葡萄糖调节蛋白78对人舌癌细胞侵袭的影响及其机制

摘要

目的：探讨葡萄糖调节蛋白78(glucose regulated protein 78，Grp78)对人舌癌细胞系Tca-8113侵袭的影响及其分子机制。方法应用pcDNA3.1(+)-Grp78重组质粒转染舌癌细胞系Tca-8113筛选得到稳定表达Grp78的克隆(78C6)，再应用siRNA-Grp78转染78C6敲除Grp78表达，应用Transwell实验分析其侵袭能力，免疫荧光和细胞伸展实验检测细胞骨架排列和细胞伸展情况，GST-pulldown技术对Rac1和RhoA活性进行分析，Western blot检测Rac1、RhoA、MMP-9和MMP-2的表达。结果 Transwell实验结果显示，特异性上调Grp78表达明显促进舌癌细胞的侵袭，并且这种促进作用可以被siRNA-Grp78转染抑制，差异有统计学意义(P＜0.05)。进一步的研究发现，与对照组相比，Grp78高表达可以促进舌癌细胞Tca-8113的伸展和极性形成，差异有统计学意义(P＜0.05)。细胞骨架染色结果表明特异性上调Grp78后，细胞骨架微丝主要分布于细胞边缘，而这种作用可以被siRNA-Grp78转染抑制，导致细胞皮质部位出现明显的应力纤维。GST-pulldown结果表明，与对照组相比，Grp78高表达使Rac1的活性水平增高，RhoA活性降低，而siRNA-Grp78转染的细胞中Rac1活性降低，RhoA活性升高。Western blot研究显示，与对照组相比，Tca-8113/Grp78细胞中MMP-2和MMP-9表达增高(P＜0.05)， siRNA-Grp78转染的细胞中MMP-2和MMP-9表达降低(P＜0.05)，而RhoA和Rac1的表达前后无变化。结论 Grp78通过激活Rac1抑制RhoA活性及上调MMP-2和MMP-9表达来促进舌癌Tca-8113细胞的侵袭。%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 ofGrp78 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 causedcytoskeleton 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, theactivity 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.