presence of 150 μM carbenoxolone, a gap junction antagonist. n = 4, ANOVA, P = 0.459. Error bars represent ± s.d. Post-hoc multiple comparisons are two-tailed t-tests.

Figure 2

Merged fluorescent and phase-contrast images reveal co-localization of cel-miR-67 in situ signal with eGFP 9L cells. A, No detection of cel-miR-67 in 9L-eGFP/9L-mir239 co-cultured cells (negative control). B, Detection of rno-miR-21 in 9L-eGFP/9L-mir67 co-cultured cells (positive control). C,D Detection of co-localized cel-miR-67 and eGFP in 9L-eGFP/9L-mir67 co-cultured cells. Red arrowheads indicate positive in situ signal. Bar = 25 μm.

Figure 3

U87-MG human glioma cells expressing cel-miR-67 suppress luciferase expression in neighboring cel-miR-67-negative cells, but U251 cells expressing cel-miR-67 do not. A, Luciferase detected in U87-Luc cells co-cultured with U87-MG cells transfected with an empty expression vector (U87-mirNeg), or one encoding for cel-miR-67 (U87-mir67), or cel-miR-239 (U87-mir239). n = 5, ANOVA, P = 0.003. B, Luciferase detected in U251-Luc cells co-cultured with U251 cells transfected with an empty expression vector (U251-mirNeg), or one encoding for cel-miR-67 (U251-mir67) or cel-miR-239 (U251-mir239). n = 5, ANOVA, P = 0.903. Error bars represent ± s.d. Post-hoc multiple comparisons are two-tailed t-tests.