下调Raptor表达对乳腺癌细胞侵袭能力影响及其机制探讨

Disruption of Raptor by using RNA interference influences the invasiveness of human breast cancer cells and it's relative mechanism

目的研究Raptor对乳腺癌细胞侵袭能力的作用及其机制。方法采用小RNA干扰技术,向MDA-MB-231细胞转染Raptor小RNA干扰质粒,并通过蛋白质印迹法检测转染细胞Raptor的表达水平以鉴定转染效果。癌细胞体外侵袭实验观察使用RNAi降低Raptor表达后MDA-MB-231细胞侵袭能力的变化。再进行蛋白质印迹实验检测细胞中ARK5的磷酸化情况和基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)与基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的表达。收集潍坊医学院附属医院病理科2008-01-15-2012-12-31 52例乳腺癌和8例癌旁组织,使用免疫组织化学染色检测Raptor的表达水平。结果转染成功的SiRaptor/MDA-MB-231细胞Raptor蛋白条带灰度值为529.22±81.58,明显低于MDA-MB-231组的1 345.63±215.05(F=37.799,P=0.004)和SCR/MDA-MB-231组的1 349.15±85.13,F=145.082,P<0.001。体外侵袭实验发现,实验组穿透Matrivgel基质膜的细胞数为40.00±3.61,少于对照组细胞的82.33±10.50,t=6.602,P=0.003。EGF刺激后实验组细胞中ARK5蛋白的磷酸化水平为224.72±30.14,较对照组(852.46±88.93)低,F=134.083,P<0.001;MMP-2蛋白的表达为604.32±177.06,较对照组(1 051.72±116.54)低,F=13.365,P=0.022;MMP-9蛋白的表达为347.85±84.80,较对照组(924.46±108.61)低,F=52.535,P=0.002。乳腺癌组织Raptor的表达与淋巴结转移存在相关性,χ2=9.094,P<0.001。结论 Raptor可能通过磷酸化ARK5以增加MMP-2和MMP-9的表达,从而与乳腺癌细胞的侵袭相关。

OBJECTIVE To study the effect of Raptor to the invasion ability of breast cancer cells and it＇s mecha- nism. METHODS Raptor siRNA plasmids was used to transfeet MDA-MB-231 cells. And Western blot was used to ana- lyze protein expression level of Raptor to appraisal the effects of transfection. In vitro Matrigel invasion assay was used to observe the variation of invasiveness to the cells being transfected. Western blot was used to analyze the phosphorylation of ARK5 and protein expression of matrix metalloproteinase-2（MMP-2） and matrix metalloproteinase-9（MMP-9）. Immu- nohistochemistry was used to detect the expression of Raptor in 52 cases of invasive ductal carcinoma and 8 cases of adja- cent non-tumor tissue, these paraffin blocks from breast tissue specimens were obtained from the Department of Patholo- gy, Affiliated Hospital of the Weifang Medical Uni,Jersity from January 15, 2008 to December 31, 2012. RESULTS Seventy-two hours after transfection, compared to control cells, the protein expression of Raptor in SiRaptor/ MDA-MB-231（529. 22±81. 58） was decreased（vs MDA-MB-231 1 345. 63±215. 05, F=37.799, P=0.004; vs Scr/ MDA-MB-231 1 349.15±85.13, F=145. 082, P〈9. 001）. After down regulation of Raptor the quantity of SiRaptor/ MDA-MB-231 cells which invaded Matrivgel（40. 00±3. 61） was decreased（vs Scr/MDA-MB-231 82. 33±10. 50, t= 6. 602, P=0. 003）. Stimulated with EGF, compared with control cells, SiRaptor/MDA-MB 231 showed lower levels ofthe phosphorylation of ARK5（224.72±30.14） than control cells（852.46±88.93, F=134. 083, P〈0. 001）, lower ex- pression of MMP-2（604. 32±177.06） than control cells（1 051. 72±116.54, F=13. 365, P=0. 022）, and lower expres- sion of MMP-9（347. 85±84.80） than control cells（924.46±108.61, F=52. 535, P=0. 002）. The expression of Raptor in breast cancer was significantly related with lymph node metastasis, X^2 = 9. 094,P（0. 001. CONCLUSION The Rap- tot-reduced breast cancer cells showed decreased invasion ability, ARK5 phosphorylation and MMP-2, MMP-9 expression, suggesting that Raptor can adjust the expression of MMP-2 and MMP-9 by phosphorylate ARK5 to promote breast cancer metastasis.