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. 2019 Jun;110(6):1947-1958.
doi: 10.1111/cas.14029.

Pattern of cell-to-cell transfer of microRNA by gap junction and its effect on the proliferation of glioma cells

Yuexia Peng  1 , Xiyan Wang  2 , Yunquan Guo  2 , Fuhua Peng  1 , Ningze Zheng  1 , Bo He  3 , Hui Ge  2 , Liang Tao  1 , Qin Wang  1
Affiliations

Pattern of cell-to-cell transfer of microRNA by gap junction and its effect on the proliferation of glioma cells

Yuexia Peng et al. Cancer Sci. 2019 Jun.

Abstract

MicroRNA is expected to be a novel therapeutic tool for tumors. Gap junctions facilitate the transfer of microRNA, which exerts biological effects on tumor cells. However, the length of microRNA that can pass through certain gap junctions composed of specific connexin remains unknown. To address this question, the present study investigated the permeability of gap junctions composed of various connexins, including connexin 43, connexin 32 or connexin 37, to microRNAs consisting of 18-27 nucleotides in glioma cells and cervical cancer cells. Results indicated that all of the microRNAs were able to be transferred from donor glioma cells to neighboring cells through the connexin 43 composed gap junction, but not the gap junctions composed of connexin 32 or connexin 37, in cervical cancer cells. Downregulation of the function of gap junctions comprising connexin 43 by pharmacological inhibition and shRNA significantly decreased the transfer of these microRNAs. In contrast, gap junction enhancers and overexpression of connexin 43 effectively increased these transfers. In glioma cells, cell proliferation was inhibited by microRNA-34a. Additionally, these effects of microRNA-34a were significantly enhanced by overexpression of connexin 43 in U251 cells, indicating that gap junctions play an important role in the antitumor effect of microRNA by transfer of microRNA to neighboring cells. Our data are the first to clarify the pattern of microRNA transmission through gap junctions and provide novel insights to show that antitumor microRNAs should be combined with connexin 43 or a connexin 43 enhancer, not connexin 32 or connexin 37, in order to improve the therapeutic effect.

Keywords: connexin; gap junction; glioma; microRNA; proliferation.

