Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer with high aggressive phenotype and poor prognosis. Accumulating evidence suggests that circRNAs have been identified as pivotal mediators in cancers. However, the role of circRNAs in ccRCC progression remains elusive. The differentially expressed circRNAs in 4 paired human ccRCC and adjacent noncancerous tissues ccRCC were screened using circRNA microarrays and the candidate target was selected based on circRNA expression level using weighted gene correlation network analysis (WGCNA) and the gene expression omnibus (GEO) database. CircPDHK1 expression in ccRCC and adjacent noncancerous tissues (n = 148) were evaluated along with clinically relevant information. RT-qPCR, RNase R digestion, and actinomycin D (ActD) stability test were conducted to identify the characteristics of circPDHK1. The subcellular distribution of circPDHK1 was analyzed by subcellular fractionation assay and fluorescence in situ hybridization (FISH). Immunoprecipitation-mass spectrometry (IP-MS) and immunofluorescence (IF) were employed to evaluate the protein-coding ability of circPDHK1. ccRCC cells were transfected with siRNAs, plasmids or lentivirus approach, and cell proliferation, migration and invasion, as well as tumorigenesis and metastasis in nude mice were assessed to clarify the functional roles of circPDHK1 and its encoded peptide PDHK1-241aa. RNA-sequencing, western blot analysis, immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) assays were further employed to identify the underlying mechanisms regulated by PDHK1-241aa. CircPDHK1 was upregulated in ccRCC tissues and closely related to WHO/ISUP stage, T stage, distant metastasis, VHL mutation and Ki-67 levels. CircPDHK1 had a functional internal ribosome entry site (IRES) and encoded a novel peptide PDHK1-241aa. Functionally, we confirmed that PDHK1-241aa and not the circPDHK1 promoted the proliferation, migration and invasion of ccRCC. Mechanistically, circPDHK1 was activated by HIF-2A at the transcriptional level. PDHK1-241aa was upregulated and interacted with PPP1CA, causing the relocation of PPP1CA to the nucleus. This thereby inhibited AKT dephosphorylation and activated the AKT-mTOR signaling pathway. Our data indicated that circPDHK1-encoded PDHK1-241aa promotes ccRCC progression by interacting with PPP1CA to inhibit AKT dephosphorylation. This study provides novel insights into the multiplicity of circRNAs and highlights the potential use of circPDHK1 or PDHK1-241aa as a therapeutic target for ccRCC.

中文翻译：

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circPDHK1 编码的新型肽 PDHK1-241aa 通过与 PPP1CA 相互作用抑制 AKT 去磷酸化并激活 AKT-mTOR 信号通路促进 ccRCC 进展

透明细胞肾细胞癌（ccRCC）是最常见的肾癌，具有高侵袭性表型和预后不良。越来越多的证据表明，circRNA 已被确定为癌症的关键介质。然而，circRNA 在 ccRCC 进展中的作用仍然难以捉摸。使用circRNA微阵列筛选4对人ccRCC和邻近非癌组织ccRCC中差异表达的circRNA，并使用加权基因相关网络分析（WGCNA）和基因表达综合（GEO）数据库根据circRNA表达水平选择候选靶点。我们评估了 ccRCC 和邻近非癌组织 (n = 148) 中的 CircPDHK1 表达以及临床相关信息。通过 RT-qPCR、RNase R 消化和放线菌素 D (ActD) 稳定性测试来鉴定 circPDHK1 的特征。通过亚细胞分级分离测定和荧光原位杂交（FISH）分析circPDHK1的亚细胞分布。采用免疫沉淀-质谱（IP-MS）和免疫荧光（IF）评估circPDHK1的蛋白质编码能力。采用siRNA、质粒或慢病毒方法转染ccRCC细胞，并评估细胞增殖、迁移和侵袭以及裸鼠肿瘤发生和转移，以阐明circPDHK1及其编码肽PDHK1-241aa的功能作用。进一步采用 RNA 测序、蛋白质印迹分析、免疫沉淀 (IP) 和染色质免疫沉淀 (ChIP) 测定来确定 PDHK1-241aa 调节的潜在机制。 CircPDHK1在ccRCC组织中表达上调，与WHO/ISUP分期、T分期、远处转移、VHL突变和Ki-67水平密切相关。 CircPDHK1 具有功能性内部核糖体进入位点 (IRES)，并编码新型肽 PDHK1-241aa。从功能上讲，我们证实PDHK1-241aa而不是circPDHK1促进ccRCC的增殖、迁移和侵袭。从机制上讲，circPDHK1 在转录水平被 HIF-2A 激活。 PDHK1-241aa 上调并与 PPP1CA 相互作用，导致 PPP1CA 重新定位到细胞核。这从而抑制 AKT 去磷酸化并激活 AKT-mTOR 信号通路。我们的数据表明，circPDHK1 编码的 PDHK1-241aa 通过与 PPP1CA 相互作用抑制 AKT 去磷酸化来促进 ccRCC 进展。这项研究提供了对 circRNA 多样性的新见解，并强调了 circPDHK1 或 PDHK1-241aa 作为 ccRCC 治疗靶点的潜在用途。