小干扰RNA抑制CD97的表达及其对胃癌细胞迁移和侵袭能力的影响

Effect of CD97 gene silencing by small interfering RNA on migration and invasion of gastric carcinoma cell lines

目的:设计合成有效的siRNA-CD97,体外转染胃癌细胞株,观察CD97表达改变及其与胃癌细胞株迁移、侵袭能力改变的关系。方法:采用AGS和MGC803胃癌细胞株。针对CD97基因设计siRNA,采用化学法合成,筛选出最有效的siRNA-CD97。siRNA-CD97转染胃癌细胞后分别用real-time RT-PCR、免疫荧光流式细胞术检测CD97 mRNA和蛋白表达的改变,MTT法检测细胞活性的改变,并用Transwell细胞迁移和侵袭实验检测细胞迁移和侵袭能力的变化。结果:在siRNA-CD97转染后48 h,real-time RT-PCR结果显示AGS和MGC803细胞CD97 mRNA的表达量相对于未转染的细胞分别下降了(89.34±9.95)%和(95.42±1.93)%。转染后72 h,流式细胞术结果显示AGS细胞CD97EGF和CD97stalk抗原的表达强度相对于未转染的细胞分别下降了(19.29±3.45)%和(30.11±5.93)%,MGC803细胞CD97EGF和CD97stalk抗原的表达强度相对于未转染的细胞分别下降了(26.25±5.73)%和(16.22±3.23)%。MTT法检测结果显示,siRNA-CD97转染前后细胞的吸光度值没有显著差异。迁移和侵袭实验结果显示,AGS细胞siRNA-CD97转染组迁移和侵袭细胞相对于未转染组分别下降了(67.63±12.03)%和(68.02±15.63)%,MGC803细胞转染组迁移和侵袭细胞相对于未转染组分别下降了(14.92±2.03)%和(22.09±5.43)%。结论:胃癌细胞转染siRNA-CD97能够抑制CD97 mRNA和蛋白表达,随着CD97表达的降低,细胞迁移和转移的能力也明显减弱。

AIM： To investigate the effect of CD97 gene silencing by small interfering RNA（siRNA） on migration and invasion of gastric carcinoma cell lines.METHODS： Gastric carcinoma cell lines AGS and MGC803 were used in the study.Four pairs of siRNA were designed according to the sequence of CD97 gene and synthesized chemically.The siRNAs were transfected into the gastric carcinoma cell lines.Forty-eight hours after transfection,the total RNA was extracted and the mRNA expression of CD97 was detected by real-time RT-PCR so as to screen the most effective siRNA.The protein level of CD97 was also measured by fluorescence-activated cell sorting（FACS） 72 h after Transfection.The abilities of migration and invasion were evaluated by Transwell test.The viability of the cells was measured by MTT method.RESULTS： Real-time RT-PCR and FACS revealed that CD97-siRNA notably down-regulated CD97 expression at both mRNA and protein levels.The mRNA level decreased by（89.34±9.95）% and（95.42±1.93）% in AGS and MGC803 cells,respectively.The protein levels of CD97EGF and CD97stalk in AGS cells decreased by（19.29±3.45）% and（30.11±5.93）%,respectively.The protein levels of CD97EGF and CD97stalk in MGC803 cells decreased by（26.25±5.73）% and（16.22±3.23）%,respectively.No change of the cell viability after siRNA transfection was observed.The cell number of migration and invasion in AGS cells was decreased by（67.63±12.03）% and（68.02±15.63）%,respectively.The cell number of migration and invasion in MGC803 cells was decreased by（14.92±2.03）% and（22.09±5.43）%,respectively.CONCLUSION： The siRNA effectively inhibits CD97 expression and restrains the migration and invasion capacities of gastric carcinoma cell lines,suggesting that CD97 plays an important role in the metastasis of gastric cancer.