MicroRNA-125b functions as a tumor suppressor in hepatocellular carcinoma cells - PubMed (original) (raw)

MicroRNA-125b functions as a tumor suppressor in hepatocellular carcinoma cells

Hong-Yan Jia et al. Int J Mol Sci. 2012.

Abstract

MicroRNAs (miRNAs) are important regulators of multiple cellular processes, and the deregulation of miRNA is a common event in diverse human diseases, particularly cancer. However, the mechanisms underlying the relationship between disordered miRNA expression and tumorigenesis have remained largely unknown. In this study, we demonstrated the down-regulation of miR-125b in hepatocellular carcinoma (HCC) tissues and HCC cell lines by Northern blot and quantitative RT-PCR analyses. The ectopic expression of miR-125b reduced the cellular proliferation and cell cycle progression of HCC cells by targeting Mcl-1 and IL6R. Furthermore, the miR-125b-induced inhibition of cell proliferation was rescued by the expression of Mcl-1 or IL6R variants that lacked 3' UTRs. Thus, this study revealed the differential expression of miR-125b in HCC cells and elucidated its potential as a tumor suppressor in HCC development.

Keywords: IL6R; Mcl-1; hepatocellular carcinoma; miR-125b.

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Figures

Figure 1

The expression of miR-125b is down-regulated in both primary hepatocellular carcinoma (HCC) tissues and HCC cell lines. (A) Images of HCC tissues stained with haematoxylin and eosin (H&E). (B) Northern blot analysis indicating a marked decrease in miR-125b in six pairs of HCC tissues (HCC) relative to matching controls (NC). (C) Northern blot analysis demonstrating the remarkable decrease in miR-125b expression in the HCC cell lines (HepG2, Huh7, and SMMC7721) relative to normal liver tissue (NC). (D, E) Validation of the differential expression of miR-125b in amplified HCC tissues and control cells. Panel D illustrates the expression levels of miR-125b in an independently validated set of 32 HCC patients and matched controls. The levels of miR-125b expression were quantified by real-time RT-PCR and normalized using the non-tumor control sample. Panel E provides the statistical results regarding the miR-125b expression in HCC tissues and matched controls. The P values in this panel were calculated using a nonparametric test.

Figure 2

The over-expression of miR-125b inhibits the cell proliferation and cell cycle in HepG2. (A) The CCK-8 assay used to evaluate the proliferation of HepG2 cells after transfection with the miR-125b mimic or the scrambled oligonucleotide at different culture durations. (B) The cell cycle analysis of HepG2 cells treated with either the miR-125b mimic or the scrambled oligonucleotide and cultured for 24 h after cell transfection.

Figure 3

Mcl-1 and Il6R are targets of miR-125b. (A) The sequences of the miR-125b binding sites within the human Mcl-1 and Il6R 3′ UTRs and schematic reporter constructs. In this panel, Mcl-1_WT and IL6R_WT represent the reporter constructs containing the entire 3′ UTR sequences of Mcl-1 and IL6R. Mcl-1_MUT and IL6R_MUT represent the reporter constructs containing mutated nucleotides. (B) The analysis of the relative luciferase activities of Mcl-1_WT, Il6R_WT, Mcl-1_MUT, and IL6R_MUT in 293T cells. The error bars are derived from triplicate experiments, and * indicates p < 0.01. (C) Left, the immunoblotting results for Mcl-1 and Il6R in extracts from HepG2 cells transfected with either the miR-125b mimic or the scrambled oligonucleotide; right, relative expression of Mcl-1 and Il6R mRNA from HepG2 cells transfected with miR-125b mimic or scrambled oligonucleotide.

Figure 3

Figure 4

The functional relevance of miR-125b and its targets in HCCs. (A) Results of the CCK-8 assay for the proliferation of HepG2 cells after their transfection with either specific siRNAs targeting Mcl-1 (si_Mcl-1) and IL6R (si_IL6R) or the scrambled oligonucleotide at different culture durations. (B) The cell-cycle analysis of HepG2 cells treated with either specific siRNAs targeting Mcl-1 (si_Mcl-1) and IL6R (si_IL6R) or with the scrambled oligonucleotide and cultured for 24 h after cell transfection. (C) HepG2 cells were treated under the “rescue” condition; this panel illustrates the immunoblotting results of Mcl-1 and IL6R in extracts from HepG2 cells that were either transfected with the miR-125b mimic or the scrambled oligonucleotide for 24 h and then subsequently treated for an additional 48 h with either pcDNA-Mcl-1, pcDNA-IL6R, or pcDNA-empty. (D) The cell cycle analysis of HepG2 cells treated under the “rescue” condition described in the previous panel.

Figure 4

References

1. Bartel D.P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. - PubMed
1. Kim V.N. Small RNAs: Classification, biogenesis, and function. Mol. Cells. 2005;19:1–15. - PubMed
1. Nicolas F.E., Lopez-Martinez A.F. MicroRNAs in human diseases. Recent Pat. DNA Gene Seq. 2010;4:142–154. - PubMed
1. Small E.M., Olson E.N. Pervasive roles of microRNAs in cardiovascular biology. Nature. 2011;469:336–342. - PMC - PubMed
1. Redova M., Svoboda M., Slaby O. MicroRNAs and their target gene networks in renal cell carcinoma. Biochem. Biophys. Res. Commun. 2011;405:153–156. - PubMed

MicroRNA-125b functions as a tumor suppressor in hepatocellular carcinoma cells - PubMed (original) (raw)