Effect and mechanism of Grp78 on invasion of tongue cancer cell

Objective To explore the role of Grp78 in invasion of human tongue cancer and its molecular mechanisms. Methods pcDNA3.1 (+)-Grp78 recombinant plasmid was used to transfect tongue cancer cell line Tca-8113 to get the cells that stably expressed Grp78 (78C6) and then siRNA-Grp78 was used to transfect 78C6 in order to knockout Grp78 expression. Transwell experiment was used to analyze the ability of invasion, and immunofluorescence and cell stretching experiments were used to detect cell cytoskeleton arrangement and stretch. The GST-pulldown technology was used to analyze the activity of Rac1 and RhoA. The expression of MMP-9, MMP-2, Rac1 and RhoA were detected by western blot. Results Transwell experiment results showed that overexpression of Grp78 promoted invasion of tongue cancer cells significantly when compared with the control group and this phenomenon could be inhibited by siRNA-Grp78 (P< 0.05). Furthermore, compared with the control group the overexpression of Grp78 also promoted the spread and polarity formation of tongue cancer cells Tca-8113 (P< 0.05). Cytoskeleton staining results showed that overexpression of Grp78 caused cytoskeleton microfilament to be mainly distributed in cell edge, however this effect could be suppressed by siRNA-Grp78 transfection which leaded to stress fibers appeared in cortical areas. GST-pull down results showed that the activity of Rac1 was higher and RhoA was lower in Tca-8113/Grp78 cells than the control group, while after transfection of siRNA-Grp78 in Tca-8113/Grp78 cells, the activity of Rac1 was lower and RhoA was higher than the control group (P< 0.05). Western blot analysis showed that compared with the control group, the expression of MMP-2 and MMP-9 increased in Tca-8113/Grp78 cells while the expression of MMP-2 and MMP-9 decreased in siRNA-Grp78 (P< 0.05), but the expression of RhoA and Rac1 had no difference in Tca-8113/Grp78 and siRNA-Grp78 cells (P< 0.05). Conclusion Grp78 promotes invasion of tongue cancer cells Tca-8113 by increasing activation of Rac1 and decreasing activation of RhoA as well as by raising the expression of MMP-2 and MMP-9.