Wu Huan-Lei, Li Sen-Mao, Huang Yao-Chen, Xia Qi-Dong, Zhou Peng, Li Xian-Miao, Yu Xiao, Wang Shao-Gang, Ye Zhang-Qun, Hu Jia
Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Department of Urology, Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Liberalization Ave, No. 1095, Wuhan, 430030, P.R. China.
Cancer Cell Int. 2021 Dec 2;21(1):641. doi: 10.1186/s12935-021-02331-x.
Heterogeneous nuclear ribonucleoprotein K (HnRNPK) is a nucleic acid-binding protein that regulates diverse biological events. Pathologically, HnRNPK proteins are frequently overexpressed and clinically correlated with poor prognosis in various types of human cancers and are therefore pursued as attractive therapeutic targets for select patients. However, both the transcriptional regulation and degradation of HnRNPK in prostate cancer remain poorly understood.
qRT-PCR was used to detect the expression of HnRNPK mRNA and miRNA; Immunoblots and immunohistochemical assays were used to determine the levels of HnRNPK and other proteins. Flow cytometry was used to investigate cell cycle stage. MTS and clonogenic assays were used to investigate cell proliferation. Immunoprecipitation was used to analyse the interaction between SPOP and HnRNPK. A prostate carcinoma xenograft mouse model was used to detect the in vivo effects of HnRNPK and miRNA.
In the present study, we noted that HnRNPK emerged as an important player in the carcinogenesis process of prostate cancer. miR-206 and miR-613 suppressed HnRNPK expression by targeting its 3'-UTR in PrCa cell lines in which HnRNPK is overexpressed. To explore the potential biological function, proliferation and colony formation of PrCa cells in vitro and tumor growth in vivo were also dramatically suppressed upon reintroduction of miR-206/miR-613. We have further provided evidence that Cullin 3 SPOP is a novel upstream E3 ubiquitin ligase complex that governs HnRNPK protein stability and oncogenic functions by promoting the degradation of HnRNPK in polyubiquitination-dependent proteolysis in the prostate cancer setting. Moreover, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of HnRNPK proteins.
Our findings reveal new posttranscriptional and posttranslational modification mechanisms of HnRNPK regulation via miR-206/miR-613 and SPOP, respectively. More importantly, given the critical oncogenic role of HnRNPK and the high frequency of SPOP mutations in prostate cancer, our results provide a molecular rationale for the clinical investigation of novel strategies to combat prostate cancer based on SPOP genetic status.
异质性核核糖核蛋白K（HnRNPK）是一种核酸结合蛋白，可调节多种生物学事件。在病理情况下，HnRNPK蛋白在多种人类癌症中经常过度表达，且在临床上与预后不良相关，因此被视为特定患者有吸引力的治疗靶点。然而，前列腺癌中HnRNPK的转录调控和降解仍知之甚少。
采用qRT-PCR检测HnRNPK mRNA和miRNA的表达；免疫印迹和免疫组化分析用于测定HnRNPK和其他蛋白的水平。流式细胞术用于研究细胞周期阶段。MTS和克隆形成试验用于研究细胞增殖。免疫沉淀用于分析SPOP与HnRNPK之间的相互作用。使用前列腺癌异种移植小鼠模型检测HnRNPK和miRNA的体内效应。
在本研究中，我们发现HnRNPK是前列腺癌发生过程中的一个重要参与者。miR-206和miR-613通过靶向HnRNPK过表达的前列腺癌细胞系中的3'-UTR来抑制其表达。为了探索其潜在的生物学功能，重新引入miR-206/miR-613后，体外前列腺癌细胞的增殖和集落形成以及体内肿瘤生长也受到显著抑制。我们进一步证明，Cullin 3 SPOP是一种新型的上游E3泛素连接酶复合物，通过在前列腺癌环境中促进HnRNPK在多聚泛素化依赖性蛋白水解中的降解来控制HnRNPK蛋白的稳定性和致癌功能。此外，前列腺癌相关的SPOP突变体无法与HnRNPK蛋白相互作用并促进其降解。
我们的研究结果分别揭示了通过miR-206/miR-613和SPOP对HnRNPK进行调控的新的转录后和翻译后修饰机制。更重要的是，鉴于HnRNPK的关键致癌作用以及前列腺癌中SPOP突变的高频率性，我们的结果为基于SPOP基因状态对抗前列腺癌新策略的临床研究提供了分子依据。
