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Reduction in gap junction intercellular communication promotes glioma migration.

Aftab Q, Sin WC, Naus CC - Oncotarget (2015)

Bottom Line: In this study we examine whether a decrease in Cx43 protein expression has a role in enhanced cell migration, a key feature associated with increased tumorigenicity.In addition gap junction intercellular communication (GJIC) played a more prominent role in mediating migration than the cytoplasmic interactions of the C-terminal tail.Live imaging revealed that reducing Cx43 expression enhanced relative migration by increasing the cell speed and affecting the direction of migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular & Physiological Sciences, Life Sciences Institute, The University of British Columbia, Vancouver, BC.

ABSTRACT
Glioblastoma Multiforme (GBM), an aggressive form of adult brain tumor, is difficult to treat due to its invasive nature. One of the molecular changes observed in GBM is a decrease in the expression of the gap junction protein Connexin43 (Cx43); however, how a reduction in Cx43 expression contributes to glioma malignancy is still unclear. In this study we examine whether a decrease in Cx43 protein expression has a role in enhanced cell migration, a key feature associated with increased tumorigenicity. We used a 3D spheroid migration model that mimics the in vivo architecture of tumor cells to quantify migration changes. We found that down-regulation of Cx43 expression in the U118 human glioma cell line increased migration by reducing cell-ECM adhesion, and changed the migration pattern from collective to single cell. In addition gap junction intercellular communication (GJIC) played a more prominent role in mediating migration than the cytoplasmic interactions of the C-terminal tail. Live imaging revealed that reducing Cx43 expression enhanced relative migration by increasing the cell speed and affecting the direction of migration. Taken together our findings reveal an unexplored role of GJIC in facilitating collective migration.

No MeSH data available.


Related in: MedlinePlus

Reducing Cx43 expression increases migration in a spheroid migration assay(A) Western blot analysis shows successful expression of exogenous full-length Cx43 cDNA lacking the 3′UTR in U118-ShRNA6 and U118-ShRNA7 cells. Using densitometry we found the Cx43 expression in control and rescue cells to be 4 times higher than ShRNA6 and ShRNA7 cells (average of 3 Western blots). (B) Expression of Cx43 in the knockdown cells lowered migration level comparable to control cells. Scale bar = 100 μm. (C) Compared to control cells ShRNA6 and ShRNA7 cells increased migration 46% and 30%, respectively. The experiments were repeated three times with control (n = 81spheroids), ShRNA 6 (n = 81 spheroids), ShRNA 7 (n = 88 spheroids), ShRNA 6-Cx43 (n = 54 spheroids) and ShRNA 7-Cx43 (n = 54 spheroids). *p < 0.05 determined by One Way Anova method followed by Dunn's Method.
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Figure 6: Reducing Cx43 expression increases migration in a spheroid migration assay(A) Western blot analysis shows successful expression of exogenous full-length Cx43 cDNA lacking the 3′UTR in U118-ShRNA6 and U118-ShRNA7 cells. Using densitometry we found the Cx43 expression in control and rescue cells to be 4 times higher than ShRNA6 and ShRNA7 cells (average of 3 Western blots). (B) Expression of Cx43 in the knockdown cells lowered migration level comparable to control cells. Scale bar = 100 μm. (C) Compared to control cells ShRNA6 and ShRNA7 cells increased migration 46% and 30%, respectively. The experiments were repeated three times with control (n = 81spheroids), ShRNA 6 (n = 81 spheroids), ShRNA 7 (n = 88 spheroids), ShRNA 6-Cx43 (n = 54 spheroids) and ShRNA 7-Cx43 (n = 54 spheroids). *p < 0.05 determined by One Way Anova method followed by Dunn's Method.

Mentions: In the wound healing assay, U118 cells expressing ShRNA6 and ShRNA7 migrated faster than the control cells after 8 hours (Figures 5A and 5B). This result was confirmed with a spheroid migration assay that had the advantage of providing a 3D architecture similar to in vivo tumors with a core and a defined border [36]. In this assay, cells from ShRNA6 and ShRNA7 glioma spheroids migrated faster than control cells on fibronectin (Figures 6B and 6C). To confirm the increase in migration was due to the decrease in Cx43, we expressed full-length Cx43 in ShRNA6 and ShRNA7 cells (Figure 6A) and observed a reduction in the level of migration comparable to control cells (Figures 6B and 6C). Exogenous Cx43 proteins localized in intracellular vesicles and at cell-cell contacts (Supplementary Figure 1A), forming functional gap junctions (Figure 10). The ShRNA constructs were designed to bind to the 3′UTR region of the endogenous mRNA so they only target the endogenous Cx43 but have no effect on exogenous wild-type Cx43 expressed from the cDNA plasmid.


