SATB1在甲状腺癌中的表达及促进甲状腺癌侵袭能力的实验研究

Expression pattern and function of SATB1 in the invasiveness of thyroid carcimoma

目的研究富含AT序列的特异结合蛋白1(special AT rich sequence binding protein 1,SATB1)对甲状腺癌细胞侵袭转移的影响,探讨SATB1促进甲状腺癌侵袭转移的分子机制。方法构建针对SATB1基因的过表达质粒,转染甲状腺癌SW-579细胞,qRT-PCR和Western blotting方法测定转染效率及上皮细胞标志蛋白E-钙粘素(E-cadherin)和间质细胞标志蛋白波形蛋白(vimentin)的表达变化;qRT-PCR进一步检测转录因子Twist1和Snail1,基质金属蛋白酶MMP2和MMP9,以及炎性因子IL-6和IL-8的表达变化;倒置显微镜观察细胞形态改变;细胞创伤愈合实验及Transwell方法检测过表达SATB1后甲状腺癌细胞迁移、侵袭能力变化。Western blotting检测SATB1增强表达后Akt磷酸化表达变化,PI3K/Akt通路抑制剂LY294002处理细胞后观察过表达SATB1的SW579细胞迁移侵袭能力变化。结果细胞迁移实验结果显示,过表达SATB1后每个视野下迁移细胞数(196±10)个,较对照组(107±12)个明显增加(P<0.01);侵袭实验结果显示,过表达SATB1后穿过基质胶的细胞数(158±12)个,较对照组(86±15)个明显增加(P<0.01)。Western blotting结果显示转染SATB1后,在总Akt蛋白无改变的情况下,SW-579细胞磷酸化Akt(p-Akt)明显增加。而通过加入PI3K/Akt通路抑制剂LY294002后,细胞迁移及侵袭能力明显减弱。结论 SATB1与甲状腺癌侵袭转移有关;其高表达具有促进甲状腺癌细胞上皮细胞间质转化(epithelial-mesenchymal transition,EMT)的作用;PI3K/Akt信号通路可能参与了SATB1的促转移过程。

Objective To study the effect of SATB1 on invasiveness and metastasis of thyroid carcinoma cells,and to explore its molecular mechanisms to promote invasion and metastasis in thyroid carcinoma. Methods Construction of over expression plasmid for SATB1 gene,transfection of thyroid carcinoma SW-579 cells,transfection efficiency and epithelial cell marker protein of E-cadherin and Western blotting method for determination of qRT-PCR( E-cadherin) and mesenchymal cell marker vimentin( Vimentin) expression; qRT-PCR further detection of transcription factor Twist1 and Snail1,matrix metalloproteinases MMP2 and MMP9,as well as the the expression of inflammatory factors IL-6 and IL-8 changes; cell morphology was observed under an inverted microscope; cell wound healing assay and Transwell were used to detect the expression of SATB1 after thyroid cancer cell migration,invasion ability changes. Western blotting for detection of SATB1 enhanced the expression changes of Akt phosphorylation,PI3 K /Akt pathway inhibitor LY294002 cells was observed after overexpression of SATB1 in SW579 cell migration and invasion ability changes. Results The experimental results showed that cell migration,over expression of SATB1 after each perspective transfer cells( 196 ± 10),compared with the control group( 107 ±12),had a significantly increased( P < 0. 01); invasion and experimental results showd that,after overexpression of SATB1 through the Matrigel cell number( 158 ± 12),compared with the control group( 86 ± 15),which significantly increased( P < 0. 01). Western blotting found that after the transfection of SATB1 in total Akt protein,no change of circumstances,SW-579 cells,the phosphorylation of Akt( p-Akt) increased significantly. But suppressed by PI3 K /Akt signaling pathway inhibitor LY294002,observed cell migration and invasion ability decreased significantly. Conclusion SATB1 is associated with invasion and metastasis of thyroid carcinoma; its high expression is the promotion of thyroid carcinoma cell EMT in vitro; PI3 K /Akt signaling pathway may be involved in promoting the transfer process of SATB1. Further confirmation and molecular SATB1 in metastatic thyroid cancer cells in the mechanism of action in vivo test,may provide the experimental basis for potential intervention targets for inhibition of metastasis of thyroid cancer.