蛋白酶体抑制剂RA190对口腔腺样囊性癌的调节作用

Regulatory effect of the proteasome inhibitor RA190 on oral adenoid cystic carcinoma

摘要:

目的 探讨蛋白酶体抑制剂双苄基吡啶酮（RA190）通过非ATP酶依赖性调节颗粒13（Rpn13）抑制泛素-蛋白酶体通路途径对口腔腺样囊性癌的作用，揭示RA190对腺样囊性癌ACC-2细胞增殖、凋亡的调节作用和可能机制。方法 确定ACC-2细胞表达Rpn13后，选择RA190对ACC-2细胞的合适剂量（600 nmol/L）；实验分为ACC-2组、ACC-2-Rpn13-siRNA组、RA190-ACC-2-Rpn13-siRNA组；敲除ACC-2细胞中Rpn13的表达；用RA190处理ACC-2细胞；用RA190处理敲除Rpn13的ACC-2细胞；MTT法观察各组细胞增殖、凋亡情况；Western blotting检测各组细胞Survivin、TIP30、泛素化蛋白相对表达量；qRT-PCR检测Survivin和TIP30 mRNA相对表达量。结果 敲除Rpn13后的细胞组中Rpn13蛋白相对表达量较ACC-2细胞组降低（P <0.05）。MTT法检测显示，不同剂量的RA190作用ACC-2细胞后光密度（OD）比较，差异有统计学意义（P <0.05）。与ACC-2组比较，其余3组OD值均降低（P <0.05）。与ACC-2组比较，其余3组Survivin蛋白相对表达量均降低（P <0.05），TIP30蛋白相对表达量均升高（P <0.05），RA190-ACC-2组TIP30蛋白相对表达量最高，泛素化蛋白聚集最明显（P <0.05）。与ACC-2组比较，其余3组Survivin mRNA的相对表达量均降低（P <0.05），TIP30 mRNA相对表达量均升高（P <0.05）。结论 RA190促凋亡的活动主要取决于Rpn13，Rpn13是口腔腺样囊性癌的致癌基因。RA190的调节作用可能改善口腔腺样囊性癌的预后，Rpn13可能成为治疗口腔腺样囊性癌的靶点。

Abstract:

Objective To investigate the effect of the proteasome inhibitor dibenzylpyridone RA190 on ACC-2 (oral adenoid cystic carcinoma) by inhibiting UPP (ubiquitin-26S proteasome pathway) via Rpn13 (non-ATP-dependent regulatory granule 13), and thus revealing its effect on ACC-2 cell proliferation，the regulatory role, and possible mechanism of apoptosis.Methods After determining the expression of Rpn13 in ACC-2 cells, the appropriate dose of RA190 for ACC-2 cells were selected. Finally, the expression of survivin, TIP30, ubiquitinated protein of four groups (A: ACC-2 cell group; group B: ACC-2-Rpn13-siRNA (small interfering RNA) group knockdown the expression of Rpn13 in ACC-2 cells; group C: group RA190-ACC-2 group, treated A group with RA190; group D: RA190-AC2-Rpn13-SiRNA treated B group with RA190) were detected. The proliferation and apoptosis of each group were observed by MTT.Results Compared to the ACC-2 group, the OD Values of another 3 groups were decreased; compared to the ACC-2 group, the relative expression of Survivin was reduced, and the relative expression of TIP30 was increased in the RA190-ACC-2 group; compared to the ACC-2-Rpn13-siRNA group, in the RA190-ACC-2-Rpn13-siRNA group, the relative expression of TIP30 was reduced, and the relative expression of Survivin was increased; the aggregation of ubiquitinated protein in the RA190- ACC-2 group was the most obviously; the Survivin mRNA valus in another 3 groups were lower than the ACC-2 group, and the TIP30 mRNA valus in another 3 groups were higher than the ACC-2 group. Above all, the differences were significantly (all P < 0.05).Conclusion RA190 promoted ACC-2 cell apoptosis and inhibited proliferation through Rpn13, may improve the prognosis of oral adenoid cystic carcinoma, and can be used as a target for the treatment of oral adenoid cystic carcinoma.