The linker of nucleoskeleton and cytoskeleton complex is required for X-ray-induced epithelial-mesenchymal transition

Abstract

The linker of nucleoskeleton and cytoskeleton (LINC) complex has been implicated in various functions of the nuclear envelope, including nuclear migration, mechanotransduction and DNA repair. We previously revealed that the LINC complex component Sad1 and UNC84 domain containing 1 (SUN1) is required for sublethal-dose X-ray-enhanced cell migration and invasion. This study focused on epithelial-mesenchymal transition (EMT), which contributes to cell migration. Hence, the present study aimed to examine whether sublethal-dose X-irradiation induces EMT and whether LINC complex component SUN1 is involved in low-dose X-ray-induced EMT. This study showed that low-dose (0.5 Gy or 2 Gy) X-irradiation induced EMT in human breast cancer MDA-MB-231 cells. Additionally, X-irradiation increased the expression of SUN1. Therefore, SUN1 was depleted using siRNA. In SUN1-depleted cells, low-dose X-irradiation did not induce EMT. In addition, although the SUN1 splicing variant SUN1_916-depleted cells (containing 916 amino acids [AA] of SUN1) were induced EMT by low-dose X-irradiation like as non-transfected control cells, SUN1_888-depleted cells (which encodes 888 AA) were not induced EMT by low-dose X-irradiation. Moreover, since the Wnt/β-catenin signaling pathway regulates E-cadherin expression via the expression of the E-cadherin repressor Snail, the expression of β-catenin after X-irradiation was examined. After 24 hours of irradiation, β-catenin expression increased in non-transfected cells or SUN1_916-depleted cells, whereas β-catenin expression remained unchanged and did not increase in SUN1- or SUN1_888-depleted cells. Therefore, in this study, we found that low-dose X-irradiation induces EMT, and LINC complex component SUN1, especially SUN1_888, is required for X-ray-induced EMT via activation of the Wnt/β-catenin signaling pathway.

Keywords: SUN1; epithelial-mesenchymal transition (EMT); linker of nucleoskeleton and cytoskeleton (LINC) complex; sublethal-dose X-ray.

Fig. 1

Sublethal-dose X-irradiation induced changes in cell migration, cell invasion, cell morphology and EMT-associated protein expression. A, Clonogenic survival curves after X-irradiation. MDA-MB-231 cells were exposed to X-ray, and colony formation assays were performed. Each bar represents the mean ± standard deviation. ^**: P < 0.01 vs non-irradiated cells. B, Cells were exposed to the indicated doses of radiation and incubated for 24 hours. The expression of γH2AX was examined using Western blotting. C, Cells were exposed to the indicated doses of X-ray, and migration activities were examined 24 hours later. Each bar represents the mean ± standard deviation. ^**: P < 0.01 vs non-irradiated cells. D, Cells were exposed to the indicated doses of X-ray, and invasion activities were examined 24 hours later. Each bar represents the mean ± standard deviation. ^**: P < 0.01 vs non-irradiated cells. E, Cells were exposed to the indicated doses of X-ray, and morphologies of MDA-MB-231 cells were examined with phase-contrast microscopy. The scale bar represents 50 μm. F and G, Cells were exposed to the indicated doses of X-ray. Twenty-four hours later, the expression of EMT-associated proteins (E-cadherin, N-cadherin, Slug and Snail) was examined by Western blotting. Each bar represents the mean ± standard deviation. ^**: P < 0.01 vs non-irradiated cells.

Fig. 2

The expression of SUN1 was increased after X-irradiation in MDA-MB-231 cells. A, MDA-MB-231 cells were exposed to the indicated doses of X-ray. Twenty-four hours after irradiation, the expression of SUN1 was examined by immunofluorescence staining. B and C, Cells were exposed to the indicated doses of radiation and incubated for 24 hours. The expression of SUN1 was assessed by Western blotting. Each bar represents the mean ± standard deviation. ^**: P < 0.01 vs non-irradiated cells.

Fig. 3

SUN1 was required for X-ray-induced EMT. A, MDA-MB-231 cells were transfected with siRNA targeting SUN1 (siSUN1) and incubated for 48 hours. The SUN1 expression level was analyzed by Western blotting. B, Cells were transfected with siSUN1 and incubated for 48 hours. These cells were then exposed to X-ray, and after 24 hours of incubation, cell morphologies were examined with phase-contrast microscopy. The scale bar represents 50 μm. C and D, Cells were transfected with siSUN1 and incubated for 48 hours. These cells were then exposed to X-ray, and after 24 hours of incubation, the expression of EMT-associated proteins (E-cadherin, N-cadherin, Slug and Snail) was examined by Western blotting. Each bar represents the mean ± standard deviation. ^**: P < 0.01 vs non-irradiated cells.

Fig. 4

SUN1 splicing variant SUN1_888 was required for X-ray-induced EMT. A, Each indicated siRNA was transfected into MDA-MB-231 cells for 48 hours. SUN1_916 and SUN1_888 mRNA expression levels were analyzed by PCR. B, Each indicated siRNA was transfected into cells and incubated for 48 hours. These cells were then exposed to X-ray, and after 24 hours of incubation morphologies of cells were examined with phase-contrast microscopy. The scale bar represents 50 μm. C and D, Each indicated siRNA was transfected into cells for 48 hours. These cells were exposed to X-ray, and after 24 hours of incubation, the expression of EMT-associated proteins (E-cadherin, N-cadherin, Slug and Snail) was analyzed by Western blotting. Each bar represents the mean ± standard deviation. ^**: P < 0.01 vs non-irradiated cells.

Fig. 5

SUN1 was important for X-ray-induced β-catenin expression. A and B, Each indicated siRNA was transfected into MDA-MB-231 cells for 48 hours. These cells were then exposed to X-ray, and after 24 hours of incubation the expression of β-catenin was examined by Western blotting. Each bar represents the mean ± standard deviation. ^**: P < 0.01 vs non-irradiated cells. C, Each indicated siRNA was transfected into cells for 48 hours. These cells were then exposed to X-ray. The expression and distribution of β-catenin were analyzed by immunofluorescence staining 24 hours later. The scale bar represents 10 μm. D, Schematic illustration showing the relationship between SUN1 and X-ray-induced EMT.