Department of Obstetrics and Gynecology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Cancer Sci. 2021 Nov;112(11):4515-4525. doi: 10.1111/cas.15130. Epub 2021 Sep 21.
We aimed to identify whether Rho GTPase activating proteins (RhoGAPs) were downregulated in cervical cancers and might be targeted to reduce the growth of cervical cancer using the GEO database and immunohistochemical analysis to identified changes in transcription and protein levels. We analyzed their proliferation, clone formation ability, and their growth as subcutaneous tumors in mice. To detect ARHGAP30 localization in cells, immunofluorescence assays were conducted. Mass spectrometry combined with immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co-immunoprecipitation assays. Western-blot and q-PCR were applied to analyze candidate binding proteins that were associated with ribosome biogenesis. Puromycin incorporation assay was used to detect the global protein synthesis rate. We identified that ARHGAP30 was the only downregulated RhoGAP and was related to the survival of cervical cancer patients. Overexpression of ARHGAP30 in cervical cancer cells inhibited cell proliferation and migration. ARHGAP30 immunoprecipitated proteins were enriched in the ribosome biogenesis process. ARHGAP30 was located in the nucleous and interacted with nucleolin (NCL). Overexpression of ARHGAP30 inhibited rRNA synthesis and global protein synthesis. ARHGAP30 overexpression induced the ubiquitination of NCL and decreased its protein level in Hela cells. The function of ARHGAP30 on cervical cancer cell ribosome biogenesis and proliferation was independent of its RhoGAP activity as assessed with a RhoGAP-deficient plasmid of ARHGAP30 . Overall, the findings revealed that ARHGAP30 was frequently downregulated and associated with shorter survival of cervical cancer patients. ARHGAP30 may suppress growth of cervical cancer by reducing ribosome biogenesis and protein synthesis through promoting ubiquitination of NCL.
我们旨在确定 Rho GTPase 激活蛋白 (RhoGAPs) 是否在宫颈癌中下调，并可能通过 GEO 数据库和免疫组织化学分析来靶向降低宫颈癌的生长，以确定转录和蛋白水平的变化。我们分析了它们的增殖、克隆形成能力以及在小鼠中的皮下肿瘤生长情况。为了检测 ARHGAP30 在细胞中的定位，进行了免疫荧光分析。通过质谱结合免疫沉淀实验鉴定结合蛋白。通过共免疫沉淀实验验证蛋白相互作用。应用 Western-blot 和 q-PCR 分析与核糖体生物发生相关的候选结合蛋白。嘌呤霉素掺入实验用于检测全局蛋白质合成率。我们确定 ARHGAP30 是唯一下调的 RhoGAP，与宫颈癌患者的生存有关。在宫颈癌细胞中过表达 ARHGAP30 抑制细胞增殖和迁移。ARHGAP30 免疫沉淀的蛋白富含核糖体生物发生过程。ARHGAP30 位于核内并与核仁蛋白 (NCL) 相互作用。过表达 ARHGAP30 抑制 rRNA 合成和全局蛋白质合成。ARHGAP30 过表达诱导 NCL 的泛素化并降低其在 Hela 细胞中的蛋白水平。ARHGAP30 对宫颈癌细胞核糖体生物发生和增殖的功能与其 RhoGAP 活性无关，如通过 ARHGAP30 的 RhoGAP 缺陷质粒评估。总体而言，研究结果表明 ARHGAP30 频繁下调，与宫颈癌患者的生存时间较短有关。ARHGAP30 可能通过促进 NCL 的泛素化来减少核糖体生物发生和蛋白质合成，从而抑制宫颈癌的生长。
