Jingjing Huang, Hongna Huang, Wenfu Zhang, Jianlin Lv, Guochu Huang, Yuanjia Lin, Songlin Chen, Yueqiang Hu
Department of Spleen, Stomach and Liver Diseases, The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning, China.
Guangxi Key Laboratory of Translational Medicine for Treating High-Incidence Infectious Diseases with Integrative Medicine, Nanning, China.
Int J Stem Cells. 2021 Aug 30;14(3):275-285. doi: 10.15283/ijsc20157.
Cancer stem cells (CSCs) with tumorigenic potential are reported as the crucial factors of hepatocellular carcinoma (HCC) recurrence and therapy resistance. Bone mesenchymal stem cells (BMSCs) are documented to play an important role in the protection of hepatocytes. Bie Jia Jian pill (BJJP), a Traditional Chinese Medicine, has been used to treat liver fibrosis and liver cancer. This study aimed to explore the potential role of combined use of BJJP with BMSCs in HCC cell lines.
Flow cytometry was used to identify BMSCs isolated from BALB/c mice and CSCs enriched from Huh7 cells by measuring CD24, CD133, CD44, CD73, CD105, CD166, CD29, CD14 and CD34. Differentiation potential of BMSCs was also determined. Cell viability and proliferation ability of CSCs were determined by CCK-8 assay and clone formation assay. The expressions of CSCs biomarkers and Wnt/-catenin signal pathway related proteins were determined by PCR and western blot. TOP-Flash/FOP-Flash luciferase assay was applied to measure the activity of -catenin/TCF. Compared with untreated CSCs, BJJP or BMSCs treatment alone on CSCs lead to increased miR-140 expression and cell apoptosis, as well as decreased expressions of CD24, CD133, EpCAM and cell viability. Downregualted expressions of Wnt/-catenin signal pathway related proteins, Wnt3a and -catenin were found in response to BJJP or BMSCs treatment alone. The combination of BJJP＋BMSCs treatment on CSCs could further enhance the suppressive effect on CSCs. Down-regulation of miR-140 in CSCs partially blocked the effects of BMSCs or BMSCs＋BJJP on the expressions of Wnt3a and -catenin as well as the cell viability and apoptosis of CSCs. Reversed expression pattern was found in CSCs transfected with miR-140 overexpression.
Taken together, we demonstrate that BJJP＋BMSCs together could further enhance the suppressive effect on CSCs through regulating miR-140 and suppressing Wnt/-catenin signal pathway. This study demonstrated the potential of BJJP＋BMSCs in therapeutic treatment of HCC.
具有致瘤潜能的癌症干细胞（CSCs）被认为是肝细胞癌（HCC）复发及治疗耐药的关键因素。有文献记载，骨髓间充质干细胞（BMSCs）在保护肝细胞方面发挥重要作用。鳖甲煎丸（BJJP）作为一种中药，已被用于治疗肝纤维化和肝癌。本研究旨在探讨BJJP与BMSCs联合应用于肝癌细胞系的潜在作用。
采用流式细胞术，通过检测CD24、CD133、CD44、CD73、CD105、CD166、CD29、CD14和CD34来鉴定从BALB/c小鼠分离的BMSCs以及从Huh7细胞富集的CSCs。同时也测定了BMSCs的分化潜能。采用CCK-8法和克隆形成试验测定CSCs的细胞活力和增殖能力。通过PCR和蛋白质印迹法测定CSCs生物标志物及Wnt/β-连环蛋白信号通路相关蛋白的表达。应用TOP-Flash/FOP-Flash荧光素酶试验检测β-连环蛋白/TCF的活性。与未处理的CSCs相比，单独用BJJP或BMSCs处理CSCs可导致miR-140表达增加和细胞凋亡，以及CD24、CD133、EpCAM表达降低和细胞活力下降。单独用BJJP或BMSCs处理可使Wnt/β-连环蛋白信号通路相关蛋白Wnt3a和β-连环蛋白的表达下调。BJJP＋BMSCs联合处理CSCs可进一步增强对CSCs的抑制作用。CSCs中miR-140的下调部分阻断了BMSCs或BMSCs＋BJJP对Wnt3a和β-连环蛋白表达以及CSCs细胞活力和凋亡的影响。在转染miR-140过表达的CSCs中发现了相反的表达模式。
综上所述，我们证明BJJP＋BMSCs可通过调节miR-140和抑制Wnt/β-连环蛋白信号通路进一步增强对CSCs的抑制作用。本研究证明了BJJP＋BMSCs在肝癌治疗中的潜力。
