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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 investigate the effect and mechanism of Raptor on invasiveness of breast cancer cells. Methods Raptor small interfering plasmid was transfected into MDA-MB-231 cells using small RNA interference technique. The expression level of Raptor in transfected cells was detected by Western blotting to identify the transfection efficiency. In Vitro Invasion of Cancer Cells The invasive ability of MDA-MB-231 cells after Raptor expression was reduced by RNAi was observed. The phosphorylation of ARK5 and the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were detected by Western blotting. We collected 52 cases of breast cancer and 8 cases of paracancerous tissues from Department of Pathology, Affiliated Hospital of Weifang Medical College. The expression of Raptor was detected by immunohistochemical staining. Results The gray value of Raptor protein in transfected SiRaptor / MDA-MB-231 cells was 529.22 ± 81.58, which was significantly lower than that of MDA-MB-231 cells (1 345.63 ± 215.05, F = 37.799, P = 0.004) 1 349.15 ± 85.13 for the MDA-MB-231 group, F = 145.082, P <0.001. In vitro invasion assay showed that the number of cells penetrating Matrivgel stroma in experimental group was 40.00 ± 3.61, which was less than that in control group (82.33 ± 10.50, t = 6.602, P = 0.003). The phosphorylation level of ARK5 in the experimental group was 224.72 ± 30.14, which was lower than that of the control group (852.46 ± 88.93), F = 134.083, P <0.001; the expression of MMP-2 protein was 604.32 ± 177.06, (1051.72 ± 116.54), F = 13.365, P = 0.022; The expression of MMP-9 protein was 347.85 ± 84.80, lower than the control group (924.46 ± 108.61), F = 52.535, P = 0.002. There was a correlation between the expression of Raptor and lymph node metastasis in breast cancer (χ2 = 9.094, P <0.001). Conclusion Raptor may be related to the invasion of breast cancer cells through phosphorylation of ARK5 to increase the expression of MMP-2 and MMP-9.