中期因子诱导上皮间质转化促进结直肠癌细胞增殖和侵袭的分子机制

目的:探讨中期因子（midkine，MK）诱导肠癌细胞上皮间质转化（epithelial-mesenchymal transition，EMT）促进结直肠癌增殖和侵袭的分子机制。方法:MTT法检测MK对肠癌细胞增殖及侵袭的影响，倒置显微镜下观察其形态学变化，Western blot检测其EMT标志物、NF-κB(p65)、Zbe1和MMP-9蛋白表达变化。结果:在10ng/ml、50ng/ml、100ng/ml和200ng/ml的MK作用下，SW480细胞的增殖率分别为:（106.33±4.57）%、（119.83±6.01）%、（148.52±8.31）%和(162.51±8.98)%，（F=70.954，P＜0.01）；HCT116细胞的增殖率分别为:(116.49±9.77）%、（129.79±2.31）%、（155.38±8.98）%和（158.11±6.75）%，（F=55.027，P＜0.01）。100ng/ml MK作用下，SW480细胞的侵袭数为每视野（40.34±2.52）个，而NF-κB信号通路抑制剂5μmol/L Bay11-7082预处理后的侵袭数为每视野（14.13±2.06）个，（F=41.391，P＜0.01）；HCT116细胞的侵袭数为每个视野（42.21±3.25）个，而Bay11-7082预处理后的细胞侵袭数为每视野（14.32±2.52）个，（F=69.206，P＜0.01）。MK诱导SW480和HCT116呈现梭形或长条形改变，E-cad蛋白表达下调，Vimentin、β-catenin、NF-κB (p65)和MMP-9蛋白表达上调，而Zbe1蛋白表达无明显变化。结论:MK通过上调NF-κB(p65)信号蛋白诱导肠癌细胞上皮间质转化，促进下游靶蛋白MMP-9表达上调，从而促进肠癌细胞增殖和侵袭。

The molecular mechanism of midkine-induced epithelial-mesenchymal transition to promote proliferation and invasion in CRC cells

Objective:To discuss the molecular mechanismof midkine-induced epithelial-mesenchymal transition to promote proliferation and invasion in CRC cells.Methods:MTT assay was used to analyze the proliferation and invasion rates of SW480 and HCT116 treating with MK.We observed the morphology of CRC cells which was treated with or without MKby a microscope and analyzed the expression of EMT markers,NF-kappaB (p65),Zeb1,and MMP-9 in these cells by Western blot.Results:By treating with MK at concentrations of 10ng/ml,50ng/ml,100ng/ml and 200ng/ml,the proliferation rates of SW480 cells respectively were（106.33±4.57）%,（119.83±6.01）%,（148.52±8.31）% and (162.51±8.98）%（F=70.954，P＜0.01）.The proliferation rates of HCT116 cells were (116.49±9.77）%,（129.79±2.31）%,（155.38±8.98）% and (158.11±6.75)%（F=55.027，P＜0.01）.When MK at the concentration of 100ng/ml was used，the numbers of SW480 cells that passed through the membrane were（40.34±2.52）.But in pre-treatment with NF-κB (p65) signaling- pathway inhibitor Bay11-7082 of 5 μmol/L,the numbers were （14.13±2.06），（F=41.391，P＜0.01）.The numbers of HCT116 cells that passed through the membrane were（42.21±3.25）,but（14.32±2.52）when pre-treatmenting with Bay11-7082 of 5 μmol/L（F=69.206，P＜0.01）.The SW480 and HCT116 cells loosed association and changed from spindle-shaped to elongated morphology.E-cadherin downregulated,but vimentin and beta-catenin upregulated.MK activated NF-κB signaling pathway and MMP-9 upregulated,but the expression of Zbe1 had no change.Conclusion:The mechanism was probably that MK activated NF-κB (p65) signaling pathway,mediatedthe epithelial-mesenchymal transition of CRC cells,promoted the upregulation of MMP-9 expression,and eventually leaded to the proliferation and invasion of CRC cells.