MicroRNA-200a suppresses metastatic potential of side population cells in human hepatocellular carcinoma by decreasing ZEB2

Abstract

Although microRNA-200a (miR-200a) is frequently downregulated in cancer, its role in side population (SP) has not been investigated. In this study, 101 pairs of primary hepatocellular carcinoma (HCC) tissues and matched normal control tissues were analyzed for miR-200a expression and its clinicopathological value was determined. We found that miR-200a was downregulated in HCC/SP and this was associated metastasis. MiR-200a suppressed metastasis of SP cells. Overexpression of miR-200a in SP cells decreased metastasis-related markers and expression of ZEB2. The associations between miR-200a, SP cells and ZEB2 were validated in HCC. These findings reveal that miR-200a suppresses metastasis of SP cells by downregulating ZEB2.

Keywords: hepatocellular carcinoma; metastasis; microRNAs; side population.

Figure 1. MiR-200a is down-regulated in HCC

(A) The expression of miR-200a in HCC tissue specimens and in corresponding non-tumor tissues was measured by qRT-PCR. (B) The expression status of miR-200a in 4 human HCC cell lines and one normal human hepatocyte was measured by qRT-PCR. (C) The relative expression level of miR-200a in primary tumor samples with or without clinically confirmed metastasis was measured by qRT-PCR. (D) Kaplan-Meier analysis of the correlation between miR-200a expression and the overall survival of 101 patients with HCC. Patients in the low miR-200a expression group had a significantly shorter overall survival (P < 0.01); * P < 0.05, t test.

Figure 2. The correlation between miR-200a expression and EMT

(A) An immunohistochemical analysis of miR-200a, ZEB2, and E-cadherin expression in HCC tissues and in adjacent non-tumor tissues. (B) The association between the expression of miR-200a and either ZEB2 or E-cadherin in human patients with HCC.

Figure 3. Identification of side population in HCC cell lines

(A) A subpopulation was shown in a representative sample (red color-nuclear staining with Hoechst33342 dye; SP-side population; NSP-non side population). (B) SP cells of MHCC-97H, Huh7, SMMC-7721 and HepG2 were analyzed by dual wavelength FACS after incubation with Hoechst 33342. (C) A differentiation analysis of SP cells in a subpopulation of SP and NSP cells after four days in culture.

Figure 4. Characteristic of SP and NSP cells

(A) The expression of ALBU (a marker of mature hepatocytes) and KRT14 (a marker of liver stem cells) in side population cells and in non-side population cells that were isolated from HCC cell lines. (B) Sphere formation assay: SP and non-SP cells were cultured in serum-free media for two weeks (magnification×100). (C) Cell invasion and migration assays: (Left panel, representative images of migration assay and quantification of cell number; Right panel, representative images of invasion assay and quantification of cell number). (D) Images of cells stained with Rhodamine Phalloidin and DAPI reflect the different morphologies (magnification×400). *p < 0.05, **p < 0.01, t test.

Figure 5. Functional analysis of miR-200a in vitro

(A) MiR-200a expression in a subpopulation in human HCC cell lines. (B) Expression of miR-200a following transfection was confirmed by qRT-PCR. (C) Relative mRNA expression of metastasis-related markers in groups with and without miR-200a induction. (D) Cell invasion assay: (upper panel, representative images of invasion assay; bottom panel, quantification of cell number); Cell migration assay: (upper panel, representative images of migration assay; bottom panel, quantification of cell number). *p < 0.05, **p < 0.01, t test.

Figure 6. In vivo metastasis assays

(A) Representative bioluminescent imaging (BLI) at 8 weeks is shown for the different groups. (B) The incidence of lung metastases in nude mice. (C) The intensity of luminescence in the different groups. (D) The number of metastatic nodules on the surface of the lungs in mice from the different groups. (E) Representative H&E staining of lung tissues is shown. (F) The overall survival of the nude mice. (G) An immunohistochemical analysis of miR-200a, ZEB2, and E-cadherin expression in metastatic nodules. *p < 0.05, **p < 0.01, t test.

Figure 7. MiR-200a induces the metastasis of SP cells through the transactivation of ZEB2 expression

(A) The miR-200a target site in the 3′UTR of ZEB2. (B) The up-regulation or down-regulation of miR-200a in MHCC-97H demonstrated an effect on ZEB2 according to western-blot. (C) Luciferase activity of ZEB2 after transfection with miR-200a mimics or an inhibitor. (D) Functional evaluation of miR-200a induction on its validated target, ZEB, in different groups by qRT-PCR. (E) Real-time PCR and (F) western-blot were used to detect the expression of EMT markers. (G) Following the infection of the MHCC-97H-SP-miR-200a cells and Huh7-SP-KD-miR-200a cells with ZEB2 siRNA and the designated SP-ZEB2 and SP-anti-ZEB2. The cell invasion and migration capacities were assessed with a transwell assay. *p < 0.05, **p < 0.01, t test.