Department of Gynecologic Oncology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Present address: Dong-Cheng District, Qi-He-Lou Street No.17, Beijing, 100006, China.
Radiat Oncol. 2020 May 29;15(1):131. doi: 10.1186/s13014-020-01580-w.
Radiosensitivity is limited in cervical cancer (CC) patients due to acquired radiation resistance. In our previous studies, we found that immediate-early response 5 (IER5) is upregulated in CC cells upon radiation exposure and decreases cell survival by promoting apoptosis. The details on the transcriptional regulation of radiation-induced IER5 expression are unknown. Studies in recent years have suggested that Pol II-associated factor 1 (PAF1) is a pivotal transcription factor for certain genes "induced" during tumor progression. In this study, we investigated the role of PAF1 in regulating IER5 expression during CC radiotherapy.
PAF1 expression in CC cells was measured by western blotting, immunohistochemistry, and qRT-PCR, and the localization of PAF1 and IER5 was determined by immunofluorescence. The effect of PAF1 and IER5 knockdown by siRNA in Siha and Hela cells was studied by western blotting, qRT-PCR, CCK-8 assay, and flow cytometry. The physical interaction of PAF1 with the IER5 promoter and enhancers was confirmed using chromatin immunoprecipitation and qPCR with or without enhancers knockout by CRISPR/Cas9.
We confirmed that PAF1 was highly expressed in CC cells and that relatively low expression of IER5 was observed in cells with highly expressed PAF1 in the nucleus. PAF1 knockdown in Siha and Hela cells was associated with increased expression of IER5, reduced cell viability and higher apoptosis rate in response to radiation exposure, while simultaneous PAF1 and IER5 knockdown had little effect on the proportion of apoptotic cells. We also found that PAF1 hindered the transcription of IER5 by promoting Pol II pausing at the promoter-proximal region, which was primarily due to the binding of PAF1 at the enhancers.
PAF1 reduces CC radiosensitivity by inhibiting IER5 transcription, at least in part by regulating its enhancers. PAF1 might be a potential therapeutic target for overcoming radiation resistance in CC patients.
由于获得性辐射抵抗，宫颈癌（CC）患者的放射敏感性受到限制。在我们之前的研究中，我们发现 IER5（立即早期反应 5）在受到辐射后在 CC 细胞中上调，并通过促进细胞凋亡来降低细胞存活率。关于辐射诱导 IER5 表达的转录调节的细节尚不清楚。近年来的研究表明，聚合酶 II 相关因子 1（PAF1）是肿瘤进展过程中某些基因“诱导”的关键转录因子。在这项研究中，我们研究了 PAF1 在调节 CC 放疗期间 IER5 表达中的作用。
通过 Western blot、免疫组化和 qRT-PCR 测量 CC 细胞中的 PAF1 表达，通过免疫荧光测定 PAF1 和 IER5 的定位。通过 siRNA 敲低 Siha 和 Hela 细胞中的 PAF1 和 IER5，通过 Western blot、qRT-PCR、CCK-8 测定和流式细胞术研究其作用。使用染色质免疫沉淀和 qPCR 以及通过 CRISPR/Cas9 敲除增强子来确认 PAF1 与 IER5 启动子和增强子的物理相互作用。
我们证实 PAF1 在 CC 细胞中高度表达，而在核内高表达 PAF1 的细胞中观察到 IER5 的相对低表达。Siha 和 Hela 细胞中 PAF1 的敲低与 IER5 的表达增加、辐射暴露后细胞活力降低和凋亡率升高相关，而同时敲低 PAF1 和 IER5 对凋亡细胞的比例几乎没有影响。我们还发现，PAF1 通过促进 Pol II 在启动子近端区域暂停来抑制 IER5 的转录，这主要是由于 PAF1 在增强子上的结合。
PAF1 通过抑制 IER5 转录降低 CC 的放射敏感性，至少部分是通过调节其增强子。PAF1 可能是克服 CC 患者辐射抵抗的潜在治疗靶点。
