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. 2017 Oct 5;8(10):e3059.
doi: 10.1038/cddis.2017.447.

miR-23a/b Promote Tumor Growth and Suppress Apoptosis by Targeting PDCD4 in Gastric Cancer

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Free PMC article

miR-23a/b Promote Tumor Growth and Suppress Apoptosis by Targeting PDCD4 in Gastric Cancer

Xiuting Hu et al. Cell Death Dis. .
Free PMC article

Abstract

MicroRNAs (miRNAs) are short non-coding RNAs of 21-23 nucleotides that play important roles in virtually all biological pathways in mammals and in other multicellular organisms. miR-23a and miR-23b (miR-23a/b) are critical oncomiRs (miRNAs that are associated with human cancers) of gastric cancer, but their detailed roles in the initiation and progression of gastric cancer remain to be elucidated. In this study, we found that miR-23a/b were consistently upregulated in gastric cancer tissues. We then investigated the molecular mechanisms through which miR-23a/b contribute to gastric cancer and identified programmed cell death 4 (PDCD4) as a direct target gene of miR-23a/b. In contrast to the upregulated expression levels of miR-23a/b, PDCD4 protein levels were dramatically downregulated and inversely correlated with miR-23a/b in gastric cancer tissues. Moreover, we observed that cell apoptosis was increased by miR-23a/b inhibitors and decreased by miR-23a/b mimics in gastric cancer cells and that the restoration of PDCD4 expression attenuated the anti-apoptotic effects of miR-23a/b in gastric cancer cells, indicating that PDCD4 is a direct mediator of miR-23a/b functions. Finally, we showed that miR-23a/b significantly suppressed PDCD4 expression and enhanced tumor growth in a gastric cancer xenograft mouse model. Taken together, this study highlights an important role for miR-23a/b as oncomiRs in gastric cancer through the inhibition of PDCD4 translation. These findings may shed new light on the molecular mechanism of gastric carcinogenesis and provide a new avenue for gastric cancer treatment.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression levels of miR-23a/b in gastric cancer tissues. (a,b) Quantitative RT-PCR analysis of the individual alteration of miR-23a/b in 10 pairs of gastric cancer tissue (GC) compared with matched normal adjacent tissue (GN) samples. (c,d) Quantitative RT-PCR analysis of the mean expression levels of miR-23a/b in 10 pairs of gastric cancer tissue (GC) and matched normal adjacent tissue (GN) samples. Each bar represents the mean±SD values. (*P<0.05; **P<0.01)
Figure 2
Figure 2
PDCD4 was predicted as a target of miR-23a/b and was downregulated in gastric cancer tissues. (a) Schematic description of the hypothetical duplexes formed by the interaction between the binding sites in the PDCD4 3′-UTR (top) and miR-23a/b (bottom). The predicted free energy value of each hybrid is indicated. The seed sequences and seed recognition sites are indicated in red and blue, respectively, and all nucleotides in these regions are highly conserved in several species. (b,c) Western blotting analysis of PDCD4 protein levels in 10 pairs of GC and GN samples. (b) representative image; (c) quantitative analysis. (d) Quantitative RT-PCR analysis of PDCD4 mRNA levels in the same 10 pairs of GC and GN samples. (e) Representative H&E-stained, PDCD4-stained and Ki-67-stained sections of the GC and GN samples. (***P<0.001)
Figure 3
Figure 3
PDCD4 was a direct target of miR-23a/b. (a) Quantitative RT-PCR analysis of miR-23a/b levels in MKN-45 and AGS cells transfected with equal doses of pre-miR-23a/b, anti-miR-23a/b or scrambled negative control RNAs (pre-miR-control or anti-miR-control). (b,c) Western blotting analysis of PDCD4 protein levels in MKN-45 and AGS cells transfected with equal doses of the pre-miR-23a/b, anti-miR-23a/b or scrambled negative control RNAs. (b) representative image; (c) quantitative analysis. (d) Quantitative RT-PCR analysis of PDCD4 mRNA levels in MKN-45 and AGS cells transfected with equal doses of pre-miR-23a/b, anti-miR-23a/b or scrambled negative control RNAs. (e) Direct recognition of the PDCD4 3′-UTR by miR-23a/b. Firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-23a/b binding sites in the PDCD4 3′-UTR were co-transfected into HEK293T cells along with equal doses of pre-miR-23a/b or pre-miR-control. The cells were assayed using a luciferase assay kit 24 h post-transfection. Firefly luciferase values were normalized to β-gal activity, and the results were calculated as the ratio of firefly luciferase activity in the miR-23a/b-transfected cells normalized to the pre-miR-control-transfected cells. (*P<0.05; **P<0.01; ***P<0.001)
Figure 4
Figure 4
Effects of miR-23a/b and PDCD4 on the apoptosis of gastric cancer cells. (a,b) Apoptosis assays were performed 24 h after the transfection of MKN-45 cells with equal doses of pre-miR-23a/b, anti-miR-23a/b or scrambled negative control RNAs (pre-miR-control or anti-miR-control), or with equal doses of pre-miR-control plus control plasmid, pre-miR-23a/b plus control plasmid, pre-miR-control plus PDCD4-overexpression plasmid or pre-miR-23a/b plus PDCD4-overexpression plasmid. (a) representative image; (b) quantitative analysis. (c,d) Western blotting analysis of PDCD4 protein levels in MKN-45 cells transfected with equal doses of pre-miR-control plus control plasmid, pre-miR-23a/b plus control plasmid, pre-miR-control plus PDCD4-overexpression plasmid or pre-miR-23a/b plus PDCD4-overexpression plasmid. (c) representative image; (d) quantitative analysis. (e,f) Western blotting analysis of the levels of cleaved CASP9, 3, 6, 7 and PARP in MKN-45 cells transfected with equal doses of pre-miR-23a/b, anti-miR-23a/b or scrambled negative control RNAs (pre-miR-control or anti-miR-control), or with equal doses of pre-miR-control plus control plasmid, pre-miR-23a/b plus control plasmid, pre-miR-control plus PDCD4-overexpression plasmid or pre-miR-23a/b plus PDCD4-overexpression plasmid. (e) representative image; (f) quantitative analysis. (*P<0.05; **P<0.01; ***P<0.001)
Figure 5
Figure 5
Effects of miR-23a/b on tumor growth in a gastric cancer xenograft mouse model. (a) Flow chart of the experimental procedure. MKN-45 cells were infected with a control lentivirus or lentiviruses to overexpress miR-23a or miR-23b. After infection, MKN-45 cells (2 × 107 cells per 0.1 ml) were subcutaneously implanted into 6-week-old SCID mice (8 mice per group), and tumor growth was evaluated on day 25 after cell implantation. (b) Representative images of the implanted mice and tumors from the implanted mice. (c) Quantitative analysis of the tumor weights. (d) Quantitative RT-PCR analysis of miR-23a/b 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) Quantitative RT-PCR analysis of PDCD4 mRNA levels in the tumors from implanted mice. (h) Representative H&E-stained, PDCD4-stained, Ki-67-stained and cleaved-CASP3-stained sections of the tumors from implanted mice. (i) Quantitative analysis of the sections. (*P<0.05; **P<0.01; ***P<0.001)

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