Bie Jia Jian Pill Combined with Bone Mesenchymal Stem Cells Regulates microRNA-140 to Suppress Hepatocellular Carcinoma Stem Cells

Abstract

Background and objectives: 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.

Methods and results: 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/β-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 β-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/β-catenin signal pathway related proteins, Wnt3a and β-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 β-catenin as well as the cell viability and apoptosis of CSCs. Reversed expression pattern was found in CSCs transfected with miR-140 overexpression.

Conclusions: Taken together, we demonstrate that BJJP＋BMSCs together could further enhance the suppressive effect on CSCs through regulating miR-140 and suppressing Wnt/β-catenin signal pathway. This study demonstrated the potential of BJJP＋BMSCs in therapeutic treatment of HCC.

Keywords: Bie Jia Jian pill; Bone mesenchymal stem cells; Cancer stem cells; Hepatocellular carcinoma; microRNA 140.

Fig. 1

Identification of isolated CSCs and BMSCs and their potential of osteogenic differentiation and adipogenic differentiation ability. (A) CSCs sphere; (B, C) measurement on CSCs biomarker, CD24 and CD133 by Flow cytometry in spheres and in Huh 7 cells; (D) surface antigens of BMSCs, CD44, CD73, CD105, CD166, CD29, CD14, CD34 and CD45 were measured by Flow cytometry; (E) alizarin red staining for determination of osteogenic differentiation; (F) oil red O staining for determination of adipogenic differentiation. CSCs, cancer stem cells; BMSCs, bone mesenchymal stem cells; BJJP, Bie Jia Jian pill.

Fig. 2

Combination use of BJJP and BMSCs further suppress the cell growth of CSCs in addition to enhancing cell apoptosis. (A, B) the expressions of CSCs biomarkers CD24, CD133 and EpCAM were measured with RT-qPCR and western blot; (C) cell proliferation ability was assessed by cell clone formation assay; (D) cell viability was determined by CCK-8 assay; (E) cell apoptosis rate was assessed by Flow cytometry. N=3, *p<0.05, **p<0.01; EpCAM, epithelial cell adhesion molecule; CSCs, cancer stem cells; BMSCs, bone mesenchymal stem cells; BJJP, Bie Jia Jian pill.

Fig. 3

miR-140 expression was increased in response to BJJP or/and BMSCs treatment as determined by RT-qPCR. N=3, *p<0.05, **p<0.01. BMSCs, bone mesenchymal stem cells; BJJP, Bie Jia Jian pill.

Fig. 4

BJJP and BMSCs suppress CSCs cell proliferation and enhance cell apoptosis through regulating miR-140. (A) Transfection efficiency was verified by RT-qPCR; (B, C) RT-qPCR and western blot were applied to detect the expression of CD24, CD133 and EpCAM after cell transfection; (D) cell proliferation ability was assessed by cell clone formation; (E) cell viability was measured by CCK-8 assay; (F) cell apoptosis was determined by Flow cytometry. N=3, *, vs control group, p<0.05; **, vs control group, p<0.01; #, vs BJJP group, p<0.05; *, vs BMSCs group, p<0.05; *, vs BJJP＋BMSCs group, p<0.05; EpCAM, epithelial cell adhesion molecule; CSCs, cancer stem cells; BMSCs, bone mesenchymal stem cells; BJJP, Bie Jia Jian pill.

Fig. 5

BJJP and BMSCs could suppress the activation of Wnt/β-ca-tenin signal pathway through regulating miR-140. (A) Western blot was applied to measure the proteins related to Wnt/β-catenin signal path-way. (B) The activity of β-catenin was measured by TOP-Flash/FOP-Flash luciferase assay. N=3, * vs control group, p<0.05; ** vs control group, p<0.01; # vs BJJP group, p<0.05; * vs BMSCs group, p<0.05; *, vs BJJP＋BMSCs group, p<0.05. BMSCs, bone mesenchymal stem cells; BJJP, Bie Jia Jian pill.