结肠癌细胞中组织因子/活性凝血因子Ⅶ-表皮生长因子受体通路间交互作用机制

Mechanism of cross talk between tissue factor/active coagulation factor Ⅶ and epidermal growth factor receptor signalings in colon cancer cells in culture

目的:探讨结肠癌细胞中组织因子/活性凝血因子Ⅶ(tissue factor/active coagulation factorⅦ,TF/FⅦa)通路与表皮生长因子受体(epidermal growth factor receptor,EGFR)通路之间是否存在交互作用。方法:在KRAS野生型的HT-29及KRAS突变型的Lo Vo结肠癌细胞中,以FⅦa活化TF/FⅦa通路,采用qRT-PCR、Western blot检测EGFR配体双调蛋白(amphiregulin,AREG)及表皮调节素(epiregulin,EREG)基因、蛋白表达改变;利用RNA干扰技术敲低TF表达后活化TF/FⅦa通路,检测其对AREG、EREG基因表达的影响,以证实FⅦa对AREG、EREG表达调节作用依赖TF。以表皮生长因子(epidermal growth factor,EGF)激活两细胞EGFR通路后,检测TF/FⅦa通路关键分子TF、FⅦ基因表达改变。结果:TF/FⅦa通路活化后,HT-29细胞AREG、EREG基因表达及EREG蛋白表达水平均较对照组显著下调(AREG、EREG基因表达量分别为0.55±0.09 vs.0.99±0.09、0.67±0.10 vs.1.02±0.02,EREG蛋白表达量0.54±0.09 vs.1.04±0.13,P均<0.05);Lo Vo细胞AREG基因表达及AREG、EREG蛋白表达水平较对照组显著上调(AREG基因表达量1.87±0.39 vs.0.93±0.23,AREG、EREG蛋白表达量3.09±0.73 vs.1.11±0.21、1.53±0.19 vs.0.97±0.23,P均<0.05);TF敲低后均可部分阻断FⅦa对两细胞AREG、EREG表达调节作用;EGFR通路激活后,HT-29细胞TF基因表达较对照组无显著变化,FⅦ基因表达未检测到,而Lo Vo细胞的FⅦ及TF基因表达均较对照组显著上调,表达量分别为1.53±0.23 vs.1.00±0.23、53.20±6.08 vs.1.00±0.15(P均<0.05)。结论:结肠癌Lo Vo细胞TF/FⅦa通路与EGFR通路的活化可分别上调另一通路的关键分子表达并发生交互作用,KRAS基因突变可能对该交互作用的发生发挥关键作用。

Objective： To preliminarily verify the cross talk between tissue factor/active coagulation factor Ⅶ（ TF/FⅦa） and epidermal growth factor receptor（ EGFR） pathways in human colon cancer cells in culture. Methods： FⅦ a was treated to HT-29（ KRAS-wild type） and Lo Vo（ KRAS-mutant） colon cancer cells to activate TF/FⅦa pathway,qRT-PCR and Western blot were used to detect the expressions of amphiregulin（ AREG） and epiregulin（ EREG）,ligands of EGFR on mRNA and protein levels,respectively. After knocking down expression of TF by TF-targeted siRNA transfection,FⅦa was treated and mRNA expressions of AREG and EREG were detected to see whether the FⅦa-induced effects were dependent on TF. Expressions of mRNA of TF and FⅦ were detected by qRT-PCR following the activation of EGFR pathway by treatment with epidermal growth factor（ EGF） to HT-29 and Lo Vo cells. Results： After TF/FⅦa pathway was activated,for HT-29 cells,expressions of AREG（ on mRNA level）and EREG（ both on mRNA and protein level） were significantly down-regulated versus those of control group,gene expressions of AREG and EREG were 0. 55 ± 0. 09 vs. 0. 99 ± 0. 09,0. 67 ± 0. 10 vs. 1. 02 ±0. 02,protein expressions of EREG were 0. 54 ± 0. 09 vs. 1. 04 ± 0. 13,all P 0. 05. For Lo Vo cells,expressions of AREG（ both on mRNA and protein level） and EREG（ on protein level） were significantly up-regulated versus those of control group,gene expression of AREG were 1. 87 ± 0. 39 vs. 0. 93 ± 0. 23,protein expressions of AREG and EREG were 3. 09 ± 0. 73 vs. 1. 11 ± 0. 21,1. 53 ± 0. 19 vs. 0. 97 ±0. 23,all P 0. 05. The regulating effect of AREG and EREG mRNA expression by FⅦa in HT-29 and Lo Vo cells could both be partly blocked by knocking down TF expression. For HT-29 cells,activation of EGFR pathway induced no significant TF mRNA expression,FⅦ mRNA expression was not detected.However,for Lo Vo cells,activation of EGFR pathway induced significantly higher mRNA expressions of both TF and FⅦ,expressions were 1. 53 ± 0. 23 vs. 1. 00 ± 0. 23,53. 20 ± 6. 08 vs. 1. 00 ± 0. 15,all P 0. 05. Conclusion： In colon cancer cell Lo Vo,when activated,TF/FⅦa pathway and EGFR pathway could interact through upregulating the other pathway＇s effectors,and mutant KRAS might play a critical role in the two pathways＇ cross talk.