Wang Yinghui, Xie Yihang, Sun Boxuan, Guo Yuwei, Song Ling, Mohammednur Dawit Eman, Zhao Chunyan
College of Laboratory Medicine, Dalian Medical University, 9 West Section, Lvshun South Road, Dalian, Liaoning, China.
Liaoning Provincial Center for Disease Control and Prevention, Shenyang, China.
Infect Agent Cancer. 2021 Dec 24;16(1):71. doi: 10.1186/s13027-021-00409-9.
Cervical cancers are closely associated with persistent high-risk human papillomaviruses (HR HPV) infection. The main mechanism involves the targeting of tumor suppressors, such as p53 and pRB, for degradation by HR HPV-encoded oncoproteins, thereby leading to tumorigenesis. Rap1GAP, a tumor suppressor gene, is down-regulated in many cancers. Previous studies have revealed that down-regulation of Rap1GAP is correlated with HPV16/18 infection in cervical cancer. However, the molecular mechanism remains unclear. In this study, we aimed to address the degradation pathway of Rap1GAP in HPV-positive cervical cancer cells.
HPV-positive (HeLa and SiHa) and negative (C33A) cervical cancer cells were used to analyze the pathways of Rap1GAP degradation. MG132 (carbobenzoxy-leucyl-leucyl-leucine) was used to inhibit protein degradation by proteasome. Co-immunoprecipitation (co-IP) was used to detect the interaction between Rap1GAP and E6AP. siRNA for E6AP was used to silence the expression of E6AP. Rapamycin was used to induce cell autophagy. Western blotting was used to check the levels of proteins.
Following treatment with MG132, the levels of Rap1GAP were increased in the HR HPV-positive HeLa and SiHa cells, but not in the HPV-negative C33A cells. Co-immunoprecipitation assay revealed ubiquitinated Rap1GAP protein in HeLa and SiHa cells, but not in C33A cells. E6-associated protein (E6AP) mediated the ubiquitination of Rap1GAP by binding to it in HeLa and SiHa cells, but not in C33A cells. However, the levels of Rap1GAP were decreased in HeLa and SiHa cells after knocking down E6AP by siRNA. Silencing of E6AP did not affect the levels of Rap1GAP in C33A cells. Autophagy marker p62 was decreased and LC3 II/LC3 I was increased after knocking down E6AP in HeLa cells, but not in C33A cells. The levels of Rap1GAP were decreased after treating the cells with rapamycin to induce cell autophagy in HeLa and C33A cells.
Rap1GAP may be degraded by autophagy in cervical cancer cells, but HPV infection can switch the degradation pathway from autophagy to E6AP-mediated ubiquitin-proteasome degradation. E6AP may be a key component of the switch.
宫颈癌与持续性高危型人乳头瘤病毒（HR HPV）感染密切相关。主要机制包括HR HPV编码的癌蛋白靶向降解肿瘤抑制因子，如p53和pRB，从而导致肿瘤发生。Rap1GAP作为一种肿瘤抑制基因，在许多癌症中表达下调。先前的研究表明，宫颈癌中Rap1GAP的下调与HPV16/18感染相关。然而，其分子机制仍不清楚。在本研究中，我们旨在探讨Rap1GAP在HPV阳性宫颈癌细胞中的降解途径。
使用HPV阳性（HeLa和SiHa）及阴性（C33A）宫颈癌细胞分析Rap1GAP的降解途径。MG132（苄氧羰基-亮氨酰-亮氨酰-亮氨酸）用于抑制蛋白酶体介导的蛋白质降解。免疫共沉淀（co-IP）用于检测Rap1GAP与E6相关蛋白（E6AP）之间的相互作用。针对E6AP的小干扰RNA（siRNA）用于沉默E6AP的表达。雷帕霉素用于诱导细胞自噬。蛋白质印迹法用于检测蛋白质水平。
用MG132处理后，HR HPV阳性的HeLa和SiHa细胞中Rap1GAP水平升高，而HPV阴性的C33A细胞中未升高。免疫共沉淀分析显示，HeLa和SiHa细胞中存在泛素化的Rap1GAP蛋白，而C33A细胞中不存在。在HeLa和SiHa细胞中，E6相关蛋白（E6AP）通过与Rap1GAP结合介导其泛素化，而C33A细胞中则不然。然而，用siRNA敲低E6AP后，HeLa和SiHa细胞中Rap1GAP水平降低。沉默E6AP对C3A细胞中Rap1GAP水平无影响。在HeLa细胞中敲低E6AP后，自噬标志物p62水平降低，LC3 II/LC3 I水平升高，而C33A细胞中则无此现象。用雷帕霉素处理细胞诱导自噬后，HeLa和C33A细胞中Rap1GAP水平均降低。
Rap1GAP在宫颈癌细胞中可能通过自噬降解，但HPV感染可使降解途径从自噬转变为E6AP介导的泛素-蛋白酶体降解。E6AP可能是这一转变的关键成分。
