MicroRNA-200a Suppresses Cell Invasion and Migration by Directly Targeting GAB1 in Hepatocellular Carcinoma

Abstract

MicroRNA-200a (miR-200a) is frequently downregulated in most cancer types and plays an important role in carcinogenesis and cancer progression. In this study, we determined that miR-200a was downregulated in hepatocellular carcinoma (HCC) tissues and cell lines, consistent with the results of our previous study. Because a previous study suggested that downregulation of miR-200a is correlated with HCC metastasis, we aimed to elucidate the mechanism underlying the role of miR-200a in metastasis in HCC. Here we observed that overexpression of miR-200a resulted in suppression of HCC metastatic ability, including HCC cell migration, invasion, and metastasis, in vitro and in vivo. Furthermore, bioinformatics and luciferase reporter assays indicated that GAB1 is a direct target of miR-200a. Inhibition of GAB1 resulted in substantially decreased cell invasion and migration similar to that observed with overexpression of miR-200a in HCC cell lines, whereas restoration of GAB1 partially rescued the inhibitory effects of miR-200a. Taken together, these data provide novel information for comprehending the tumor-suppressive role of miR-200a in HCC pathogenesis through inhibition of GAB1 translation.

Figure 1

Expression of miR-200a in HCC tissues and cell lines. (A) Expression of miR-200a in HCC tissue and corresponding adjacent noncancerous tissues. (B) Relative expression of miR-200a in four HCC cell lines (Huh7, HepG2, SMMC-7721, and MHCC97-H) and a human normal liver cell line (HL-7702) as analyzed by RT-PCR. The data represent the mean ± SD. *p < 0.05.

Figure 2

miR-200a inhibits HCC cell migration and invasion in vitro. (A) Expression of miR-200a following transfection in MHCC97-H and SMMC-7721 cells was confirmed by RT-PCR. (B and C) Overexpression of miR-200a significantly inhibited cell migration and invasion in MHCC97-H and SMMC-7721 cells. Migrated and invaded cells were counted in five randomly selected areas under a 200× microscope field. *p < 0.05 compared to the control.

Figure 3

miR-200a suppresses tumor metastasis in vivo. (A) Incidence of lung metastasis in mice injected with MHCC97-H cells transfected with miR-NC or miR-200a cells through the lateral tail vein. (B) The number of metastatic nodules on the surface of the lungs in mice from the different groups. (C) Representative H&E staining of lung metastatic nodules. *p < 0.05 compared to the control.

Figure 4

GAB1 is a direct downstream target of miR-200a. (A) GAB1 was bioinformatically predicted as a target of miR-200a using online software (http://www.microrna.org/). (B) A miR-200a mimic or negative control and a luciferase vector encoding the wild-type or mutant GAB1 3′-UTR region were introduced into 239T cells, and the relative luciferase activity was measured. (C) Quantitation of GAB1 mRNA levels in MHCC97-H and SMMC-7721 cells by RT-PCR after transfection with LV-miR-NC or LV-miR-miR-200a. (D) Detection of GAB1 protein in MHCC97-H and SMMC-7721 cells by Western blot analysis after transfection with LV-miR-NC or LV-miR-200a. *p < 0.05 compared to the control.

Figure 5

Alterations of GAB1 levels influence the effects of miR-200a on HCC cells. (A) Quantitation of GAB1 mRNA levels in SMMC-7721 cells by RT-PCR after transfection with siRNA targeting GAB1 (si-GAB1) or a negative control siRNA (si-NC). (B) Detection of GAB1 protein in SMMC-7721 cells by Western blot analysis after transfection with GAB1 siRNAs or si-NC. (C) GAB1 knockdown inhibited SMMC-7721 cell migration and invasion. (D) GAB1 reintroduction into SMMC-7721 cells partially rescued the miR-200a-mediated inhibition of cell migration and invasion. Migrated and invaded cells were counted in five randomly selected areas under a 200× microscope field. *p < 0.05 compared to the control.