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目的构建核糖核酸酶抑制因子(RI)基因siRNA干扰质粒,并检测其对人膀胱癌细胞BIU-87迁移和侵袭能力的影响。方法设计并构建针对RI基因mRNA的2个特异性siRNA表达载体和1个无同源性的阴性对照载体,经酶切和DNA测序鉴定后,转染BIU-87细胞,通过RT-PCR、Western blot和免疫细胞化学法检测干扰的有效性。光镜及HE染色观察细胞形态的改变,黏附试验、划痕试验和Transwell法检测细胞黏附、迁移和侵袭能力的变化,激光共聚焦显微镜观察细胞骨架微丝结构。结果酶切和测序结果证明干扰质粒构建正确;转染特异性siRNA表达载体的BIU-87细胞RI蛋白的表达水平明显降低,对照组未发生明显改变;转染干扰质粒的细胞堆叠严重,细胞黏附能力显著降低,迁移和侵袭能力显著提高,细胞骨架微丝聚集,伸展出片状伪足。结论已成功构建RI基因siRNA干扰质粒,其可显著提高膀胱癌细胞的迁移和侵袭能力,说明RI可作为抑制肿瘤迁移和侵袭的有效靶点。
Objective To construct a siRNA plasmid targeting ribonuclease inhibitor (RI) gene and study its effect on the migration and invasion of human bladder cancer cell BIU-87. Methods Two specific siRNA expression vectors targeting RI gene mRNA and one negative control vector without homology were designed and constructed. After identification by restriction enzyme and DNA sequencing, BIU-87 cells were transfected into BIU-87 cells and transfected into BIU-87 cells by RT-PCR, Western blot and immunocytochemistry to detect the effectiveness of the interference. Light microscopy and HE staining were used to observe the change of cell morphology, adhesion test, scratch test and Transwell assay. The changes of cell adhesion, migration and invasion were observed. The microfilament structure of cytoskeleton was observed by laser scanning confocal microscopy. Results The results of enzyme digestion and sequencing showed that the interference plasmids were constructed correctly. The expression of RI protein in BIU-87 cells transfected with specific siRNA expression vector was significantly lower than that in the control group. The transfected plasmids had severe cell stacking and cell adhesion Ability to significantly reduce the migration and invasion ability was significantly increased, cytoskeletal microfilament aggregation, stretching out of pseudopodia. Conclusion The siRNA plasmid of RI gene has been successfully constructed, which can significantly enhance the migration and invasion ability of bladder cancer cells, indicating that RI can be used as an effective target for inhibiting tumor migration and invasion.