Expression and regulatory function of miRNA-34a in targeting survivin in gastric cancer cells |
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Authors: | Weiguo Cao Rong Fan Lifu Wang Shidan Cheng Hao Li Jinsong Jiang Mei Geng Yening Jin Yunlin Wu |
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Affiliation: | 1. Department of Oncology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China 2. Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, No. 197, Ruijin Er Rd, Shanghai, 200025, China
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Abstract: | We aimed to investigate the expression of microRNA-34a (miR-34a) in human gastric cancer cells and to evaluate the effects of miR-34a, acting via its gene survivin, on gastric cancer cell HGC-27 to provide potential new strategies for treating gastric cancer. In vitro cultures of the human gastric cancer cell lines MGC80-3, HGC-27, NCI-N87, and SGC-7901 and the normal human gastric epithelial cell line GES-1 were established. The expression of miR-34a in each gastric cancer cell line and GES-1 normal human gastric epithelial cell line was detected using quantitative real-time polymerase chain reaction (qRT-PCR). After the HGC-27 cells were transfected with a miR-34a mimic for 48 h, the changes in the expression levels of miR-34a were detected using qRT-PCR. The effect of miR-34a on HGC-27 cell viability was measured using a tetrazolium-based colorimetric [?(4,5)-dimethylthiahiazo-(?z-y1)-3,5-di-phenytetrazoliumromide (MTT)] assay. Flow cytometry was used to analyze the effects of miR-34a on HGC-27 cell proliferation. Annexin V/propidium iodide double staining and flow cytometry were used to analyze the effects of miR-34a on HGC-27 cell apoptosis. A Transwell invasion chamber was used to detect the effects of miR-34a on HGC-27 cell invasion. Finally, western blotting was used to analyze the effects of miR-34a on survivin protein expression. The qRT-PCR test determined that miR-34a expression in gastric cancer cells was significantly reduced compared to the normal gastric epithelial cell line GES-1 (p?0.01). Compared to the control group, cellular miR-34a expression levels were significantly increased in HGC-27 human gastric carcinoma cells after transfection with a miR-34a mimic for 48 h (p?0.01). The MTT assay demonstrated that after overexpressing miR-34a in HGC-27 cells, cellular viability was significantly reduced (p?0.05). Flow cytometry analysis determined that upon miR-34a overexpression, the proliferation index decreased significantly (p?0.05), and cellular apoptosis was significantly increased (p?0.01). The Transwell invasion chamber assay illustrated that after increasing the expression of miR-34a, the number of cells passing through the Transwell chamber was significantly reduced (p?0.01). Based on western blotting, compared with the control group, survivin protein expression levels were significantly decreased in the HGC-27 cells transfected with the miR-34a mimic for 48 h (p?0.01). In conclusion, the expression level of miR-34a was downregulated in human gastric cancer cell lines. miR-34a can negatively regulate survivin protein expression and inhibit gastric cancer cell proliferation and invasion. Therapeutically enhancing miR-34a expression or silencing the survivin gene may benefit patients with gastric cancer. |
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