Ribosomal L1 domain-containing protein 1 coordinates with HDM2 to negatively regulate p53 in human colorectal Cancer cells.
Ribosomal L1 domain-containing protein 1 coordinates with HDM2 to negatively regulate p53 in human colorectal Cancer cells.
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含核糖体 L1 结构域的蛋白 1 与 HDM2 协同负向调节人结直肠癌细胞中的 p53
DOI：
10.1186/s13046-021-02057-8
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发表时间：
2021-08-06
期刊：
影响因子：
--
通讯作者：
Zhang X
中科院分区：
文献类型：
--
作者：
Ding L;Zhang Z;Zhao C;Chen L;Chen Z;Zhang J;Liu Y;Nie Y;He Y;Liao K;Zhang X
核糖体L1结构域含蛋白1 （RSL1D1）是一种在细胞增殖中至关重要的核核蛋白。目前认为，RSL1D1在核核胁迫下易位到核质中，通过直接相互作用抑制HDM2的E3连接酶活性，从而稳定p53。通过siRNA转染，在HCT116p53+/+、HCT116p53−/−和HCT-8人结直肠癌（CRC）细胞中实现了基因敲低。用慢病毒表达系统建立过表达感兴趣基因的细胞株。采用qRT-PCR和western blot分析细胞mRNA和蛋白水平。MTT法、PI染色法和Annexin V-FITC/PI双染色法分别检测细胞增殖、细胞周期和细胞凋亡。用IP法检测p53泛素化蛋白水平。用RIP研究了蛋白与rna的相互作用。目的蛋白的亚细胞定位用IFA测定。通过gst -pull - down、BiFC和co-IP检测蛋白-蛋白相互作用。在HCT116荷瘤裸鼠中评价RSL1D1沉默对肿瘤生长的治疗效果。RSL1D1在人结直肠癌细胞中分布于整个细胞核。RSL1D1基因的沉默以p53依赖的方式诱导细胞周期阻滞在G1/S和细胞凋亡。RSL1D1直接与p53相互作用并募集到HDM2，形成三元RSL1D1/HDM2/p53蛋白复合物，从而增强p53的泛素化和降解，导致p53蛋白水平降低。三元复合物的破坏增加了p53蛋白的水平。RSL1D1也通过稳定HDM2 mRNA间接降低p53蛋白水平。因此，在裸鼠模型中，RSL1D1对p53的负调控促进了细胞的增殖和存活，RSL1D1的下调显著抑制了HCT116p53+/+肿瘤的生长。我们首次报道，RSL1D1是人类结直肠癌细胞中p53的一种新的负调节因子，更重要的是，它是抗癌药物开发的潜在分子靶点。在线版本包含补充资料，网址为10.1186/s13046-021-02057-8。
Ribosomal L1 domain-containing protein 1 (RSL1D1) is a nucleolar protein that is essential in cell proliferation. In the current opinion, RSL1D1 translocates to the nucleoplasm under nucleolar stress and inhibits the E3 ligase activity of HDM2 via direct interaction, thereby leading to stabilization of p53. Gene knockdown was achieved in HCT116p53+/+, HCT116p53−/−, and HCT-8 human colorectal cancer (CRC) cells by siRNA transfection. A lentiviral expression system was used to establish cell strains overexpressing genes of interest. The mRNA and protein levels in cells were evaluated by qRT-PCR and western blot analyses. Cell proliferation, cell cycle, and cell apoptosis were determined by MTT, PI staining, and Annexin V-FITC/PI double staining assays, respectively. The level of ubiquitinated p53 protein was assessed by IP. The protein-RNA interaction was investigated by RIP. The subcellular localization of proteins of interest was determined by IFA. Protein-protein interaction was investigated by GST-pulldown, BiFC, and co-IP assays. The therapeutic efficacy of RSL1D1 silencing on tumor growth was evaluated in HCT116 tumor-bearing nude mice. RSL1D1 distributed throughout the nucleus in human CRC cells. Silencing of RSL1D1 gene induced cell cycle arrest at G1/S and cell apoptosis in a p53-dependent manner. RSL1D1 directly interacted with and recruited p53 to HDM2 to form a ternary RSL1D1/HDM2/p53 protein complex and thereby enhanced p53 ubiquitination and degradation, leading to a decrease in the protein level of p53. Destruction of the ternary complex increased the level of p53 protein. RSL1D1 also indirectly decreased the protein level of p53 by stabilizing HDM2 mRNA. Consequently, the negative regulation of p53 by RSL1D1 facilitated cell proliferation and survival and downregulation of RSL1D1 remarkably inhibited the growth of HCT116p53+/+ tumors in a nude mouse model. We report, for the first time, that RSL1D1 is a novel negative regulator of p53 in human CRC cells and more importantly, a potential molecular target for anticancer drug development. The online version contains supplementary material available at 10.1186/s13046-021-02057-8.
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