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目的探讨人α2,6-唾液酸转移酶(ST6GalI)小分子干扰RNA(siRNA)及其与相同或不同靶点的反义寡核苷酸(ASO)联合应用,对ST6GalI高表达的结肠癌SW480细胞的粘附和侵袭力的影响。方法设计并合成靶向ST6GalI的siRNA及ASO,用脂质体Lipofectamine2000包裹后转染SW480细胞。实验分为空白对照组、脂质体对照组、siRNA组、ASO1组(与siRNA不同靶点)、ASO2组(与siRNA相同靶点)、siRNA+ASO1组及siRNA+ASO2组,应用RT-PCR测定细胞中ST6GalImRNA水平,流式细胞术检测细胞表面α2,6-唾液酸含量,分别用CytoMatrixTM细胞粘附试剂盒及细胞侵袭分析试剂盒,检测细胞对细胞外基质的粘附与侵袭力。结果siRNA组、ASO1组、ASO2组、siRNA+ASO1组、siRNA+ASO2组中,SW480细胞的ST6GalImRNA表达水平、细胞表面α2,6-唾液酸含量及细胞对ECM的粘附与侵袭力均明显低于空白对照组、脂质体对照组(P<0.05),以siRNA+ASO1组最明显,且siRNA+ASO1组与siRNA组比较,差异有统计学意义(P<0.05),而siRNA+ASO2组与siRNA组比较,差异无统计学意义(P>0.05)。结论化学合成的靶向ST6GalI的siRNA能够下调SW480细胞中ST6GalI基因的表达,继而降低细胞对ECM的粘附和侵袭力,并且与不同靶点的ASO联合应用具有相加和协同效应。
Objective To investigate the effect of ST6GalI small interfering RNA (siRNA) and its antisense oligonucleotide (ASO) on the same or different targets in the treatment of colon cancer SW480 overexpressing ST6GalI Effect of cell adhesion and invasiveness. Methods siRNA and ASO targeting ST6GalI were designed and synthesized, and then transfected into SW480 cells with Lipofectamine2000. The experiment was divided into blank control group, liposome control group, siRNA group, ASO1 group (different targets with siRNA), ASO2 group (same target with siRNA), siRNA + ASO1 group and siRNA + ASO2 group. The level of ST2GalI mRNA in cells was determined. The cell surface α2,6- sialic acid content was detected by flow cytometry. CytoMatrixTM Cell Adhesion Kit and Cell Invasion Assay Kit were used to detect the cell adhesion and invasion to extracellular matrix. Results The expression of ST6GalI mRNA, the content of α2,6- sialic acid and the adhesion and invasiveness of ECM in SW480 cells were significantly lower in siRNA group, ASO1 group, ASO2 group, siRNA + ASO1 group and siRNA + ASO2 group There were significant differences between siRNA + ASO1 group and siRNA + ASO1 group (P <0.05), but no difference between siRNA + ASO1 group and siRNA + ASO1 group (P <0.05) Compared with siRNA group, the difference was not statistically significant (P> 0.05). Conclusion Chemically synthesized siRNA targeting ST6GalI can down-regulate the expression of ST6GalI gene in SW480 cells, and then decrease the adhesion and invasiveness of ECM to SW480 cells. The combination of ST6GalI with different target sites has additive and synergistic effects.