Department of Urology, Affiliated Zhongda Hospital of Southeast University, No.87 Dingjiaqiao Road, Nanjing, 210009, People's Republic of China.
Urology Research Center, Southeast University Medical School, No.87 Dingjiaqiao Road, Nanjing, 210009, People's Republic of China.
Mol Cancer. 2022 Jul 15;21(1):146. doi: 10.1186/s12943-022-01607-8.
Increasing evidence has demonstrated that circular RNAs (circRNAs) are implicated in cancer progression. However, the aberrant expression and biological functions of circRNAs in clear cell renal cell carcinoma (cRCC) remain largely elusive.
Differentially expressed circRNAs in cRCC were filtered via bioinformatics analysis. Aberrant circPOLR2A expression was validated in cRCC tissues and cell lines via qRT-PCR. Sanger sequencing was used to identify the backsplicing site of circPOLR2A. In vitro and in vivo functional experiments were performed to evaluate the role of circPOLR2A in cRCC malignancy. RNA pull-down, mass spectrometry, RIP, FISH and immunofluorescence assays were used to identify and validate the circPOLR2A-interacting proteins. Ubiquitination modification and interaction between proteins were detected via Co-IP and western blotting. The m6A modification in circPOLR2A was validated by the meRIP assay.
Bioinformatics analysis revealed that circPOLR2A was highly expressed in cRCC tissues and metastatic cRCC tissues. CircPOLR2A expression was associated with tumor size and TNM stage in cRCC patients. In vitro and in vivo functional assays revealed that circPOLR2A accelerated cRCC cell proliferation, migration, invasion and angiogenesis, while inhibiting apoptosis. Further mechanistic research suggested that circPOLR2A could interact with UBE3C and PEBP1 proteins, and that UBE3C could act as a specific ubiquitin E3 ligase for the PEBP1 protein. The UBE3C/circPOLR2A/PEBP1 protein-RNA ternary complex enhanced the UBE3C-mediated ubiquitination and degradation of the PEBP1 protein which could inactivate the ERK signaling pathway. Rescue experiments revealed that the PEBP1 protein was the functional downstream target of circPOLR2A. Furthermore, m6A modification in circPOLR2A was confirmed, and the m6A reader YTHDF2 could regulate circPOLR2A expression.
Our study demonstrated that circPOLR2A modulated the UBE3C-mediated ubiquitination and degradation of the PEBP1 protein, and further activated the ERK pathway during cRCC progression and metastasis. The m6A reader, YTHDF2, regulated circPOLR2A expression in cRCC. Hence, circPOLR2A could be a potential target for the diagnosis and treatment of cRCC.
越来越多的证据表明，环状 RNA（circRNA）参与了癌症的进展。然而，环状 RNA 在透明细胞肾细胞癌（ccRCC）中的异常表达和生物学功能仍很大程度上难以捉摸。
通过生物信息学分析筛选出 ccRCC 中的差异表达环状 RNA。通过 qRT-PCR 验证 ccRCC 组织和细胞系中 circPOLR2A 的异常表达。通过 Sanger 测序鉴定 circPOLR2A 的反向剪接位点。进行体外和体内功能实验以评估 circPOLR2A 在 ccRCC 恶性肿瘤中的作用。使用 RNA 下拉、质谱、RIP、FISH 和免疫荧光测定来鉴定和验证 circPOLR2A 相互作用的蛋白质。通过 Co-IP 和 Western blot 检测蛋白质的泛素化修饰和相互作用。通过 meRIP 测定验证 circPOLR2A 的 m6A 修饰。
生物信息学分析显示 circPOLR2A 在 ccRCC 组织和转移性 ccRCC 组织中高表达。circPOLR2A 的表达与 ccRCC 患者的肿瘤大小和 TNM 分期有关。体外和体内功能实验表明，circPOLR2A 可加速 ccRCC 细胞的增殖、迁移、侵袭和血管生成，同时抑制细胞凋亡。进一步的机制研究表明，circPOLR2A 可以与 UBE3C 和 PEBP1 蛋白相互作用，UBE3C 可以作为 PEBP1 蛋白的特异性泛素 E3 连接酶。UBE3C/circPOLR2A/PEBP1 蛋白-RNA 三元复合物增强了 UBE3C 介导的 PEBP1 蛋白的泛素化和降解，从而使 ERK 信号通路失活。挽救实验表明，PEBP1 蛋白是 circPOLR2A 的功能下游靶标。此外，circPOLR2A 的 m6A 修饰得到了证实，m6A 读取器 YTHDF2 可以调节 circPOLR2A 的表达。
本研究表明，circPOLR2A 调节 UBE3C 介导的 PEBP1 蛋白的泛素化和降解，进而在 ccRCC 进展和转移过程中激活 ERK 通路。m6A 读取器 YTHDF2 调节 ccRCC 中的 circPOLR2A 表达。因此，circPOLR2A 可能是 ccRCC 诊断和治疗的潜在靶点。
