Objective To evaluate the effect of RNF2 gene knockdown in ECA109 cells on the radiosensitivity to esophageal cancer cell xenograft in nude mice. Methods Thirty-six male BALB/cu nude mice were randomly divided into 6 groups: control group, control+ irradiation group, NC group, NC+irradiation group, RNF2 shRNA group and RNF2 shRNA+ irradiation group. The nude mouse models with transplanted tumors were established by subcutaneous inoculation of EAC109 cells and given with irradiation at a dose of 3 Gy for 5 times. The longest ( a) and shortest ( b) diameters of the transplanted tumor were measured every 2 to 3 day since the fourteenth day after inoculation. The time of tumor formation was recorded. The tumor volume was calculated according to the formula ( ab2/2 ) . The growth curve was delineated. Three nude mice were sacrificed in each group at 24 h after the initial irradiation. The expression of RNF2 at the mRNA and protein levels in transplanted tumor tissues was measured by qRT-PCR and immunohistochemistry, respectively. The growth and tumor volume of the other nude mice in each group were observed. The cell apoptosis of transplanted tumor tissues was detected by TUNEL assay. The expression of Bcl-2 and Bax at the mRNA and protein levels in transplantated tumor tissues was quantitatively measured by qRT-PCR and immunohistochemistry, respectively. Results The tumor growth rate was the highest in the control and NC groups. The knockdown of RNF2 reduced the growth rate of xenografts and the tumor growth rate was the slowest in the RNF2 shRNA+ irradiation group ( P<0.05) . TUNEL assay revealed that the cell apoptosis rates in all groups were significantly increased after irradiation ( all P<0.05) . Before and after irradiation, the apoptosis rate in the RNF2 shRNA group was markedly higher than those in the control and NC groups ( both P<0.05) . Prior to irradiation, the expression levels of RNF2 mRNA and protein in the RNF2 shRNA group were significantly lower compared with those in the control and NC groups ( all P<0.05) , and the tendency became more significant after irradiation. Compared with the control and NC groups, the expression levels of Bcl-2 mRNA and protein were significantly down-regulated in the RNF2 shRNA group before and after irradiation ( all P<0.05) , whereas those of Bax mRNA and protein were considerably up-regulated ( all P<0.05 ) . Conclusions In vivo experiment demonstrates that RNF2 knockdown effectively increases the radiosensitivity of esophageal carcinoma EAC109 cells in nude mouse models with transplanted tumors, which is intimately associated with inducing the cell apoptosis.%目的 探讨沉默食管癌ECA109细胞RNF2基因对裸鼠移植瘤放射敏感性的影响.方法 将36只BALB/cu裸鼠随机分为对照组、对照+照射组、空载组、空载+照射组、沉默RNF2组、沉默RNF2+照射组,于裸鼠皮下接种ECA109细胞构建裸鼠移植瘤模型,给予X线照射3 Gy5次.自接种后第14天开始每2～3 d测量1次瘤最大径( a)和最短径( b),记录成瘤时间并按公式计算瘤体积(ab 2/2),绘制生长曲线.自首次照射24 h后每组处死3只,qRT-PCR和免疫组织化学法分别检测各组瘤组织中 RNF2 mRNA 和蛋白表达;观察各组剩余瘤照射后生长情况和体积变化.采用TUNEL法检测各组瘤组织细胞凋亡情况,分别采用qRT-PCR和免疫组织化学法检测各组瘤组织中Bcl-2、Bax mRNA和蛋白表达情况.结果 对照组与空载组瘤生长速度最快,沉默RNF2后生长速度减慢,沉默RNF2+照射组瘤生长速度最慢( P<0.05).照射后各组凋亡水平均明显高于照射前(P<0.05),照射前、照射后RNF2沉默组凋亡水平均高于对照组、空载组(P<0.05).照射前RNF2沉默组细胞中RNF2 mRNA和蛋白表达水平均低于对照组、空载组(P<0.05),照射后这种趋势更明显.与对照组、空载组相比,照射前、照射后 RNF2 沉默组 Bcl-2 mRNA 和蛋白的表达水平均下降( P<0.05);Bax mRNA和蛋白的表达水平均上调(P<0.05).结论 体内研究显示沉默RNF2基因有效增加了食管癌ECA109细胞裸鼠移植瘤组织放射敏感性,这与诱导细胞凋亡密切相关.
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