Small interfering RNA in silencing cox-2 expression enhances radiosensitivity of esophageal cancer EC9706 cell

Abstract

To explore the effects of small interfering RNA (siRNA) specific to cox-2 gene on the radiosensitivity of esophageal cancer cell EC9706. The siRNA vector was established for cox-2 gene and then induced into esophageal cancer cell EC9706 by lipofectamine. G418 screening yielded stably transfected cells. After the irradiation of 0, 2 and 4 Gy, the cellular expression levels of cox-2, matrix metalloproteinase-2 (MMP2), Bax and Bcl-2 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and those of cox-2 protein, AKT protein and phosphorylation AKT protein (pAKT) by Western blot. Cell apoptosis was examined by flow cytometer. Invasion of cells was detected by invasive assay in vitro. The invasive and metastatic capacities of cancer cells were assessed by invasion assay in vitro. Proliferative potential was quantified by clone-forming assay. The sequencing result confirmed that siRNA vector pRNA-U6 for cox-2 gene was established. The results of 1-sinCox214, RT-PCR and Western blot showed that cox-2 gene expression of transfected EC9706 cell was silenced efficiently. After the irradiation of 0, 2 and 4 Gy, the expressions of MMP2, Bcl-2 mRNA, AKT protein and pAKT in silencing cox-2 gene expression significantly decreased. There was an inverse correlation with irradiation dose. The Bax mRNA expression evidently increased directly with irradiation dose; the apoptotic rate in cox-2 silencing groups was evidently higher than the control groups. And the difference was significant (P < 0.01); invasion cells in vitro in Cox-2 silencing groups evidently decreased with significant difference (P < 0.01). The colony formation rate of cells decreased obviously in cox-2 silencing groups after the irradiation of 0, 1, 2, 4, 6, 8 and 10 Gy (P < 0.01). Small interference RNA in silencing cox-2 gene expression can enhance significantly the radiosensitivity of esophageal cancer EC9706 cells. And the mechanism may be related with MMP2, Bax, Bcl-2, AKT protein and pAKT protein.