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Figures

Figure 1
Figure 1
MicroRNA (miRNA) mimics composed of 18‐27 nucleotides transferred between cocultured glioma U87 cells. A, U87 cells (donor cells) were transfected with Cy3‐miR‐34a mimics and cocultured with U87‐GFP (receiving cells). Then, the cocultured cells were analyzed by confocal microscopy. Scale bar, 10 μm. B, Delivery of Cy3‐miR‐34a by U87 donor cells to U87‐GFP receiving cells was analyzed using flow cytometry. a, Double negative cocultured cells; b, cocultured receiving cells, U87 cells stably transfected with GFP (U87‐GFP); c, cocultured donor cells, U87 cells transfected with Cy3‐miR‐34a; d, cocultured cells with GFP and Cy3 positive. C, Expression of miRNAs in the receiving cells was detected by qPCR before and after the coculture. Columns represent the means of four experiments; bars represent SEM. **P < .01
Figure 2
Figure 2
Gap junctions mediated the delivery of microRNAs (miRNAs) between glioma U87 cells. A, Parachute assay shows the degree of dye coupling in U87 cells. B, Expression of miRNAs in the receiving cells was assessed by qPCR after the coculture. Columns represent the means of four experiments; bars represent the SEM. *P < .05, **P < .01. 18a‐GA, 18α‐glycyrrhetinic acid; CBX, carbenoxolone; RA, retinoic acid
Figure 3
Figure 3
Effect of shRNA‐mediated knockdown of connexin 43 (Cx43) expression on the transfer of microRNAs (miRNAs) between U87 cells. A, mRNA expression of Cx43 in U87 cells after the knockdown of Cx43 expression. B,C, Expression of Cx43 in U87 cells with shRNA stable transfection. GFP labeled U87 cells as the coculture receiving cells. D,E, Function of gap junctions in Cx43‐deficient cells (U87ShCx43) was analyzed by parachute assay. Scale bar, 10 μm. F, Compared with the control group, knockdown of Cx43 markedly decreased the expression of miRNAs in the receiving cells. Columns represent the means of four experiments; bars represent the SEM. **P < .01
Figure 4
Figure 4
Effect of the overexpression of connexin 43 (Cx43) on the transfer of microRNAs (miRNAs) between U251 cells. A, Western blot analysis confirmed stable overexpression of Cx43 in U251 cells. GFP was used to label U251 cells as the coculture receiving cells. B, Function of gap junctions in U251 cells with overexpression of Cx43. Scale bar, 10 μm. C, Expression of miRNAs was not significantly different before and after coculture in the receiving cells. D, Compared with the control group, overexpression of Cx43 remarkably increased the expression of miRNAs in the receiving cells. Columns represent the means of five experiments; bars represent the SEM. **P < .01
Figure 5
Figure 5
Ability of gap junctions composed of connexin 32 (Cx32) or connexin 37 (Cx37) to transfer microRNAs (miRNAs) between cervical cancer cells. A, Expression of Cx32 in HeLa‐Cx32 cells after 48 h of doxycycline (Dox) treatment. B, Function of gap junctions in HeLa‐Cx32 cells with induction of Cx32 expression. Scale bar, 10 μm. C, Expression of miRNAs in receiving cells was analyzed by qPCR. D, Expression of Cx37 in HeLa cells with Cx37 plasmid stable transfection. GFP was labeled in HeLa cells as the coculture receiving cells. E, Function of gap junctions in HeLa cells transfected with Cx37 plasmid. Scale bar, 10 μm. F, Expression of miRNAs was not significantly changed before and after the coculture. Columns represent the means of three experiments; bars represent the SEM. **P < .01
Figure 6
Figure 6
Overexpression of miR‐34a inhibited glioma cell proliferation and induced cell cycle arrest. A,B, Dose‐ and time‐dependent effect of miR‐34a on cell proliferation in U87 and U251 cells by CCK‐8 assay. C, Cell cycle analysis to evaluate the effects of miR‐34a on cell cycle distribution. D, Expression of cell cycle‐regulated proteins cyclin D1 and cyclin‐dependent kinase 6 (CDK6) with miR‐34a overexpression. Columns represent the means of three experiments; bars represent the SEM. *P < .05, **P < .01
Figure 7
Figure 7
Effect of siRNA‐mediated knockdown of connexin 43 (Cx43) expression on miR‐34a‐induced inhibition of proliferation in U87 cells. A, Western blot analysis of the siRNA‐mediated knockdown of Cx43 expression in U87 cells. B, Histograms show the degree of dye coupling as measured by the parachute assay. C, CCK‐8 assay was carried out to evaluate the effect of miR‐34a on cell proliferation. D, The cell cycle was assessed by flow cytometry after miR‐34a or siRNA transfection. E,F, Expression of cyclin D1 and cyclin‐dependent kinase 6 (CDK6) in U87 cells transfected with miR‐34a or siRNA. β‐Tubulin was used as a loading control. G, Percentage of Cy3 miR‐34a positive cells was measured in U87 cells after Cy3‐labeled miR‐34a or siRNA transfection. Columns represent the means of four experiments; bars represent the SEM. *P < .05, **P < .01
Figure 8
Figure 8
Overexpression of connexin 43 (Cx43) enhanced miR‐34a‐mediated inhibition of proliferation in U251 cells. A, Cell proliferation of different overexpressed groups was detected by CCK‐8 assay. B, The cell cycle was analyzed in U251 cells with miR‐34a or Cx43 plasmid transfection. C,D, Expression of cyclin D1 and cyclin‐dependent kinase 6 (CDK6) in U251 cells transfected with miR‐34a or Cx43 plasmid. β‐Tubulin was used as a loading control. E,F, Percentage of Cy3 miR‐34a positive cells was measured by flow cytometry after Cy3‐labeled miR‐34a or Cx43 plasmid transfection. Columns represent the means of four experiments; bars represent the SEM. *P < .05, **P < .01
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