Cholangiocarcinoma is one of the deadly disease with poor 5-year survival and poor response to conventional therapies. Previously, we found that p27kip1 nuclear-cytoplasmic translocation confers proliferation potential to cholangiocarcinoma cell line QBC939 and this process is mediated by crm-1. However, no other post-transcriptional regulation was found in this process including sumoylation in cholangiocarcinoma. In this study, we explored the role of sumoylation in the nuclear-cytoplasmic translocation of p27kip1 and its involvement of QBC939 cells’ proliferation. First, we identified K73 as the sumoylation site in p27kip1. By utilizing plasmid flag-p27kip1, HA-RanBP2, GST-RanBP2 and His-p27kip1 and immunoprecipitation assay, we validated that p27kip1 can serve as the sumoylation target of RanBP2 in QBC939. Furthermore, we confirmed crm-1’s role in promoting nuclear-cytoplasmic translocation of p27kip1 and found that RanBP2’s function relies on crm-1. However, K73R mutated p27kip1 can’t be identified by crm-1 or RanBP2 in p27kip1 translocation process, suggesting sumoylation of p27kip1 via K73 site is necessary in this process by RanBP2 and crm-1. Phenotypically, the overexpression of either RanBP2 or crm-1 can partially rescue the anti-proliferative effect brought by p27kip1 overexpression in both the MTS and EdU assay. For the first time, we identified and validated the K73 sumoylation site in p27kip1, which is critical to RanBP2 and crm-1 in p27kip1 nuclear-cytoplasmic translocation process. Taken together, targeted inhibition of sumoylation of p27kip1 may serve as a potentially potent therapeutic target in the eradication of cholangiocarcinoma development and relapses.

中文翻译：

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通过RanBP2在p27kip1中进行糖基化可促进胆管癌细胞系QBC939中癌细胞的生长。

胆管癌是致命的疾病之一，其5年生存期较差且对传统疗法的反应较差。以前，我们发现p27kip1核细胞质易位赋予胆管癌细胞系QBC939增生潜能，该过程由crm-1介导。然而，在此过程中未发现其他转录后调控，包括胆管癌中的磺酰化。在这项研究中，我们探讨了sumoylation在p27kip1的核质细胞转运中的作用及其对QBC939细胞增殖的影响。首先，我们将K73识别为p27kip1中的磺酰化位点。通过利用质粒flag-p27kip1，HA-RanBP2，GST-RanBP2和His-p27kip1以及免疫沉淀测定，我们验证了p27kip1可以作为QBC939中RanBP2的磺酰化靶标。此外，我们证实了crm-1在促进p27kip1的核质转运中的作用，并发现RanBP2的功能依赖于crm-1。然而，在p27kip1易位过程中，crm-1或RanBP2不能识别K73R突变的p27kip1，这表明RanBP2和crm-1必须通过K73位点对p27kip1进行磺酰化。从表型上讲，在MTS和EdU分析中，RanBP2或crm-1的过表达都可以部分挽救p27kip1过表达所带来的抗增殖作用。我们首次鉴定并验证了p27kip1中的K73磺酰化位点，这对p27kip1核质转移过程中的RanBP2和crm-1至关重要。在一起