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. 2016 Oct 11;7(41):67321-67332.
doi: 10.18632/oncotarget.12006.

miR-208a-3p Suppresses Cell Apoptosis by Targeting PDCD4 in Gastric Cancer

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Free PMC article

miR-208a-3p Suppresses Cell Apoptosis by Targeting PDCD4 in Gastric Cancer

Kai Yin et al. Oncotarget. .
Free PMC article

Abstract

Programmed cell death 4 (PDCD4) is a novel tumor suppressor gene and a promising target for anticancer therapies. PDCD4 is frequently downregulated in various human cancers; however, the molecular mechanism accounting for the loss expression of PDCD4 in cancers is not fully understood. In this study, we identified specific targeting sites for miR-208a-3p in the 3'-untranslated region (3'-UTR) of the PDCD4 gene which regulated PDCD4 expression. We demonstrated that miR-208a-3p suppressed apoptosis in gastric cancer cells by targeting PDCD4. We also showed that miR-208a-3p promoted the development of tumor growth in xenograft mice by negatively regulating PDCD4. Taken together, this study revealed a critical role for miR-208a-3p as an oncogenic miRNA in gastric carcinogenesis and it may provide a potential novel target for gastric cancer diagnosis and therapy.

Keywords: PDCD4; apoptosis; gastric cancer; miR-208a-3p; microRNA.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Expression patterns of PDCD4 in human gastric cancer tissues
(A) H&E-stained sections and immunohistochemical staining for PDCD4 in gastric cancer tissue (GC) and gastric noncancerous tissue (GN) samples. (B and C) Western blotting analysis of PDCD4 protein levels in 16 pairs of GC and gastric GN samples. (A) representative image; (B) quantitative analysis. (D) Quantitative RT-PCR analysis of mRNA levels in 16 pairs of GC and GN samples (*P < 0.05; **P < 0.01).
Figure 2
Figure 2. Prediction of the miR-208a-3p binding site within the PDCD4 3′-UTR
(A) Schematic description of the hypothetical duplexes formed by the interactions between the binding sites in the PDCD4 3′-UTR (top) and miR-208a-3p (bottom). The predicted free energy value of each hybrid is indicated. The seed recognition sites are indicated in red, and all nucleotides in these regions are highly conserved in several species. (B) Quantitative RT-PCR analysis of the expression levels of miR-208a-3p in the same 16 pairs of GC and GN samples (*P < 0.05).
Figure 3
Figure 3. PDCD4 is a direct target of miR-208a-3p
(A) Quantitative RT-PCR analysis of the expression levels of miR-208a-3p in MKN45, HGC-27 and AGS cells transfected with equal doses of the pre-miR-208a-3p, anti-miR-208a-3p or scrambled negative control RNAs (pre-miR-control or anti-miR-control). (B and C) Western blotting analysis of PDCD4 protein levels in MKN45, HGC-27 and AGS cells transfected with equal doses of the pre-miR-208a-3p, anti-miR-208a-3p or scrambled negative control RNAs. B: representative image; C: quantitative analysis. (D) Quantitative RT-PCR analysis of PDCD4 mRNA levels in MKN45, HGC-27 and AGS cells transfected with equal doses of the pre-miR-208a-3p, anti-miR-208a-3p or scrambled negative control RNAs. (E) Direct recognition of the PDCD4 3′-UTR by miR-208a-3p. Firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-208a-3p binding sites in the PDCD4 3′-UTR were co-transfected into AGS cells with equal doses of the pre-miR-208a-3p, anti-miR-208a-3p or scrambled negative control RNAs. The cells were assayed using a luciferase assay kit 24 h post-transfection. Firefly luciferase values were normalized to β-galactosidase activity, and the results were calculated as the ratio of firefly luciferase activity in the miR-208a-3p-transfected cells normalized to the control RNA-transfected cells (*p < 0.05; **p < 0.01; ***p < 0.005).
Figure 4
Figure 4. Effect of miR-208a-3p and PDCD4 on the apoptosis of gastric cancer cells
(A) The apoptosis assay was performed 24 h after the transfection of MKN45 cells with equal doses of the pre-miR-208a-3p, anti-miR-208a-3p or scrambled negative control RNAs (upper panel), or with equal doses of control siRNA, PDCD4 siRNA, control plasmid or PDCD4 overexpression plasmid (middle panel), or with equal doses of pre-miR-control plus control plasmid, pre-miR-control plus PDCD4 overexpression plasmid, pre-miR-208a-3p plus control plasmid, or pre-miR-208a-3p plus PDCD4 overexpression plasmid (lower panel). (B) Quantitative analysis of the apoptotic cell ratio in panel A. (C and D) Western blotting analysis of Cleaved Caspase-3 protein levels in MKN45 cells transfected with equal doses of the pre-miR-208a-3p, anti-miR-208a-3p or scrambled negative control RNAs, or with equal doses of control siRNA, PDCD4 siRNA, control plasmid or PDCD4 overexpression plasmid. (C) representative image; (D) quantitative analysis. (*p < 0.05; **p < 0.01; ***p < 0.005).
Figure 5
Figure 5. Effects of miR-208a-3p and PDCD4 on the growth of gastric cancer cell xenografts in mice
(A) Flow chart of the experimental procedure. MKN45 cells were infected with a control lentivirus or a miR-208a-3p overexpression lentivirus, or transfected with a PDCD4 overexpression plasmid, or co-transfected with a miR-208a-3p overexpression lentivirus and a PDCD4 overexpression plasmid. MKN45 cells (2 × 106 cells per 0.1 mL) with different treatments were implanted subcutaneously into 6-week-old SCID mice (5 mice per group), and the tumor growth was evaluated on day 28 after cell implantation. (B) Representative images of the tumors from the implanted mice. (C) Quantitative analysis of the tumor weights. (D) Quantitative RT-PCR analysis of miR-208a-3p levels in the tumors from implanted mice. (E) Quantitative RT-PCR analysis of PDCD4 mRNA levels in the tumors from implanted mice. (F and G) Western blotting analysis of PDCD4 protein levels in the tumors from implanted mice. (F) representative image; (G) quantitative analysis. (HJ) H&E-stained sections and immunohistochemical staining for Ki-67 and PDCD4 in the tumors from implanted mice. (H) representative image; (I) and (J) quantitative analysis (*p < 0.05; **p < 0.01; ***p < 0.005).

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