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. 2016 Mar 29;6:23772.
doi: 10.1038/srep23772.

miR-93 Functions as an oncomiR for the Downregulation of PDCD4 in Gastric Carcinoma

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Free PMC article

miR-93 Functions as an oncomiR for the Downregulation of PDCD4 in Gastric Carcinoma

Hongwei Liang et al. Sci Rep. .
Free PMC article

Abstract

Programmed cell death 4 (PDCD4), as a tumor suppressor gene, is frequently reduced in a variety of tumors, including gastric cancer. Previous findings have indicated that PDCD4 participates in tumorigenesis through the regulation of apoptosis, but the molecular basis of this process has not been fully elucidated, and no studies have shown the upstream regulation of this gene in gastric cancer. In this study, we used bioinformatics analysis to search for miRNAs that could potentially target PDCD4 and identified miR-93 as a candidate. Moreover, we observed the inverse correlation between miR-93 and PDCD4 protein levels, but not mRNA levels, in human gastric cancer tissues. We further experimentally validated PDCD4 as the direct target of miR-93 by evaluating PDCD4 expression in gastric cancer cells after the overexpression or knockdown of miR-93. Additionally, the biological consequences of targeting PDCD4 through miR-93 were examined using cell apoptosis assays in vitro. We demonstrated that the repression of PDCD4 through miR-93 suppressed the apoptosis of gastric cancer cells. Finally, we revealed that miR-93 promoted the development of gastric tumor growth in xenograft mice by negatively regulating PDCD4. Taken together, the findings of the present study indicated the oncogenic role of miR-93 in gastric cancer tumorigenesis through targeting PDCD4, particularly in apoptosis.

Figures

Figure 1
Figure 1. PDCD4 protein and mRNA expression levels in gastric cancer tissues.
(A,B) Western blot analysis of the expression levels of the PDCD4 protein in 6 pairs of gastric cancer (GC) and normal adjacent (GN) tissue samples. (A) representative image; (B) quantitative analysis. (C) Quantitative RT-PCR analysis of the relative expression levels of PDCD4 mRNA in 6 pairs of GC and GN samples. ***P < 0.001.
Figure 2
Figure 2. Profiling of miRNA expression in gastric cancer tissues and normal adjacent tissues by miRNA microarray technology.
The expression of miRNAs is hierarchically clustered on the y axis, and GC (gastric cancer tissue samples) or GN (normal adjacent tissue samples) are hierarchically clustered on the x axis. The relative miRNA expression is depicted according to the color scale shown on the right. Red indicates upregulation; green, downregulation.
Figure 3
Figure 3. Detection of an inverse correlation between miR-93 and PDCD4 levels in gastric cancer tissue samples.
(A) Schematic depicting the hypothetical duplexes formed through interactions between the binding sites in the PDCD4 3′-UTR (top) and miR-93 (bottom). The predicted free energy of each hybrid is indicated. The seed recognition sites are denoted, and all nucleotides in these regions are highly conserved across species. (B) Quantitative RT-PCR analysis of the miR-93 expression levels in six pairs of GC and GN samples. (C) Pearson’s correlation scatter plot of the fold-change in the levels of miR-93 and PDCD4 protein in human gastric cancer tissues. (D) Pearson’s correlation scatter plot of the fold-change in the levels of miR-93 and PDCD4 mRNA in human gastric cancer tissues. ***P < 0.001.
Figure 4
Figure 4. Direct post-transcriptional regulation of PDCD4 expression through miR-93.
(A) Quantitative RT-PCR analysis of the miR-93 levels in AGS cells treated with pre-miR-control, pre-miR-93, anti-miR-control or anti-miR-93. (B,C) Western blot analysis of PDCD4 protein levels in AGS cells treated with pre-miR-control, pre-miR-93, anti-miR-control or anti-miR-93. (B) representative image; (C) quantitative analysis. (D) Quantitative RT-PCR analysis of PDCD4 mRNA levels in AGS cells treated with pre-miR-control, pre-miR-93, anti-miR-control or anti-miR-93. (E) Firefly luciferase reporters containing either wild-type (WT) or mutant (Mut) miR-93 binding sites in the PDCD4 3′-UTR were co-transfected into AGS cells along with pre-miR-control, pre-miR-93, anti-miR-control or anti-miR-93. Twenty-four hours post-transfection, the cells were assayed using a luciferase assay kit. ***P < 0.001.
Figure 5
Figure 5. The role of PDCD4 targeting through miR-93 in the regulation of apoptosis in gastric cancer cells.
AGS cells were transfected with equal doses of pre-miR-control, pre-miR-93, anti-miR-control, anti-miR-93, control siRNA, PDCD4 siRNA, control plasmid, PDCD4 plasmid, pre-miR-control plus control plasmid, pre-miR-93 plus control plasmid, pre-miR-control plus PDCD4 plasmid, or pre-miR-93 plus PDCD4 plasmid. (A,B) The cell apoptosis profiles were analyzed using Annexin V-FITC/PI staining in flow cytometry. The biparametric histogram shows cells in early (bottom right quadrant) and late apoptotic states (upper right quadrant). Viable cells are double negative (bottom left quadrant). (A) representative image; (B) quantitative analysis. (C) Representative images of apoptotic AGS cells analyzed using DAPI staining. DAPI staining is used to monitor the apoptosis of cells by exhibiting a strong fluorescence when DAPI becomes bound to natural double-stranded DNA. The normal cell nuclei are round in shape and staining is evenly distributed. When the cells become apoptotic, the cell nuclei become deformed due to the aggregation of the DNA. (D) Representative images of apoptotic AGS cells analyzed using TUNEL staining. TUNEL staining is used to monitor the apoptosis of cells by exhibiting a strong fluorescence when DNA strand breaks in the apoptotic cells and is labeled with fluorescein-12-dUTP. *P < 0.05; **P < 0.01.
Figure 6
Figure 6. Effects of miR-93 and PDCD4 on the growth of gastric cancer cell xenografts in mice.
AGS cells were infected with a control lentivirus or a miR-93 overexpression lentivirus, or transfected with a PDCD4 overexpression plasmid, or co-transfected with the miR-93 overexpression lentivirus plus PDCD4 overexpression plasmid. Then the cells (2 × 106 cells per 0.1 mL) were implanted subcutaneously into 6-week-old SCID mice (5 mice per group), and tumor growth was evaluated at day 60 after cell implantation. (A) Representative images of the tumors from the implanted mice. (B) Quantitative analysis of the tumor weights. (C) Quantitative RT-PCR analysis of miR-93 levels in the tumors from implanted mice. (D) Quantitative RT-PCR analysis of PDCD4 mRNA levels in the tumors from implanted mice. (E,F) Western blotting analysis of PDCD4 protein levels in the tumors from implanted mice. (E) representative image; (F) quantitative analysis. (G–I) H & E-stained sections and immunohistochemical staining for PDCD4 and Ki-67 in the tumors from implanted mice. (G) representative image; (H,I) quantitative analysis. **p < 0.01; ***p < 0.001.

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