MicroRNA-125b Functions as a Tumor Suppressor in Hepatocellular Carcinoma Cells
<p>The expression of miR-125b is down-regulated in both primary hepatocellular carcinoma (HCC) tissues and HCC cell lines. (<b>A</b>) Images of HCC tissues stained with haematoxylin and eosin (H&E). (<b>B</b>) Northern blot analysis indicating a marked decrease in miR-125b in six pairs of HCC tissues (HCC) relative to matching controls (NC). (<b>C</b>) 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). (<b>D</b>, <b>E</b>) 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.</p> ">
<p>The over-expression of miR-125b inhibits the cell proliferation and cell cycle in HepG2. (<b>A</b>) 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>B</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.</p> ">
<p>Mcl-1 and Il6R are targets of miR-125b. (<b>A</b>) 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>B</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 <span class="html-italic">p</span> < 0.01. (<b>C</b>) 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.</p> ">
<p>Mcl-1 and Il6R are targets of miR-125b. (<b>A</b>) 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>B</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 <span class="html-italic">p</span> < 0.01. (<b>C</b>) 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.</p> ">
<p>The functional relevance of miR-125b and its targets in HCCs. (<b>A</b>) 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>B</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. (<b>C</b>) 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. (<b>D</b>) The cell cycle analysis of HepG2 cells treated under the “rescue” condition described in the previous panel.</p> ">
<p>The functional relevance of miR-125b and its targets in HCCs. (<b>A</b>) 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>B</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. (<b>C</b>) 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. (<b>D</b>) The cell cycle analysis of HepG2 cells treated under the “rescue” condition described in the previous panel.</p> ">
Abstract
:1. Introduction
2. Results and Discussion
2.1. MiR-125b Expression is Down-Regulated in both HCC Tissues and HCC Cell Lines
2.2. MiR-125b Inhibits the Proliferation and Cell Cycle Progression of HCC Cells
2.3. Mcl-1 and IL6R Are Direct Targets of miR-125b in HCC
2.4. MiR-125b Suppresses the Expression of Mcl-1 and IL6R in HCC
3. Experimental Section
3.1. Human Liver Tissues and Cell Lines
3.2. Oligonucleotides, Constructs, Cell Transfection, and Dual Luciferase Assays
3.3. The Rescue Assays of Mcl-1 and IL6R Gene Expression
3.4. RNA Isolation, Northern Blots and Western Blots
3.5. Quantitative Real Time RT-PCR
3.6. Cell Proliferation Assay
3.7. Flow Cytometry
3.8. Statistical Analysis
4. Conclusions
Supplementary Information
ijms-13-08762-s001.pdfAcknowledgments
- Conflict of InterestThe authors declare no conflict of interest.
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Jia, H.-Y.; Wang, Y.-X.; Yan, W.-T.; Li, H.-Y.; Tian, Y.-Z.; Wang, S.-M.; Zhao, H.-L. MicroRNA-125b Functions as a Tumor Suppressor in Hepatocellular Carcinoma Cells. Int. J. Mol. Sci. 2012, 13, 8762-8774. https://doi.org/10.3390/ijms13078762
Jia H-Y, Wang Y-X, Yan W-T, Li H-Y, Tian Y-Z, Wang S-M, Zhao H-L. MicroRNA-125b Functions as a Tumor Suppressor in Hepatocellular Carcinoma Cells. International Journal of Molecular Sciences. 2012; 13(7):8762-8774. https://doi.org/10.3390/ijms13078762
Chicago/Turabian StyleJia, Hong-Yan, Yu-Xuan Wang, Wen-Ting Yan, Hui-Yu Li, Yan-Zhang Tian, Shi-Ming Wang, and Hao-Liang Zhao. 2012. "MicroRNA-125b Functions as a Tumor Suppressor in Hepatocellular Carcinoma Cells" International Journal of Molecular Sciences 13, no. 7: 8762-8774. https://doi.org/10.3390/ijms13078762