Reduction in gap junction intercellular communication promotes glioma migration.

Aftab Q, Sin WC, Naus CC - Oncotarget (2015)

Reducing Cx43 expression increases migration in a spheroid migration assay(A) Western blot analysis shows successful expression of exogenous full-length Cx43 cDNA lacking the 3′UTR in U118-ShRNA6 and U118-ShRNA7 cells. Using densitometry we found the Cx43 expression in control and rescue cells to be 4 times higher than ShRNA6 and ShRNA7 cells (average of 3 Western blots). (B) Expression of Cx43 in the knockdown cells lowered migration level comparable to control cells. Scale bar = 100 μm. (C) Compared to control cells ShRNA6 and ShRNA7 cells increased migration 46% and 30%, respectively. The experiments were repeated three times with control (n = 81spheroids), ShRNA 6 (n = 81 spheroids), ShRNA 7 (n = 88 spheroids), ShRNA 6-Cx43 (n = 54 spheroids) and ShRNA 7-Cx43 (n = 54 spheroids). *p < 0.05 determined by One Way Anova method followed by Dunn's Method.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4484468&req=5

Figure 6: Reducing Cx43 expression increases migration in a spheroid migration assay(A) Western blot analysis shows successful expression of exogenous full-length Cx43 cDNA lacking the 3′UTR in U118-ShRNA6 and U118-ShRNA7 cells. Using densitometry we found the Cx43 expression in control and rescue cells to be 4 times higher than ShRNA6 and ShRNA7 cells (average of 3 Western blots). (B) Expression of Cx43 in the knockdown cells lowered migration level comparable to control cells. Scale bar = 100 μm. (C) Compared to control cells ShRNA6 and ShRNA7 cells increased migration 46% and 30%, respectively. The experiments were repeated three times with control (n = 81spheroids), ShRNA 6 (n = 81 spheroids), ShRNA 7 (n = 88 spheroids), ShRNA 6-Cx43 (n = 54 spheroids) and ShRNA 7-Cx43 (n = 54 spheroids). *p < 0.05 determined by One Way Anova method followed by Dunn's Method.
Mentions: In the wound healing assay, U118 cells expressing ShRNA6 and ShRNA7 migrated faster than the control cells after 8 hours (Figures 5A and 5B). This result was confirmed with a spheroid migration assay that had the advantage of providing a 3D architecture similar to in vivo tumors with a core and a defined border [36]. In this assay, cells from ShRNA6 and ShRNA7 glioma spheroids migrated faster than control cells on fibronectin (Figures 6B and 6C). To confirm the increase in migration was due to the decrease in Cx43, we expressed full-length Cx43 in ShRNA6 and ShRNA7 cells (Figure 6A) and observed a reduction in the level of migration comparable to control cells (Figures 6B and 6C). Exogenous Cx43 proteins localized in intracellular vesicles and at cell-cell contacts (Supplementary Figure 1A), forming functional gap junctions (Figure 10). The ShRNA constructs were designed to bind to the 3′UTR region of the endogenous mRNA so they only target the endogenous Cx43 but have no effect on exogenous wild-type Cx43 expressed from the cDNA plasmid.

Bottom Line: In this study we examine whether a decrease in Cx43 protein expression has a role in enhanced cell migration, a key feature associated with increased tumorigenicity.In addition gap junction intercellular communication (GJIC) played a more prominent role in mediating migration than the cytoplasmic interactions of the C-terminal tail.Live imaging revealed that reducing Cx43 expression enhanced relative migration by increasing the cell speed and affecting the direction of migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular & Physiological Sciences, Life Sciences Institute, The University of British Columbia, Vancouver, BC.

ABSTRACT
Glioblastoma Multiforme (GBM), an aggressive form of adult brain tumor, is difficult to treat due to its invasive nature. One of the molecular changes observed in GBM is a decrease in the expression of the gap junction protein Connexin43 (Cx43); however, how a reduction in Cx43 expression contributes to glioma malignancy is still unclear. In this study we examine whether a decrease in Cx43 protein expression has a role in enhanced cell migration, a key feature associated with increased tumorigenicity. We used a 3D spheroid migration model that mimics the in vivo architecture of tumor cells to quantify migration changes. We found that down-regulation of Cx43 expression in the U118 human glioma cell line increased migration by reducing cell-ECM adhesion, and changed the migration pattern from collective to single cell. In addition gap junction intercellular communication (GJIC) played a more prominent role in mediating migration than the cytoplasmic interactions of the C-terminal tail. Live imaging revealed that reducing Cx43 expression enhanced relative migration by increasing the cell speed and affecting the direction of migration. Taken together our findings reveal an unexplored role of GJIC in facilitating collective migration.

No MeSH data available.


Related in: MedlinePlus