基于Wnt5a/Ca^(2+)/NFAT信号通路研究小陷胸汤调控Ca^(2+)载量抑制MGC-803细胞侵袭迁移和上皮间质转化作用

Xiao Xianxiongtang Regulates Ca^(2+) Load and Inhibits Epithelial-mesenchymal Transition,Invasion,and Migration of MGC-803 Cells:Based on Wnt5a/Ca^(2+)/NFAT Signaling Pathway

目的探讨小陷胸汤对转化生长因子-β_(1)(TGF-β_(1))诱导胃癌MGC-803细胞侵袭转移及上皮间质转化的影响,并探讨其可能的机制。方法通过CB-DOCK在线平台(http://clab.labshare.cn/cb-dock/)预测小陷胸汤与活化T细胞核转录因子(NFAT)分子对接。使用质量浓度为10μg·L^(-1)的TGF-β_(1)建立人胃癌MGC-803细胞侵袭转移模型,将MGC-803细胞分为空白组、模型组、小陷胸汤组(0.1、0.2、0.4 g·L^(-1)),为进一步探讨Wnt5a/Ca^(2+)/NFAT信号通路在小陷胸汤抑制胃癌中的关键参与作用,将Wnt5a过表达质粒转染MGC-803细胞,分为空白质粒组、Wnt5a-OE组、空白质粒+小陷胸汤(0.4 g·L^(-1))组和Wnt5a-OE+小陷胸汤(0.4 g·L^(-1))组。分别使用细胞增殖与活性检测(CCK-8)法、Transwell小室实验、划痕愈合实验、蛋白免疫印迹法(Western blot)、免疫荧光法检测MGC-803细胞活力、侵袭能力、迁移能力、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、锌指蛋白(Snail)、Wnt5a/Ca^(2+)/NFAT信号通路关键蛋白Wnt5a、钙调神经磷酸酶(CaN)、NFAT1、磷酸化(p)-NFAT1和NFAT1核蛋白表达以及细胞Ca^(2+)浓度变化。结果分子对接提示小陷胸汤作用于Wnt5a/Ca^(2+)/NFAT信号通路。与模型组比较,小陷胸汤(0.1、0.2、0.4 g·L^(-1))能明显促进MGC-803细胞活力的丧失,可通过基质凝胶侵入Transwell下室抑制细胞,并以剂量依赖性的方式减缓细胞划痕愈合,并促进E-cadherin的表达,抑制N-cadherin、Vimentin和Snail的表达(P<0.05,P<0.01)。进一步实验表明,与模型组比较,小陷胸汤可以抑制Wnt5a、CaN、NFAT1和p-NFAT1的表达,降低NFAT1核表达和NFAT1介导的转录活性,从而降低细胞Ca^(2+)浓度,且可逆转Wnt5a的作用(P<0.05,P<0.01)。结论小陷胸汤可通过Wnt5a/Ca^(2+)/NFAT通路减弱人胃癌MGC-803细胞的侵袭转移和上皮间质转化(EMT),从而减弱TGF-β_(1)诱导的促瘤作用。这提示小陷胸汤可能通过调节胃癌的侵袭、转移和EMT来预防和治疗胃癌。

Objective To explore the effect of Xiao Xianxiongtang(XXXT)on the transforming growth factor(TGF)-β_(1)-induced invasion,metastasis,and epithelial-mesenchymal transition(EMT)of gastric cancer MGC-803 cells and the underlying mechanism.Method The molecular docking between XXXT and nuclear factor of activated T cells(NFAT)was performed by CB-DOCK(http://clab.labshare.cn/cb-dock/).The invasion and metastasis model of MGC-803 cells was established with 10μg·L^(-1) TGF-β_(1).MGC-803 cells were classified into blank group,model group,0.1 g·L^(-1) XXXT group,0.2 g·L^(-1) XXXT group,and 0.4 g·L^(-1) XXXT group.For further clarifying the key role of Wnt5a/Ca^(2+)/NFAT signaling pathway in the inhibition of XXXT on gastric cancer,MGC-803 cells were transfected with Wnt5a overexpression plasmid,and then the cells were classified into blank plasmid group,Wnt5a-OE group,blank plasmid+XXXT(0.4 g·L^(-1))group,and Wnt5a-OE+XXXT(0.4 g·L^(-1))group.Cell viability was determined by cell counting kit-8(CCK-8)assay,cell invasion and migration ability by Transwell invasion assay and wound healing assay,expression of EMT-related proteins(E-cadherin,N-cadherin,Vimentin,Snail)and Wnt5a/Ca^(2+)/NFAT signaling pathway-related key proteins[Wnt5a,calcineurin(CaN),NFAT1,and p-NFAT1]by Western blot,and changes in intracellular Ca^(2+)concentration by immunofluorescence assay.Result Molecular docking suggested that XXXT acted on Wnt5a/Ca^(2+)/NFAT signaling pathway.XXXT(0.1,0.2,0.4 g·L^(-1))significantly promoted the loss of MGC-803 cell viability(P<0.05,P<0.01).It inhibited cells from invading the transwell lower chamber and slowed down the healing of cell wounds in a dose-dependent manner(P<0.05,P<0.01).Moreover,it promoted the expression of E-cadherin while suppressed the expression of N-cadherin,Vimentin,and Snail(P<0.05,P<0.01).Further experiments showed that XXXT could inhibit the expression of Wnt5a,CaN,NFAT1,and p-NFAT1,and reduce the nuclear expression of NFAT1 and the transcription activity mediated by NFAT1,so as to reduce the cellular Ca^(2+)concentration(P<0.05,P<0.01).XXXT can reverse the effect of Wnt5a(P<0.05,P<0.01).Conclusion XXXT can attenuate the invasion,metastasis,and EMT of MGC-803 cells via the Wnt5a/Ca^(2+)/NFAT pathway,thereby weakening the tumor-promoting effect of TGF-β_(1).In summary,XXXT may exert therapeutic effect on gastric cancer by regulating the invasion,metastasis,and EMT of gastric cancer cells.