TF/FⅦa体外诱导大肠癌LOVO细胞系表达MMP-7mRNA的研究

TF/F Ⅶa complex induce the expression of MMP-7mRNA via P38 signal pathway in LOVO cells of colon cancer in vitro

目的观察组织因子/活性凝血七因子(TF/FⅦa)诱导大肠癌 LOVO 细胞系表达基质金属蛋白酶7(MMP-7)mRNA 及信号传导途径。方法构建靶向组织因子基因干扰的 RNAi 质粒并稳定转染 LOVO 细胞获得 TFRNAi LOVO 细胞系,Western blot 检测干扰效率;RT-PCR 观察100 nmol/L FⅦa刺激0、4、8、12、24 h 后 LOVO 细胞中 MMP-7mRNA 的变化,Northern blot 证实时间效应及测定0、10、50、100、200 nmol/L FⅦa刺激后的 MMP-7mRNA 水平;100 nmol/L FⅦa分别刺激LOVO 细胞0、4、8、12、16、24 h,Western blot 测定 p-P38的水平;Northern blot 分别检测预先使用10 mmol/L P38阻断剂 SB203580 0.5 h 再以100 nmol/L FⅦa刺激 LOVO 细胞8 h 及100 nmol/L FⅦa刺激 TFRNAi LOVO 细胞系8 h 后 MMP-7mRNA 水平。结果 Northern blot 结果显示 FⅦa刺激MMP-7mRNA 表达存在时间、浓度依赖性;Western blot 测定 FⅦa刺激 p-P38表达存在时间依赖性。应用 P38通路阻断剂 SB203580后 MMP-7mRNA 基因表达下降59.2%,转染组织因子干扰质粒的LOVO 细胞中,MMP-7mRNA 的表达水平较 LOVO 细胞组下降48%。结论活性Ⅶ因子与组织因子结合诱导体外大肠癌 LOVO 细胞系表达 MMP-7mRNA,其机理可能与 P38通路有关。

Objective To investigate the expression of matrix metalloproteinase 7 （MMP-7） mRNA in LOVO cells of colon cancer induced by TF/F Ⅶa and its signal pathway, Methods We transfected LOVO cells stably with RNAi plasmid targeting to tissue factor to get TFRNAi LOVO cells and detected efficiency of interference in TFRNAi LOVO cells based on Western blot analysis; Expression of MMP-7 was evaluated in LOVO cells treated with 100 nmol/L Ⅶa in 0 h,4 h,8 h,12 h,24 h based on RT-PCR and Northern blot, Expression of MMP-7mRNA was determined in quiescent LOVO cells treated with different doses of Ⅶaa （0 nmol/L,10 nmol/L,50 nmol/L,100 nmol/L,200 nmol/L） for 8 h based on Northern blot. Quiescent LOVO cells were treated for 0 h,4 h,8 h,12 h,16 h,24 h with 100 nmol/L Ⅶa to evaluate the expression of p-P38 ; The expression level of MMP-7mRNA induced by 100 nmol/L Ⅶa for 8 h in LOVO cells blocked by 10mmol SB203580 0.5 h previously and in TFRNAi LOVO cells were measured by Northern blot. Results Northern blot analysis revealed that Ⅶa a markedly increased the expression of MMP-7mRNA in a time- and dose-dependent manner. Western blot analysis confirmed that FVaa stimulates p-P38 in a time-dependent manner. SB203580 block 59.2% expression of MMP-TmRNA in LOVO ceils induced by TF/FⅦa. In TFRNAi LOVO cells, the expression of MMP-7mRNA induced by TF/F Ⅶa was 48% less than that in normal LOVO cells. Conclusions TF/F Va a Complex induces the expression of MMP-7mRNA in LOVO cells in vitro, possibly through 1）38 pathway.