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Abstract
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OBJECTIVE: To investigate the effect of phospholipase C epsilon 1 (PLCε1) gene silencing on apoptosis and invasion of esophageal carcinoma Eca109 cells and its possible mechanism.
METHODS: The recombinant vectors targeting short hairpin RNA (shRNA) of PLCε1 (pGenesil-1-PLCε1-shRNA, pGenesil-1-PLCε1-shRNA2 and pGenesil-1-PLCε1-shRNA3) were transfected into Eca109cells by cationic liposome method and to screen out the expression vector with best interference effect. The apoptosis rate and invasive ability of Eca109 cells 48 h after transfection were determined by flow cytometry (FCM) and Transwell chamber method, respectively. After PLCε1 gene silencing, the expression levels of factorassociated suicide (Fas), Fas ligand (FasL), CD44, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) mRNAs were detected by semi-quantitative reverse transcriptionpolymerase chain reaction (RT-PCR).
RESULTS: The pGenesil-1-PLCε1-shRNA2 had the best interference effect on PLCε1 gene. The apoptosis rate of Eca109 cells 48 h after transfection with PLCε1-shRNA2 was (30.27±5.13)%, which was significantly higher than that of the cells transfected with pGenesil-1-NC-shRNA (NC group) [(22.06±4.47)%, P < 0.05]. The number of Eca109 cells across the polycarbonate membrane of Transwell chamber in the NC group and PLCε1-shRNA2 group 48 h after transfection were 82.00±2.00 and 62.67±3.06, respectively. The number of Eca109 cells across the polycarbonate membrane in PLCε1-shRNA2 group was lower than that of the NC group (P < 0.05). The expression level of Fas mRNA in Eca109 cells of PLCε1-shRNA2 group was significantly up-regulated as compared with that of the NC group (P < 0.05), while the expression levels of FasL, MMP-9 and VEGF mRNAs in PLCε1 - shRNA2 group were significantly down-regulated (all P < 0.05). There was no significant difference in CD44 mRNA expression level between the NC group and PLCε1-shRNA2 group.
CONCLUSION: Silencing PLCε1 gene might promote the apoptosis of Eca109 cells by up-regulating the expression of Fas and down-regulating the expression of FasL, and it can also suppress the invasive ability of Eca109 cells by down-regulating the expressions of MMP-9 and VEGF.
Language: zh |