Generation of Yes-associated protein 1 short hairpin RNA plasmid and its effects on proliferation, migration and invasion of bladder cancer T24 cells

Objective To block the expression of Yes-associated protein 1 (YAP1) gene in human bladder cancer cell line T24 using RNA interference, and to observe the changes of cell proliferation, migration and invasion. Methods The YAP1 expression was detected in T24 cells. The recombinant plasmid targeting YAP1 gene with eukaryotic transcription plasmid pGreenPuro.YAP1short hairpin RNA (shRNA) plasmids of 4 different sequences and YAP1 shRNA normal control (NC) were transfected into T24 cells respectively. After 48 h, the expression level of YAP1 was detected by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR), and the shRNA plasmid with the best silencing effect of YAP1 gene was screened for following research. The methyl thiazol tetrazolium (MTT) assay was used to detect the effect of shRNA plasmid on the proliferation of T24 cells, the effect of shRNA plasmid on cell migration ability was detected by scratch test, and the invasion ability of T24 cells was analyzed by Transwell assay. Results RT-qPCR detection showed that T24 cells expressed YAP1 gene. YAP1 shRNA plasmid was successfully transfected into T24 cells by Lipofectamine 2000. RT-qPCR detection showed that YAP1 shRNA3 plasmid had the most significant effect on silencing YAP1 of T24 cells (the ratio of silencing was 75.5%), and then T24 cells were interfered with YAP1 shRNA3 plasmid. MTT assay showed that the proliferation ability of T24 cells transfected with YAP1 shRNA3 plasmid was less than control group (the proliferation ratio of interference group was 85.9% of the control group, P=0.000). The migration ability of T24 cells transfected with YAP1 shRNA3 was weaker than that in control group [24-h scratch width was (218.50±3.50) μm and (86.5±4.50) μm respectively, P=0.003], and the invasion ability was also weaker than that in control group (the mean number of cells penetrating Transwell was 62∶345, P=0.000). Conclusion RNA interference can significantly reduce the expression of YAP1 gene in human bladder cancer cell line T24, and inhibit the proliferation, migration and invasion of tumor cells. Key words: Bladder cancer; Yes-associated protein 1; Short hairpin RNA; RNA interference; Proliferation; Migration