Compound Astragalus and Salvia miltiorrhiza extract inhibits hepatocellular carcinoma progression via miR-145/miR-21 mediated Smad3 phosphorylation
Compound Astragalus and Salvia miltiorrhiza extract inhibits hepatocellular carcinoma progression via miR-145/miR-21 mediated Smad3 phosphorylation
复制标题
复方黄芪丹参提取物通过 miR-145/miR-21 介导的 Smad3 磷酸化抑制肝细胞癌进展
DOI：
10.1016/j.jep.2018.11.007
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发表时间：
2019-03-01
影响因子：
5.4
通讯作者：
Yang, Yan
中科院分区：
文献类型：
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作者：
Wu, Chao;Chen, Weiyang;Yang, Yan
民族药理相关性：复方黄芪丹参提取物(CASE)，含黄芪总皂苷、黄芪多糖。太棒了。丹参中的丹参酚酸通过介导转化生长因子-β(TGF-β)/Smad信号转导，尤其是Smad3的磷酸化，抑制肝癌的发生。MicroRNA-145/microRNA-21(miR-145/miR-21)与Smad3磷酸化之间的相互作用与肝细胞癌的发生发展密切相关。然而，CASE对肝癌进展的影响涉及miR-145/miR-21的表达及其与转化生长因子-β/MAPK/Smad通路下游Smad3磷酸化的相互作用，目前尚不清楚。材料与方法：建立了体内[二乙基亚硝胺(DEN)诱导的大鼠肝癌模型]和体外[转化生长因子-β(1)刺激的HepG2细胞]肝癌模型，并采用不同剂量/浓度(大鼠使用60、120、240 mg/kg；HepG2细胞使用20、40、80 mU/ml)、miR-145和miR-21联合给药。将miR-145antagomir、miR-21agomir和Smad3C/L(分别与pSmad3c、pSmad3L和pSmad3c/3L表达上调相关的Smad3EPSM、Smad33S-A和Smad3WT)分别导入HepG2细胞，用CASE(80ug/m1)处理。同样，分别用miR-145、miR-21和CASE(310 mg/kg)处理HepG2细胞，分别构建稳定表达SMAD3 WT、SMAD3 EPSM和SMAD3 3S A的细胞株，建立裸鼠移植瘤模型，用CASE(310 mg/kg)处理。检测移植瘤细胞的增殖、迁移、凋亡、肿瘤生长和组织病理学特征，并检测结构域特异性Smad3磷酸化异构体(pSmad3C/pSmad3L)、活化的MAPKs(pERK1/2、pJNK1/2、pp38)和miR-145、miR-21的表达。CASE通过增加miR-145和促进miR-145调控的pSmad3L->；pSmad3C信号转导，同时抑制miR-21和miR-21调节的MAPK依赖的Smad3L磷酸化，抑制了转化生长因子-β(1)刺激的HepG2细胞和裸鼠移植瘤的细胞迁移、增殖和肿瘤生长，促进了细胞的凋亡。同时，上调的pSmad3C增强了CASE对肿瘤生长的抑制作用，促进了细胞的凋亡，参与了miR-145表达的增加和miR-21表达的降低，而pSmad3L的表达上调则相反。结论：CASE通过介导miR-145/miR-21与SMAD3磷酸化的相互作用抑制肝癌进展，尤其是miR-145/miR-21介导的SMAD3磷酸化，为CASE抗肝癌治疗提供了重要的理论依据。
Ethnopharmacological relevance: Compound Astragalus and Salvia miltiorrhiza extract (CASE), containing astragalosides, astragalus polysaccharide extracted from Astragalus membranaceus (Fisch.) Bge. and salvianolic acids from Salvia miltiorhiza Bge., has been found to inhibit hepatocarcinogenesis via mediating transforming growth factor-beta (TGF-beta)/Smad signaling, especially Smad3 phosphorylation. The crucial interaction between microRNA-145/microRNA-21 (miR-145/miR-21) and Smad3 phosphorylation is implicated in the pathogenesis and progression of hepatocellular carcinoma (HCC). However, effects of CASE on HCC progression involved in the expression of miR-145/miR-21 and their interaction with Smad3 phosphorylation downstream of TGF-beta/ MAPK/Smad pathway remain unclear. This study addressed above questions using in vitro (HepG2 cells) and in vivo (Xenografts of nude mice) models of HCC.Materials and methods: In vivo [Diethylnitrosamine (DEN)-induced HCC in rats] and in vitro [TGF-beta(1)-stimulated HepG2 cells] models of HCC were established and co-administrated using graded doses/concentrations CASE (60, 120, 240 mg/kg used in rats; 20, 40, 80 mu g/ml used in HepG2 cells), miR-145 and miR-21 were measured. HepG2 cells were transfected with miR-145 antagomir, miR-21 agomir and Smad3C/L plasmids (Smad3 EPSM, Smad3 3S-A and Smad3 WT related to up-regulated expression of pSmad3C, pSmad3L and pSmad3C/3L respectively) and then treated by CASE (80 mu g/m1). Similarly, HepG2 cell xenografted nude mice were administered with miR-145 antagomir, miR-21 agomir and CASE (310 mg/kg); Smad3 WT, Smad3 EPSM and Smad3 3S A plasmids stably transfected HepG2 cell lines were constructed respectively and their xenografted nude mice were established, and then treated by CASE (310 mg/kg). Cell proliferation, migration, apoptosis, tumor growth and histopathologic characteristics of xenografts were assessed; also, domain-specific Smad3 phosphorylation isoforms (pSmad3C/pSmad3L), activated MAPKs (pERK1/2, pJNK1/2, pp38) and miR-145, miR-21 were measured.Results: CASE up-regulated miR-145 while down-regulated miR-21 expression in both rats with DEN-induced HCC and TGF-beta(1)-stimulated HepG2 cells; CASE inhibited cell migration, proliferation and tumor growth while facilitated cell apoptosis in TGF-beta(1)-stimulated HepG2 cells and xenografts of nude mice with miR-145 antagomir/miR-21 agomir treatment via increasing miR-145 and facilitating miR-145 modulated pSmad3L -> pSmad3C signaling switch while decreasing miR-21 and inhibiting miR-21 modulated MAPK-dependent Smad3L phosphorylation. Also, up-regulated pSmad3C enhanced inhibited effect of CASE on tumor growth and facilitated effect of CASE on cell apoptosis involved in increased miR-145 while decreased miR-21 expression, however, inverse phenomena were observed when up-regulated pSmad3L.Conclusion: Our results suggest that CASE inhibits HCC progression via mediating the interaction of miR-145/ miR-21 and Smad3 phosphorylation, especially miR-145/miR-21 mediated Smad3 phosphorylation, which maybe provides an important theoretical foundation for CASE's anti-HCC therapy used for patients in a near future.