MicroRNA-424 is down-regulated in hepatocellular carcinoma and suppresses cell migration and invasion through c-Myb

Abstract

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs) are important regulators of multiple cellular processes, and the aberrant miRNAs expressions have been observed in different types of cancer including HCC. Their pathysiologic role and their relevance to tumorigenesis are still largely unknown. In this study, we demonstrated the down-regulation of miR-424 in HCC cell lines and tissues by quantitative RT-PCR analyses. Overexpression of miR-424 reduced the HCC cell prolifetation, migration, and invasion. Conversely, inhibiton of miR-424 expression significantly accelerated the cell proliferation, migration, and invasion. In addition, we further identified c-Myb as a functional downstream target of miR-424 by directly targeting the 3'UTR of c-Myb. Furthermore, overexpression of c-Myb impaired miR-424-induced inhibition of proliferation and invasion in HCC cells. Our results demonstrated that miR-424 was involved in tumorigenesis of HCC at least in part by suppression of c-Myb.

Figure 1. The expression of miR-424 is down-regulated in both primary HCC tissuess and cell lines.

(A) qRT-PCR analysis of miR-424 expression in normal hepatopcytes (HL-7792 cells) and HCC cells (HepG2, Hep3B, Bel7402, SMMC-7721). (B) qRT-PCR analysis of miR-424 expression in 80 pairs HCC tissues and their corresponding adjacent nontumorous livers. The expression of miR-424 was normalized to U6 snRNA. (C) Relative miR-424 expression levels in HCC tissues and adjacent normal regions; (D) and (E) Statistical analysis of the association between miRNA level, pTNM stage (I, II, III and IV) and pM stage (No metastasis and Metastasis, respectively); *p<0.05, and **p<0.01.

Figure 2. Overexpression of miR-424 inhibits the cell proliferation in HepG2.

(A) qRT-PCR analysis of miR-424 expression after the transfection of miR-424 mimics, inhibitors or scramble or no transfection. (B) The CCK8 assay used to evalute the proliferation of HepG2 cells after transfection with the miR-424 mimics, inhibitors or scramble or no transfection. (C) (C) miR-424 inhibited Ki-67 mRNA expression. HepG2 cells were transfected with miR-424 mimics, inhibitors or scramble or no transfection. Ki-67 mRNA levels were detected by real-time PCR. The expression of Ki-67 was normalized to GAPDH. (D) miR-424 inhibited Ki-67 protein expression. HepG2 cells were transfected with miR-424 mimics, inhibitors or scramble or no transfection. Ki-67 levels were detected by western blot. GAPDH was also detected as a loading control. Values are presented as the mean ± SD. Compared with control, *p<0.05, ** p<0.01, and ***p<0.001.

Figure 3. Ectopic expression of miR-424 suppressed HCC cell migration and invasion.

(A) Wound healing assays of HepG2 cells after treatment with miRNA mimics, inhibitors or scramble or no transfection; the relative ratio of wound closure per field is shown. (B) Transwell analysis of HepG2 cells after treatment with miRNA mimics, inhibitors or scramble or no transfection; the relative ratio of invasive cells per field is shown below, ** p<0.01, and ***p<0.001.

Figure 4. miR-424 targets at c-Myb in HCC cells.

(A) The sequences of miR-424 binding sites within the human c-Myb 3′UTRs and schematic reporter constructs, in this panel, c-Myb-WT represent the reporter constructs containing the entire 3′UTR sequences of c-Myb. C-Myb-MUT represent the reporter constructs containing mutated nucleotides. (B) The analysis of the relative luciferase activities of c-Myb-WT, c-Myb-MUT in 293T cells. The error bars are derived from triplicate expriments. (C) qRT-PCR analysis of c-Myb mRNA expression in HepG2 cells after treatment with miRNA mimics or scramble or no transfection. The expression of c-Myb was normalized to GAPDH. (D) (E) Western blot analysis of c-Myb expression in HepG2 cells transfected with miR-424 mimics or scramble or no transfection. GAPDH was also detected as a loading control. ** p<0.01.

Figure 5. Overexpression of c-Myb impairs miR-424-induced inhibition of proliferation and invasion in HCC cells.

(A) Western blot analysis of c-Myb expression in 8 miR-424 down-regulated HCC tissues. GAPDH was also detected as a loading control. (B) Western blot analysis of c-Myb expression in HepG2 cells co-transfected with either miR-424 mimics or scramble and pCDNA-c-Myb or pCDNA empty vector; GAPDH was also detected as a loading control. (C) Cell growth curves in HepG2 cells transfected with different combinations. (D) Transwell analysis of HepG2 cells treated with different combinations. The relative ratio of invasive cells per field is shown right. *p<0.05,** p<0.01, ***p<0.001.