Abstract:

Objective　To explore the mechanism of Yes associated protein 1 (YAP1) in  the regulation of the expression of Nexilin in bladder smooth muscle cells. Methods　The smooth muscle specific YAP1-knockout mice (SMMHC-Cre^ERT2/YAP^F/F) were bred; twelve of them were selected and were randomly divided into a YAP1 knockout group and a wild type group, with 6 in each group. The Yap1 knockout group were treated by the intraperitoneal injection of tamoxifen dissolved in ethanol, and the wild type group were treated with ethanol. Two weeks later, the protein levels of total YAP (tYAP) and Nexilin in the urinary bladders were detected by Western blot. The human bladder smooth muscle cells were cultured in vitro. The cells were: treated with sphingosine-1-phosphate (S1P) dissolved in sterile ultrapure water containing 4% BSA for 2 and 4 h; or transduced with recombinant adenovirus to overexpress YAP1; or transfected with siRNA to knockdown YAP/TAZ (YAP1/WWTR1) and then treated with S1P. The negative control (NC) and solvent control (BSA) were set up throughout the experiments. The expression level of NEXN was detected by real-time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). t test and one-way analysis of variance were applied. Results　The expression levels of tYAP and Nexilin in the bladder smooth muscle of the YAP1 knockout group were (0.19±0.05) time and (0.34±0.04) time those of the wild-type group, respectively, with statistical differences (both P<0.05). In human bladder smooth muscle cells, the expression levels of NEXN mRNA in the S1P group were (1.32±0.13) times and (2.11±0.44) times those in the BSA group at hour 2 and 4, respectively, with statistical differences (both P<0.05). The expression level of NEXN in the YAP1 overexpression group was (1.78±0.15) times that in the empty vector group (P<0.05). The expression level of NEXN in the siYAP/TAZ+BSA group was (0.75±0.05) time that in the NC+BSA group (P<0.05). The expression level of NEXN in the siYAP/TAZ+S1P group was significantly lower than that in the NC+S1P group (P<0.05), and there was no statistical difference in the NEXN expression between the siYAP/TAZ+S1P group and the siYAP/TAZ+BSA group (P>0.05). Conclusions　YAP1, a transcription coactivator, plays a regulatory role in the expression of Nexilin in bladder smooth muscle cells. YAP1 may affect the contractile properties of bladder smooth muscle by regulating the expression of Nexilin.

Key words:

Bladder, Smooth muscle, Yes-associated protein1, Nexilin, Smooth muscle specific Yap1-knockout

摘要：

目的　探索在膀胱平滑肌细胞中Yes相关蛋白1（Yes-associated protein1，YAP1）对Nexilin表达的调控机制。方法　构建平滑肌特异性YAP1敲除小鼠（SMMHC-Cre^ERT2/YAP^F/F）；选取其中12只，随机分为YAP1敲除组及野生型组，每组6只，分别以他莫昔芬或乙醇溶剂腹腔注射；2周后以免疫印迹试验（Western blot）检测膀胱组织总YAP（tYAP）及Nexilin的蛋白表达水平。体外培养人膀胱平滑肌细胞，以溶解于含4% BSA无菌超纯水的鞘氨醇-1-磷酸（S1P）处理2 h及4 h；或通过重组腺病毒过表达YAP1，或通过siRNA敲低YAP/TAZ（YAP1/WWTR1）后再以S1P处理，同时设置相应的阴性对照（NC）及溶剂（BSA）对照，逆转录实时定量聚合酶链反应（RT-qPCR）检测NEXN的表达水平。采用t检验、单因素方差分析。结果　YAP1敲除组小鼠膀胱平滑肌组织中tYAP和Nexilin表达水平分别为野生型组的（0.19±0.05）倍和（0.34±0.04）倍，差异均有统计学意义（均P<0.05）；在人膀胱平滑肌细胞中，S1P组NEXN的mRNA表达水平于2 h和4 h时分别为BSA组的（1.32±0.13）倍和（2.11±0.44）倍，差异均有统计学意义（均P<0.05）；YAP1过表达组的NEXN表达水平为空载体组的（1.78±0.15）倍（P<0.05）；siYAP/TAZ+BSA组的NEXN表达水平为NC+BSA组的（0.75±0.05）倍（P<0.05）；siYAP/TAZ+S1P组的NEXN表达水平与NC+S1P组比较显著下调（P<0.05），且与siYAP/TAZ+BSA组比较差异无统计学意义（P>0.05）。结论　在膀胱平滑肌细胞中，转录共激活因子YAP1对Nexilin的表达具有调控作用；YAP1可能通过对Nexilin的表达调控影响膀胱平滑肌的收缩特性。

关键词:

膀胱, 平滑肌, Yes相关蛋白1, Nexilin, 平滑肌特异性基因敲除