作者：刘巧丽1,2，陈自喜1，相芬芬1，范嫣1，朱兆伟1，张孟哲1，吴蓉1，康向东1

单位：1.上海中医药大学附属普陀医院 检验科， 上海 200062；2.上海交通大学附属上海市第一人民医院 中医科， 上海 200080

Authors:  Liu Qiaoli1,2，Chen Zixi1，Xiang Fenfen1，Fan Yan1，Zhu Zhaowei1，Zhang Mengzhe1，Wu Rong1，Kang Xiangdong1

Unit:  1.Clinical Laboratory, Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China；2.Department of Traditional Chinese Medicine, Shanghai General Hospital, Shanghai 200080, China

摘要：

目的 探讨熊果酸通过下调MyD88抑制结肠癌细胞侵袭转移的作用及可能的机制。方法 在细胞增殖实验中以人结肠癌HCT-116细胞和人结肠癌Lovo细胞作为本次研究对象,分为对照组、熊果酸组,熊果酸组给予不同的熊果酸药物浓度处理,继续培养48 h后再应用CCK-8法检测其增殖曲线;细胞凋亡实验则将人结肠癌HCT-116细胞分为对照组、熊果酸组,熊果酸组予以20μmol/L的浓度处理48 h,收集细胞后应用流式细胞术检测其凋亡水平。后期的侵袭转移及机制研究包括2部分,一部分为HCT-116细胞为研究对象,分组及处理方法同前;另一部分以MyD88稳转人结肠癌HCT-116细胞为研究对象进行实验,分为MyD88-NC组、MyD88-KD组、MyD88-OE组。划痕实验则分别于0、48 h观察各组HCT-116细胞的迁移情况,比较细胞迁移能力;Transwell实验分别观察0、24 h各组HCT-116细胞的迁移情况,比较细胞迁移能力;蛋白质印迹法检测上述各组细胞的MyD88、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9 (MMP-9)蛋白的表达水平差异。结果 熊果酸可以显著抑制结肠癌细胞的增殖,且呈明显的剂量依赖性;熊果酸可以显著促进结肠癌细胞的凋亡,对照组相比差异有统计学意义(P=0.02)。MyD88-KD组的迁移率(划痕实验、Transwell实验)明显低于对照组(P=0.04, P=0.03),而MyD88-OE组的迁移率(划痕实验、Transwell实验)则显著高于对照组,两者差异均有统计学意义(P=0.004, P=0.002);熊果酸组的迁移率也显著低于对照组,两者差异有统计学意义(P=0.009);MyD88-KD组及熊果酸组MMP-2、MMP-9的蛋白表达均明显降低,而MyD88-OE组则明显升高,与对照组比较差异均有统计学意义(P<0.01)。结论 熊果酸可显著降低MyD88蛋白的表达,并通过对MMP-2和MMP-9蛋白表达的下调实现对人结肠癌细胞侵袭转移能力的抑制作用。

关键词：髓样分化因子88；结肠癌；自噬；凋亡

Abstract：

Objective To investigate the effect and the possible mechanism of ursolic acid on inhibiting the invasion and metastasis of colon cancer cells by down regulating MyD88. Methods In the cell proliferation experiment, human colon cancer HCT-116 cells and human colon cancer Lovo cells were divided into control group and ursolic acid group. The ursolic acid group were treated with different concentrations of ursolic acid. When cultured 48 hours, the proliferation curve were detected by CCK-8 method. In the apoptosis experiment, human colon cancer HCT-116 cells were divided into control group and ursolic acid group. The concentration of the ursolic acid group was 20 μmol, cells were collected after 48 h and the apoptosis level were detected by flow cytometry. The mechanism research of invasion and metastasis includes two parts. One part is HCT-116 cells, the grouping and treatment methods are the same as before. The other part took MyD88 stably transformed human colon cancer HCT-116 cells as the research objects. And they were divided into MyD88-NC group, MyD88-KD group and myd88-OE group. The migration of HCT-116 cells in each group was observed at 0 h and 48 h respectively. Transwell experiment was used to observe the migration of HCT-116 cells in each group at 0 h and 24 h, then compare the cell migration ability. The protein expression levels of MyD88, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were detected by Western blot. Results ursolic acid could significantly inhibit the proliferation of colon cancer cells in a dose -dependent manner. Meanwhile, ursolic acid could significantly promote the apoptosis of colon cancer cells, and the difference was statistically significant compared with the control group (P=0.02). The mobility of MyD88-KD group (scratch test and Transwell test) was significantly lower than the control group (P=0.04, P=0.03), while the mobility of MyD88-OE group (scratch test and Transwell test) was significantly higher than the control group (P=0.004, P=0.002); The mobility of ursolic acid group was also significantly lower than the control group (P=0.009); The protein expressions of MMP-2 and MMP-9 in MyD88-KD group and ursolic acid group decreased significantly, while those in MyD88-OE group increased significantly, which was statistically significant compared with the control group (P<0.01). Conclusion ursolic acid can significantly reduce the expression of MyD88 protein and inhibit the invasion and metastasis of human colon cancer cells by down regulating the expression of MMP-2 and MMP-9 protein.

Key Words:  Myeloid differentiation factor 88; Colon cancer; Autophagy; Apoptosis