外泌体介导miR-106a靶向Smad7调控间皮细胞MMT对胃癌腹膜转移的影响

Exosomes mediated miR-106a targeting Smad7 on peritoneal metastasis of gastric cancer by regulating MMT of mesothelial cells

目的探讨肿瘤源性外泌体(tumor-derived exosomes,TDEs)通过miR-106a-Smad7调控间皮细胞间质性转化(mesothelial-to-mesenchymal transition,MMT)影响胃癌腹膜转移的机制。方法人胃癌细胞系AGS分离外泌体,转染miR-106a mimic,与人腹膜间皮细胞HMrSV5共培养,EdU检测HMrSV5细胞增殖活力,Western blot法检测Smad7、E-cadherin、N-cadherin蛋白表达。给予TGF-β抑制剂GW788388,EdU检测HMrSV5细胞增殖变化,Western blot法检测SMA、Fibronectin表达。给予Smad7过表达质粒,Western blot法检测MMT相关蛋白表达,Transwell实验检测HMrSV5迁移能力。结果AGS外泌体与HMrSV5共培养后,HMrSV5增殖活力下降,合并miR-106a mimic处理后,HMrSV5增殖活力进一步下降。Western blot显示,外泌体处理后Smad7表达下降,合并miR-106a mimic处理后,Smad7表达进一步下降;与此同时,E-cadherin表达下降,N-cadherin表达升高。TGF-β干预后,EdU检测显示HMrSV5细胞增殖活力下降,但给予GW788388后,HMrSV5细胞增殖活力有所恢复,Western blot显示TGF-β干预后SMA、Fibronectin表达升高,但给予GW788388后,SMA,Fibronectin表达逆转。给予Smad7过表达,Western blot显示SMA、Fibronectin表达下降,Transwell显示HMrSV5迁移能力下降(P<0.001)。结论胃癌细胞外泌体影响腹膜转移的机制可能有间皮细胞MMT转化,而间皮细胞MMT转化可能与外泌体介导的miR-106a转运靶向Smad7激活TGF-β信号通路相关。

Purpose To investigate the mechanism of tumor derived exosomes regulating mesothelial mesenchymal transition through miR-106a-Smad7 on peritoneal metastasis of gastric cancer.Methods The exosomes were isolated from human gastric cancer cell line AGS and transfected with miR-106a mimic.The cells were co-cultured with human peritoneal mesothelial cell HMrSV5.The proliferation of HMrSV5 cells were detected by EdU.The expression of Smad7,E-cadherin and N-cadherin were detected by Western blot.After administration of TGF-βinhibitor GW788388,the activity of HMrSV5 cells was detected by EdU,and the expression of SMA and Fibronectin were detected by Western blot.After giving Smad7 over-expression plasmid,the expression of MMT related proteins were detected by Western blot,and the migratory ability of HMrSV5 was detected by Transwell chamber.Results After co-culture with AGS-exosomes,the proliferation activity of HMrSV5 decreased,and further decreased after combined with miR-106a mimic.Western blot showed that the expression of Smad7 decreased after treatment of exosomes,and further decreased after combined with miR-106a mimic,in the mean time,the expression of E-cadherin decreased,and the expression of N-cadherin increased.After treatment with TGF-β,EdU showed that the proliferation of HMrSV5 cells decreased,but restored with the treatment of GW788388.Western blot showed that the expression of SMA and Fibronectin increased.However,the expression of SMA and Fibronectin reversed with the treatment of GW788388.When Smad7 was over-expressed,Western blot showed that SMA and Fibronectin expression decreased,Transwell showed HMrSV5 migratory ability decreased(P<0.001).Conclusion The mechanism of gastric cancer cell exosomes affecting peritoneal metastasis may include mesothelial cell MMT transformation,which may be related to exosomes mediated miR-106a transport targeting Smad7 and activating TGF-βsignaling pathway.