SUMO1介导的p65核转运在肝癌细胞恶性进展中的作用

The nuclear import of p65 plays an important role in the activation of NF-κB pathway and the promotion of hepatocellular carcinoma (HCC) development. A recent study indicated that nuclear localization signal (NLS)-mutated p65 retained strong transcriptional activity compared with the vector, which suggests that p65 may be imported into the nucleus independently of its NLS. However, the underlying mechanism was still limited. We found that both SUMO1- and p65-positive immunoreactivities were remarkably increased in the nuclei of tumor tissues in HCC patients compared with non-tumor tissues, and further analysis suggested a correlation between them (R=0.851, P=0.002, n=10). The knockdown of SUMO1 inhibited p65 nuclear translocation. We also verified the interaction of p65 and SUMO1 in the liver tissues of HCC patient. Based on these results, we hypothesize that p65 may be SUMOylated by SUMO1 protein and SUMO1-related p65 SUMOylation increases p65 nuclear translocation, and promotes HCC development. To test this hypothesis, we firstly detect p65 and SUMO1 expressions in the cancer tissues from 50 patients by using immunohistochemistry assay and search for the correlation of them by using Pearson's correlation tests. Secondly, SUMO1-related p65 SUMOylation in vivo and in vitro are detected by using co-immunoprecipitation and in vitro SUMOylation assay, and we search for the SUMOylation site of p65 according to the prediction results of SUMO sp 2.0 software. Thirdly, in order to get rid of the effect of other NF-κB subunits SUMOylation on NF-κB transcriptional activity, we construct the p65 mutants of SUMOylation and compare the effect of p65-WT and p65-mutant on the nuclear import of exogenous p65, the transcriptional activity of NF-κB and the proliferation and migration of hepatoma cells. Fourthly, we construct p65-WT and p65-mutant stable cell lines. The xenograft tumor growth containing p65-WT or p65-mutant is observed in nude mice. This study will find a new mechanism of p65 nuclear import.

p65核转运引起的NF-κB激活能促进肝癌进展，但p65不依赖于核定位序列的核转移机制未明。前期发现HCC中SUMO1高表达，并与胞核p65共定位，且p65与SUMO1有相互作用。故推测：SUMO1可能通过介导p65的SUMO化促进p65核转运及HCC恶性进展。为此，进一步扩大临床样本，确定SUMO1与胞核p65的相关性；在病人肝组织和肝癌细胞株上证实p65-SUMO1相互作用；体外SUMO化实验证明SUMO1能介导p65的SUMO化，并找到p65的SUMO化位点；在肝癌细胞中过表达和敲低SUMO1，观察SUMO1是否促进p65核转运及NF-κB活性，同时观察炎症和低氧对这一作用的影响；构建SUMO1结合位点缺陷的p65突变体，建立稳转细胞株，裸鼠成瘤实验中观察野生型p65与SUMO1结合位点突变的p65对肿瘤生长及器官转移的影响。该研究结果将揭示p65核转运的全新机制及其对HCC的影响。

p65核转移激活NF-κB信号通路、促进肝癌发展，但p65不依赖于核定位序列的核转移机制未明。SUMO1具有调节靶蛋白核转移的作用，且在肝癌中表达增多。本项目研究发现：1.SUMO1与细胞核p65在人肝癌组织中存在密切正相关。2.p65与SUMO1存在相互作用，且肿瘤微环境（炎症及缺氧）上调SUMO1蛋白表达及SUMO1蛋白相关的p65的SUMO化修饰水平。3.SUMO1促进p65核转移，但过表达SUMO1却抑制NF-κB转录活性。推测，SUMO1可能通过调节其他靶蛋白对NF-κB活性产生抑制作用。4.中脑星形胶质细胞源性神经营养因子（MANF）与p65在肝癌细胞的胞核中存在相互作用，且MANF抑制NF-κB信号通路。那么无核定位序列的MANF是否依赖于SUMO1发生核转移、进而在胞核内发挥抑制NF-κB活性的作用，引起了我们的兴趣。5.在人肝癌组织及肝癌细胞株中证实MANF与SUMO1存在相互作用，SUMO1促进MANF核转移。6.敲低SUMO1，MANF与p65在细胞核中相互作用明显减少。7.寻找到p65蛋白的第37位、122位、123位和221位赖氨酸是SUMO化修饰位点，并发现p65发生SUMO化修饰后招募MANF，MANF在SUMO1协助下核转移，抑制NF-κB信号通路。8.MANF在肝癌组织中表达明显低于癌旁组织，且MANF高表达的肝癌患者生存期较好，说明MANF抑制肝细胞癌。9.构建肝细胞特异性MANF敲除鼠。与野生鼠相比，肝细胞特异性MANF敲除鼠采用DEN造肝癌模型后，肝肿瘤体积更大、肝细胞恶性程度更高、肝组织上皮-间质转化和肝肿瘤肺转移更明显。10.与癌旁组织相比，肝癌组织中SUMO1蛋白相关的p65的SUMO化修饰水平增高、MANF的SUMO化修饰水平降低。综上，炎症和缺氧上调SUMO1蛋白的表达，SUMO1促进p65核转移、激活NF-κB信号通路、促进肝癌发展。胞核内p65发生SUMO化修饰后，招募抑制性蛋白MANF。癌旁组织中MANF水平较高、且MANF的SUMO化修饰水平较高，MANF在SUMO1协助下发生核转移，与p65结合抑制NF-κB信号通路，发挥抑制肝细胞癌上皮-间质转化的作用；肝癌组织中MANF表达较低，抑制NF-κB/Snail信号通路作用较弱。故MANF高表达的病人，肝癌预后较好。