胃肠安通过靶向ARHGAP25抑制裸鼠结肠癌原位移植瘤侵袭转移的机制研究

Mechanism of Weichang’an inhibiting invasion and metastasis of orthotopic xenografts of colon cancer in nude mice by targeting ARHGAP25

目的研究胃肠安对裸鼠结肠癌原位移植瘤Rho鸟苷三磷酸酶激活蛋白25(ARHGAP25)表达的影响,探索胃肠安抑制结肠癌侵袭转移的分子机制。方法采用人结肠癌HCT-116细胞制作原位移植瘤模型,将造模后的裸鼠随机分为空载体组、ARHGAP25过表达组、空载体+胃肠安低浓度组和空载体+胃肠安高浓度组,每组6只。比较各组原位移植瘤的瘤质量、体积、抑瘤率;免疫组织化学法检测原位移植瘤中ARHGAP25阳性表达;RT-PCR法检测原位移植瘤中ARHGAP25 mRNA表达,Western blot法检测ARHGAP25、基质金属蛋白酶7(MMP-7)、基质金属蛋白酶9(MMP-9)、E盒结合锌指蛋白1(ZEB1)及β-连环蛋白(β-catenin)的表达。结果各组裸鼠原位移植成瘤率为100%。ARHGAP25过表达组、空载体+胃肠安低浓度组和空载体+胃肠安高浓度组的抑瘤率分别为88.03%、22.79%和51.48%。ARHGAP25过表达组的原位移植瘤质量较空载体组显著减少(P<0.05);ARHGAP25过表达组和空载体+胃肠安高浓度组的原位移植瘤体积较空载体组显著减小(P<0.05)。与空载体组相比,ARHGAP25过表达组、空载体+胃肠安低浓度组原位移植瘤中ARHGAP25阳性表达面积显著升高(P<0.05);ARHGAP25过表达组、空载体+胃肠安低浓度组、空载体+胃肠安高浓度组的原位移植瘤ARHGAP25 mRNA的表达显著上调(P<0.05);ARHGAP25过表达组、空载体+胃肠安低浓度组、空载体+胃肠安高浓度组的原位移植瘤ARHGAP25蛋白表达显著上调(P<0.05),MMP-7、MMP-9、ZEB1及β-catenin蛋白表达明显下调(P<0.05)。结论胃肠安可抑制裸鼠结肠癌原位移植瘤的生长,促进ARHGAP25表达,降低上皮-间充质转化相关分子ZEB1、β-catenin、MMP-7及MMP-9的表达,抑制结肠癌裸鼠原位移植瘤的侵袭转移。

Objective To study the effect of Weichang’an on the expression of Rho guanosine triphosphatase activating protein 25(ARHGAP25)in orthotopic xenografts of colon cancer in nude mice,and to explore the molecular mechanism of Weichang’an inhibiting the invasion and metastasis of colon cancer.Methods The orthotopic xenograft model was made by human colon cancer HCT-116 cells.The nude mice were randomly divided into empty vector group,ARHGAP25 overexpression group,empty vector+Weichang’an low concentration group and empty vector+Weichang’an high concentration group with 6 mice in each group.The body mass,volume and tumor inhibition rate of orthotopic xenografts were compared.The positive expression of ARHGAP25 was detected by immunohistochemistry,the expression of ARHGAP25 mRNA was detected by RT-PCR,and the protein expressions of ARHGAP25,matrix metalloproteinase-7(MMP-7),matrix metalloproteinase-9(MMP-9),E-box binding zinc finger protein 1(ZEB1)andβ-catenin(β-catenin)were detected by Western blot.Results The rate of tumor formation in nude mice was 100%.The tumor inhibition rates of ARHGAP25 overexpression group,empty vector+Weichang’an low concentration group and empty vector+Weichang’an high concentration group were 88.03%,22.79%and 51.48%,respectively.The weight of orthotopic xenografts in ARHGAP25 overexpression group was significantly lower than that in empty vector group(P<0.05);the volume of orthotopic xenografts in ARHGAP25 overexpression group and empty vector+Weichang’an high concentration group was significantly smaller than that in empty vector group(P<0.05).Compared with those in the empty vector group,the positive expression area of ARHGAP25 was significantly increased in ARHGAP25 overexpression group and empty vector+Weichang’an low concentration group(P<0.05);the expression of ARHGAP25 mRNA was significantly up-regulated in ARHGAP25 overexpression group,empty vector+Weichang’an low concentration group and empty vector+Weichang’an high concentration group(P<0.05);the expression of ARHGAP25 protein was significantly up-regulated(P<0.05),while the expression of MMP-7,MMP-9,ZEB1 andβ-catenin were significantly down-regulated(P<0.05)in ARHGAP25 overexpression group,empty vector+Weichang’an low concentration group and empty vector+Weichang’an high concentration group.Conclusion Weichang'an can inhibit the growth of orthotopic xenografts of colon cancer in nude mice,promote the expression of ARHGAP25,reduce the expression of epithelial-mesenchymal transition-related molecules including ZEB1,β-catenin,MMP-7 and MMP-9,restrict the invasion and metastasis of orthotopic colon cancer in nude mice.