Salido-Guadarrama I, Romero-Cordoba S, Peralta-Zaragoza O, Hidalgo-Miranda A, Rodriguez-Dorantes M

Salido-Guadarrama I, Romero-Cordoba S, Peralta-Zaragoza O, Hidalgo-Miranda A, Rodriguez-Dorantes M. transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity. = 4, * Strontium ranelate (Protelos) 0.05. D. 18= 5, * 0.05. F. Expression of dominant-negative mutant Cx43-T154A in U87MG cells decreased gap junction dye coupling. Scale bar, 10 = 4, * 0.05. Glioma-astrocyte and astrocyte-astrocyte gap junctions promote glioma invasion Having established the inhibitory role of glioma-glioma gap junctions in invasion, we next investigated the contribution of glioma-astrocyte and astrocyte-astrocyte gap junctions to glioma invasion. To mimic the glioma microenvironment in which glioma cells are surrounded by astrocytes, normal human astrocytes were co-cultured with the glioma cells in the matrigel transwell, and the invasive behavior of the glioma cells was assessed. Compared to glioma monoculture, the invasive index of glioma cells is significantly increased when co-cultured with astrocytes (Figure ?(Figure2A).2A). This pro-invasive effect could be mediated by a variety of mechanisms, including glioma-astrocyte gap junctional contacts. Open in a separate window Figure 2 Effects of glioma-astrocyte and astrocyte-astrocyte gap junctions on glioma invasionA. Co-culture with astrocytes (AST) promotes U87MG cell invasion. Mean SEM, = 5, * 0.05. B. Knockdown of Cx43 in U87MG cells by siRNAs (which inhibit glioma-glioma and glioma-astrocyte communication) did not affect glioma invasion in astrocyte co-culture. Mean SEM, = 4, * 0.05. C. Strontium ranelate (Protelos) Expression of Cx43-T154A in U87MG cells did not affect glioma invasion in astrocyte co-culture. Mean SEM, = 4, * 0.05. D. 18= 3, * 0.05. I. Summary of different treatments to manipulate gap junction function and the final effect on glioma invasion. To explore the latter, we downregulated gap junction function in U87MG cells by siRNA-Cx43 or Cx43-T154A in this co-culture system. In addition to glioma-glioma gap junctions, glioma-astrocyte gap junctions are also blocked by these methods, but astrocyte-astrocyte Strontium ranelate (Protelos) gap junctions are unaffected. In contrast to the effects in glioma monoculture, the siRNAs and T154A expression had no effect on glioma invasion in the co-culture system (Figure ?(Figure2B2B and Figure ?Figure2C).2C). Since our previous results demonstrated that inhibition of glioma-glioma gap junctions promotes glioma invasion (Figure ?(Figure1),1), the combined null effect on glioma invasion when, in addition, glioma-astrocyte gap junctions are TNFRSF4 blocked indicates that inhibiting glioma-astrocyte communication counteracted the pro-invasive effect of blocking glioma-glioma gap junctions. Therefore, we infer that glioma-astrocyte gap junctions promote glioma invasion. To investigate the effect of astrocyte-astrocyte coupling in glioma invasion, we applied 18that is not found in human cells, was employed as a tracer. U87MG cells were pre-loaded with cel-miR-67 by electroporation and co-cultured with astrocytes in a ratio of 1 1:1. The two types of cells were labeled with different Vybrant? cell-labeling dyes, co-cultured for 24 h, and then separated by flow cytometry (Figure ?(Figure3A).3A). We detected a significant level of cel-miR-67 in astrocytes after co-culture, which was blocked by the gap junction inhibitor 18= 3, * 0.05. C. Normalized cel-miR-67 level in U87MG cells after co-culture with astrocytes loaded with cel-miR-67. The Cx43-T154A mutant greatly reduces transfer of cel-miR-67 to astrocytes. Mean SEM, = 3, * 0.05. MicroRNA profile of astrocytes is altered by glioma cells The above results raise the possibility that glioma cells deliver miRNAs to astrocytes via gap junctions, and that these miRNAs or their downstream effects subsequently spread among astrocytes via gap junctions, which amplify the pro-invasive effect. To identify potential candidate miRNAs that may be transferred to astrocytes, we first identified the miRNAs that are increased in astrocytes after co-culture with U87MG glioma cells by miRNA profiling. Total RNA from astrocytes before and after co-culture were analyzed using the 0.01; log2 fold change range from 7.74-0.40) (Figure ?(Figure4A).4A). We selected 25 of the 54 miRNAs for validation by real-time qPCR. The criteria for the selected miRNAs are: 1) fold change after co-culture; 2) deep sequencing data from miRBase, which indicates the annotated confidence of each miRNA [34]; 3) the number of.