沉默癌胚抗原相关细胞黏附分子1基因抑制SHG44人胶质瘤细胞增殖并促进其凋亡

Silencing of carcinoembryonic antigen-related cell adhesion molecule 1 inhibits proliferation and induces apoptosis in human glioma SHG44 cells

目的采用RNA干扰技术(RNAi)沉默癌胚抗原相关细胞黏附分子1(CEACAM1)基因的表达,观察沉默效果及沉默CEACAM1表达后对SHG44人胶质瘤细胞增殖和凋亡的影响。方法设计并化学合成针对CEACAM1的3对小干扰RNA(siRNA),脂质体法瞬时转染SHG44细胞。流式细胞术(FCM)检测转染效率,采用实时定量PCR(qRT-PCR)检测siRNA转染前后SHG44细胞中CEACAM1 mRNA的表达,Western blot法检测CEACAM1蛋白的表达。CCK-8法检测SHG44细胞细胞增殖活性,annexin V-FITC/PI染色结合FCM检测SHG44细胞凋亡,Western blot法检测cleaved caspase-3和裂解型多聚腺苷酸二磷酸核糖聚合酶(cleaved PARP)的变化。结果 CEACAM1 siRNA转染效率达85%。与空白对照组和阴性对照组比较,qRT-PCR及Western blot结果显示,3组特异性siRNA转染48 h后,在mRNA和蛋白水平上CEACAM1的表达均降低,以CEACAM1-siRNA3效果最明显。siRNA组SHG44细胞的增殖能力较正常对照组明显下降,凋亡细胞比例增加。沉默CEACAM1表达可以上调cleaved caspase-3和cleaved PARP的表达。结论沉默CEACAM1基因表达可有效抑制SHG44胶质瘤细胞的增殖,促进细胞凋亡。

Objective To investigate the effect of siRNA-induced silencing of carcinoembryonic antigen-related cell adhesion molecule 1 （CEACAM1） on proliferation and apoptosis in human glioma SHG44 cells. Methods Three pairs of specific siRNA targeting CEACAM1 were designed and synthesized, and then transiently transfected into SHG44 cells via cationic liposome transfection method. Transfection efficiency was examined by flow cytometry （FCM）. Real-time quantitative PCR （qRT-PCR） and Western blotting were respectively used to detect CEACAM] expression at mRNA and protein levels, and the proliferation ability and apoptosis of SHG44 cells after transfection were assessed by CCK-8 assay and FCM in combination with annexin V-FITC/PI staining, respectively. The expression levels of cleaved caspase-3 and cleaved poly（ADP-ribose） polymerase （PARP） proteins were detected by Western blotting. Results The efficiency of CEACAM1 siRNA transfection into human SHG44 cells were 85%. Forty-eight hours after the three pairs of specific CEACAM1 siRNA were transfected into SHG44 cells, qRT-PCR and Western blot results showed that the expression of CEACAM1 mRNA and protein were significantly inhibited compared with those in the blank control and the negative control groups, and the most significant interference effect was CEACAMI-siRNA3. The proliferation of SHC_.-~ cells was inhibited and the apoptosis rate was raised by the CEACAMI-siRNA transfection. The expression levels of cleaved caspase-3 and cleaved PARP proteins were up-regulated after silencing of CEACAMI. Conclusion The siRNA-mediated CEACAM1 silencing can inhibit the proliferation and promote the apoptosis in human glioma SHG44 cells. CEACAM1 gene may be used as a potential therapeutic target of glioma.