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, 19 (1), 294-300

miR-629 Targets FOXO3 to Promote Cell Apoptosis in Gastric Cancer

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miR-629 Targets FOXO3 to Promote Cell Apoptosis in Gastric Cancer

Ming Li et al. Exp Ther Med.

Abstract

Gastric cancer (GC) is one of the most aggressive types of human tumor worldwide, and the 5-year survival rate is less than 25%. The transcriptional factor, forkhead box O3 (FOXO3), is regulated by various micro (mi)RNAs and has been reported to be associated with multiple regulatory signaling pathways involved in tumor development. The current study therefore assessed the impact of miR-629 and FOXO3 on gastric cancer. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to assess the expression of mRNA and protein, respectively. Additionally, the cell proliferation and apoptosis rate were determined via an MTT assay and flow cytometry, respectively. The online database TargetScan predicted that FOXO3 was a target of miR-629. A luciferase reporter assay was also performed to verify that FOXO3 was the direct target of miR-629. The results demonstrated that miR-629 and FOXO3 was upregulated and downregulated in GC tissue, respectively. Furthermore, following transfection with a miR-629 inhibitor, SGC-7901, cell proliferation and apoptosis rate were inhibited and promoted when compared with the control group, respectively. Moreover, after the treatment with SGC-7901, the expression of FOXO3, Bax, Caspase 3 was upregulated, and Bcl-2 was downregulated. Furthermore, the luciferase reporter assay revealed that FOXO3 was the target of miR-629. The results demonstrated that miR-629 and FOXO3 serve vital roles in the development of gastric cancer and may be a future therapeutic target.

Keywords: apoptosis; forkhead box O3; gastric cancer; miRNA-629; proliferation.

Figures

Figure 1.
Figure 1.
Downregulated FOXO3 and upregulated miR-629 expression in tissues with gastric cancer. (A) FOXO3 mRNA and protein expression were determined using RT-qPCR and western blotting, respectively. A sample from one patient is presented. (B) miR-629 expression was determined using RT-qPCR. **P<0.01. FOXO3, forkhead box O3; miR, miRNA; control, paracarcinoma tissues; tumor, tumor tissues.
Figure 2.
Figure 2.
miR-629 is downregulated in SGC-7901 cells following inhibitor transfection. The expression of miR-629 was determined via reverse transcription-quantitative polymerase chain reaction. **P<0.01 vs. the control group. miR, microRNA; control, non-transfected group; NC, transfected with negative control plasmids; inhibitor, transfected with miR-629 inhibitor plasmids.
Figure 3.
Figure 3.
SGC-7901 cell proliferation rate is inhibited following transfection with a miR-629 inhibitor. The proliferation of SGC-7901 cells was determined via an MTT assay. **P<0.01 vs. the control group. miR, microRNA; control, non-transfected group; NC, transfected with negative control plasmids; inhibitor, transfected with miR-629 inhibitor plasmids; OD, optical density.
Figure 4.
Figure 4.
SGC-7901 cell apoptosis is promoted following transfection with the miR-629 inhibitor. Flow cytometry was utilized to assess SGC-7901 cell apoptosis. (A) Flow cytometric scatter plots and (B) quantification of apoptosis rate. **P<0.01 vs. the control group. miR, microRNA; control, non-transfected group; NC, transfected with negative control plasmids; inhibitor, transfected with miR-629 inhibitor plasmids; PI, propidium iodide; FITC, fluorescein isothiocyanate.
Figure 5.
Figure 5.
miR-629 inhibitor transfection upregulates FOXO3, Bax and caspase-3, and downregulates Bcl-2 in SGC-7901 cells. The expression of (A) FOXO3, (B) Bax, (C) Bcl-2 and (D) caspase-3 mRNA was determined using a reverse transcription-quantitative polymerase chain reaction assay. (E) The protein level of FOXO3, Bax, Bcl-2 and caspase-3 was determined using western blotting. (F) Quantification of western blotting results. **P<0.01 vs. the control group miR, microRNA; FOXO3, forkhead box O3; Bax, Bcl-2 associated x; Bcl-2, B-cell lymphoma 2; control, non-transfected group; NC, transfected with negative control plasmids; inhibitor, transfected with miR-629 inhibitor plasmids.
Figure 6.
Figure 6.
miR-629 target verification via a luciferase reporter assay. (A) TargetScan predicted the binding site (underlined) of miR-629 and the 3′-UTR of FOXO3 mRNA. (B) Luciferase activity following plasmid transfection. **P<0.01 vs. the negative control group. miR, microRNA; FOXO3, forkhead box O3; UTR, untranslated region; NC, negative control; miR-629 group, miR-629 overexpression plasmid; WT (wild-type), transfected with wild-type FOXO3 3′UTR; MUT (mutant), transfected with mutated FOXO3 3′UTR.

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