Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China; Department of Urology, Zhujiang Hospital, Southern Medical University, Guangzhou, China.
Cancer. 2014 Oct 15;120(20):3208-18. doi: 10.1002/cncr.28796. Epub 2014 Jun 12.
Nuclear factor κB (NFκB) signaling is strongly associated with tumor progression, and studies have shown that SHANK-associated RH domain interacting protein (SHARPIN) is crucial for NFκB pathway activation. However, the expression and functions of SHARPIN in prostate cancer (PCa) have not yet been defined.
The expression of SHARPIN in PCa cell lines and tissues was evaluated with western blotting, quantitative real-time polymerase chain reaction, and immunohistochemistry. After SHARPIN was silenced in the PCa cell lines, western blots were used to confirm that SHARPIN physically associated with components of the NFκB pathway and the downstream targets (survivin and livin). The functions of SHARPIN in cell proliferation, migration, and invasion in vitro were measured with 5-(3-carboxymethoxyphenyl)-2-(4,5-dimenthylthiazoly)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS), Transwell, and invasion assays, respectively. Flow cytometry was employed to evaluate cell apoptosis. Furthermore, tumorigenesis in vivo was examined with tumorigenicity assays.
SHARPIN expression was upregulated in PCa cell lines and tissues. The knockdown of SHARPIN or incubation with Bay 11-7082 (an NFκB inhibitor) led to dramatically decreased levels of phosphorylated IκBα and phosphorylated p65 in comparison with the control group. Downregulation of survivin and livin due to SHARPIN inhibition was attributable to transcriptional repression (P < .05). Decreases in cell viability, migration, invasion, and survival with a higher sensitivity to docetaxel in vitro and with repressed tumorigenesis in vivo were observed upon SHARPIN silencing, and this was consistent with the results from inhibition of the NFκB pathway and its downstream targets.
The current study demonstrates that overexpression of SHARPIN promotes activation of the NFκB pathway and downstream targets survivin and livin, which potentially contributes to PCa development.
核因子 κB（NFκB）信号与肿瘤进展密切相关，研究表明 SHANK 相关 RH 结构域相互作用蛋白（SHARPIN）对于 NFκB 通路的激活至关重要。然而，SHARPIN 在前列腺癌（PCa）中的表达和功能尚未确定。
通过 Western blot、实时定量聚合酶链反应和免疫组织化学评估 SHARPIN 在 PCa 细胞系和组织中的表达。沉默 PCa 细胞系中的 SHARPIN 后，Western blot 用于证实 SHARPIN 与 NFκB 通路的组成部分及其下游靶标（survivin 和 livin）物理结合。通过 5-（3-羧基甲氧基苯基）-2-（4,5-二甲基噻唑基）-3-（4-磺苯基）四唑鎓，内盐（MTS）、Transwell 和侵袭试验分别测量 SHARPIN 在体外细胞增殖、迁移和侵袭中的功能。流式细胞术用于评估细胞凋亡。此外，通过致瘤性试验评估体内肿瘤发生情况。
SHARPIN 在 PCa 细胞系和组织中表达上调。与对照组相比，沉默 SHARPIN 或用 NFκB 抑制剂 Bay 11-7082 孵育导致磷酸化 IκBα 和磷酸化 p65 的水平明显降低。SHARPIN 抑制导致 survivin 和 livin 的下调归因于转录抑制（P <.05）。体外沉默 SHARPIN 后观察到细胞活力、迁移、侵袭和存活降低，对多西他赛的敏感性更高，体内肿瘤生成受到抑制，与 NFκB 通路及其下游靶标的抑制结果一致。
本研究表明，SHARPIN 的过表达促进 NFκB 通路及其下游靶标 survivin 和 livin 的激活，这可能有助于 PCa 的发展。
