调控Cox-2基因表达与食管癌细胞放射敏感性机制的初步探讨

目的 通过调控环氧合酶-2(Cox-2)基因表达来探讨影响食管癌细胞放射敏感性的机制.方法 通过构建C ox-2特异性siRNA,转染EC9706细胞,调控细胞内Cox-2表达,检测不同辐射剂量后MMP-2 、Bcl-2 mRNA、AKT蛋白和磷酸化AKT的表达,以及观察集落形成、细胞增殖、细胞凋亡、体外细胞侵袭能力,结果采用单因素方差分析进行统计学处理.结果 2和4 Gy照射后,上调组Bcl-2 mRNA表达量升高(F=3.36、4.32,P＜0.05);下调组MMP-2 mRNA表达量降低(F=3.86、8.09,P＜0.05),Bcl-2 mRNA表达量降低(F=3.73、5.64,P＜0.05),Bax mRNA表达量升高(F=7.03、7.42,P＜0.05).而各组细胞总AKT蛋白和磷酸化AKT蛋白印迹呈现上调组最强印迹,下调组最浅印迹.下调组细胞随照射量的增加凋亡率上升陡度最大,且差异有统计学意义(F=317.40,P＜0.05).G0～G1期细胞比例逐渐升高,S和G2～M期细胞比例逐渐降低,细胞增殖抑制率明显升高,体外侵袭实验穿透细胞数下降陡度最大.结论 调控C ox-2基因表达影响食管癌细胞放射敏感性的机制可能是,下调细胞内Cox-2 mRNA表达继而下调MMP-2、Bcl-2 mRNA表达,上调Bax表达,导致肿瘤细胞的侵袭及转移能力的降低,促进其向G0 ～G1期细胞转换,诱导细胞凋亡.同时使AKT和磷酸化AKT(pAKT)水平下降,影响PI3 K/Akt信号转导途径降低其放疗抗拒的能力.

Preliminary study on the correlation between regulation of Cox-2 gene expression and radiosensitivity of esophageal cancer

Objective To investigate the mechanism of the radiosensitivity effect of Cox-2 gene in esophageal cancer. Methods Cox-2 specific siRNA was constructed and transfected to EC9706 cells to downregulate intracellular Cox-2 expression. The expressions of MMP-2, Bcl-2 mRNA, AKT and phosphorylated AKT proteins were assayed after radiation. Colony formation, cell proliferation, apoptosis and cell invasion in vitro were examined as well. One-way ANOVA method was used to analyze the data. Results After 2 and 4 Gy irradiation, a significant increase in the mRNA expression of Bcl-2 was observed in the Cox-2 up-regulation group(F=3.36,4.32,P<0.05). In the group of Cox-2 downregulation, the expression of MMP-2 mRNA was significantly reduced(F=3.86,8.09,P<0.05). After irradiation, a significant decrease of Bcl-2 mRNA(F=3.73,5.64,P<0.05) as well as an increase of Bax(F=7.03,7.42,P<0.05)was detected, and the levels of total and phosphorylated AKT proteins had the highest level in the Cox-2 upregulation group and had the lowest level in the Cox-2 downregulation group. In the Cox-2 downregulation group, the apoptosis induction obviously increased with dose(F=317.40,P<0.05), and the proportion of cells in G₀-G₁ phase gradually increased but the proportion of cells in S and G₂-M phases decreased, concomitant with the obvious suppression of cell proliferation, in addition, cell invasion was decreased. Conclusions Downregulation of intracellular Cox-2 mRNA expression, concomitant with subsequent downregulation of MMP-2 and Bcl-2 and upregulation of Bax, resulted in reduction of the invasion and metastatic capabilities of tumor cells, and induction of G₀-G₁ phase arrest and apoptosis. Downregulation of AKT and phosphorylated AKT (pAKT) protein expression might also interfere with the capability of the PI3K/Akt signal transduction pathway to resist radiotherapy.