In Vitro Anti-MicroRNA-21 Effect on Apoptosis, Proliferation and Infiltration of Gastric Carcinoma MKN28 Cells

Abstract

Background: Gastric carcinoma (GC) is the third leading cause of malignant tumor mortality worldwide. The morbidity of gastric carcinoma is noticeably increased in Eastern Asia. MicroRNA-21 is a specific member of microRNAs, which is involved in carcinogenesis. This study aimed to analyze and investigate the influence of anti-microRNA-21 on the apoptosis and proliferation of gastric carcinoma MKN28 cells to determine the effect of anti-microRNA-21 on the development and progression of gastric carcinoma. Methods: Human gastric carcinoma MKN-28 cell lines were obtained and microRNA-21 inhibitor and mismatched sequence negative control were synthesized by GenePharma (B03001, Shanghai, China). After the MKN-28 cells were cultured, the total RNA was extracted from cells and reversely transcribed using the Moloney Murine Leukemia Virus (M-MLV) First Strand cDNA Synthesis kits. A quantitative PCR (qPCR) was performed to detect maspin, Bcl-2-associated X protein (Bax) and B-cell lymphoma-2 (Bcl-2) protein levels. Flow cytometry was applied at 72 h after transfection to detect the MKN28 cell apoptosis. Western blot was completed following the standard procedures to determine the effects of downregulated microRNA-21 on the expression of proteins, including maspin, Bcl-2, Bax, FAS-associated death domain protein (FADD), and pro-caspase3 in the MKN28 cells. Transwell® assays were also performed for cell infiltration. Results: The proliferation of MKN28 cells was prominently decreased in the anti-microRNA-21 transfected groups relative to the control group, and apoptosis was increased dramatically in MKN28 cells 72 h after transfection with anti-microRNA-21 (p < 0.05). The protein expression levels of Bax and pro-caspase3 increased, whereas the Bcl-2 levels were markedly decreased in MKN28 cells when transfected with anti-microRNA-21 (p < 0.01), but there were insignificant differences in the levels of FADD between the two groups (p > 0.05). Relative to the negative control group, the levels of mRNA and maspin were increased in the anti-microRNA-21 groups (p < 0.05). The MKN28 cells transfected with anti-microRNA-21 migrated and invaded more slowly than those in the negative control (p < 0.05). Conclusions: This study shows that inhibition of microRNA-21 at the transcriptional level can inhibit the proliferation and metastasis of gastric cancer cells by regulating the mitochondrial apoptosis pathway. Since the transcription of the tumor suppressor gene, maspin, may be blocked by microRNA-21 in gastric cancer, microRNA-21 inhibition is a promising treatment for gastric cancer patients.