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. 2017 Apr 25;8(17):28711-28724.
doi: 10.18632/oncotarget.15646.

MicroRNA-143-3p, up-regulated in H. pylori-positive gastric cancer, suppresses tumor growth, migration and invasion by directly targeting AKT2

Fang Wang  1 , Jiatao Liu  1   2 , Yanfeng Zou  3 , Yang Jiao  1 , Yawei Huang  1 , Lulu Fan  1 , Xiaoqiu Li  1 , Hanqing Yu  1 , Chengqun He  4 , Wei Wei  5 , Hua Wang  1   6 , Guoping Sun  1
Affiliations

MicroRNA-143-3p, up-regulated in H. pylori-positive gastric cancer, suppresses tumor growth, migration and invasion by directly targeting AKT2

Fang Wang et al. Oncotarget. .

Abstract

Our previous studies have suggested a protective role for H. pylori infection in the prognosis of gastric cancer. Based on those findings, we hypothesized that H. pylori-positive and -negative gastric cancers may exhibit different growth patterns and pathobiological behaviors, indicating different mechanisms of cancer progression. By microarray analysis, we studied miRNAs expression profiles in 42 gastric cancer patients, comparing 21 H. pylori-positive and 21 H. pylori-negative groups. Luciferase reporter assay and western blot were used to examine the potential target genes of the interested miRNA. In the present study, 53 miRNAs were significantly differentially expressed in H. pylori-positive and -negative gastric cancer tissues. We investigated the expression and function of one candidate, miR-143-3p, which was the most significantly increased miRNA in H. pylori-positive gastric cancer tissues. We observed that miR-143-3p expression was significantly decreased in gastric cancer tissues and cells, which correlated with late stage and lymph node metastasis. Using gain- and loss-of-function experiments in vitro, we demonstrate that miR-143-3p negatively regulated cell growth, apoptosis, migration and invasion. We further characterized AKT2 as a novel direct target of miR-143-3p. Knockdown of AKT2 expression mimicked the effects of miR-143-3p restoration. In conclusion, our data suggest that miR-143-3p acts as a novel tumor suppressive miRNA by regulating tumor growth, migration and invasion through directly targeting AKT2 gene. Further investigation is warranted to characterize the mechanisms underlying gastric cancer progression and may eventually contribute to its therapy.

Keywords: AKT2; Helicobacter pylori; gastric cancer; microRNA-143-3p; progression.

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Conflict of interest statement

CONFLICTS OF INTEREST

All authors declare that there are no conflicts of interest.

Figures

Figure 1
Figure 1. Differentially expressed miRNAs in H. pylori-positive and H. pylori-negative gastric cancer tissues
Figure 2
Figure 2. Down-regulation of miR-143-3p in gastric cancer tissues and gastric cancer cells
A. The miR-143-3p expression level is increased in H. pylori-positive tumor tissues compared with the H. pylori-negative tumor tissues. B. Expression of miR-143-3p in 42 human gastric cancer (GC) samples. Data are shown as -△△CT values. C. Paired comparison of miR-143-3p expression level between tumor and adjacent non-tumor tissues. D. miR-143-3p is differentially expressed in the early stage (stages I and II) group compared with the advanced-stage (stages III and IV) group. E. MiR-143-3p is differentially expressed in the lymph node-negative (LN-negative) group compared with the lymph node-positive (LN-positive) group. F. Expression of miR-143-3p in different human gastric cancer cell lines and human gastric epithelial cell GES-1 cells. Expression of miR-143-3p was detected by qRT-PCR and was normalized against the expression of the endogenous control RNU48 (A-E) or U6 RNA (F). The horizontal lines indicate the median, 2.5% percentile and 97.5% percentile values (A, C, D, E). *P<0.05, *** P<0.001.
Figure 3
Figure 3. Ectopic expression of miR-143-3p in gastric cancer cells suppresses cell proliferation, induces apoptosis and inhibites cell migration and invasion
A. MiR-143-3p expression was significantly increased by transfecting miR-143-3p mimics into BGC-823 cells and reduced by transfecting miR-143-3p inhibitor into SGC-7901 cells. As compared with their respective negative controls 48 hours after transfection and normalized to U6 snRNA. B. Cell viability of gastric cancer cells was examined with CCK-8 assays. Quantified relative proliferation rates are presented as a histogram in the lower panel. C-D. Effect of miR-143-3p on apoptosis of gastric cancer cells was examined with flow cytometry analysis. Figure is representative of 3 experiments with similar results. Late apoptosis rate, early apoptosis rate and total apoptosis rate are presented as a histogram in the lower panel. E-F. The transwell assay was performed to analyze the effect of miR-143-3p on the migration and invasion of BGC-823 and SGC-7901 cells. Magnification, ×200. Scale bar represents 100μm. Quantified results are presented as a histogram in the lower panel. Data represent the means±SD from 3 independent experiments. *P < 0.05, ** P<0.01, *** P<0.001.
Figure 4
Figure 4. AKT2 is a direct target of miR-143-3p in gastric cancer
A. Schematic of the construction of wild-type or mutant pGL3-AKT2 3′UTR vectors is indicated. B. Relative luciferase activities were analysed in HEK293T cells. Renilla luciferase vector was used as an internal control. C. The effect of miR-143-3p on the expression of AKT2 mRNA by qRT-PCR. β-actin served as an internal control. Data represent the mean±SD from 3 independent experiments. D. Related expression of AKT2 protein in cells treated with miR-143-3p mimics or its inhibitor was determined by western blot.. All *P<0.05, **P<0.01.
Figure 5
Figure 5. Fuctional effects of AKT2 siRNA on gastric cancer cell proliferation, apoptosis, migration and invasion
A. Suppression of AKT2 significantly inhibited gasric cancer cell proliferation. B-C. Suppression of AKT2 significantly induced cell apoptosis. D-E. Suppression of AKT2 significantly inhibited the migration and invasion of BGC-823 and SGC-7901 cells. Data represent the means ± SD. *P < 0.05, ** P<0.01, *** P<0.001.
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