Xu Fenghua, Xiao Zhifeng, Fan Liqin, Ruan Guangcong, Cheng Yi, Tian Yuting, Chen Minjia, Chen Dongfeng, Wei Yanling
Department of Gastroenterology, Daping Hospital, Army Medical University (Third Military Medical University), Chongqing, China.
Front Cell Dev Biol. 2021 Oct 12;9:675356. doi: 10.3389/fcell.2021.675356. eCollection 2021.
Colorectal cancer (CRC) is one of the most common human malignancies. It was reported that the alterations in the DNA damage response (DDR) pathways are emerging as novel targets for treatment across different cancer types including CRC. RFWD3 plays a critical role in replication protein A (RPA)-mediated DNA damage in cancer cells. More importantly, RFWD3 can response to DNA damage by positively regulating p53 stability when the G1 cell cycle checkpoint is activated. However, the functional significance of RFWD3 in CRC has not been reported in the existing documents. Here, we revealed high expression of RFWD3 in CRC tissues by IHC analysis and The Cancer Genome Atlas (TCGA) database. Besides, overexpression of RFWD3 in CRC cell lines was also confirmed by qRT-PCR and western blot assay. The Celigo cell counting method and wound-healing/transwell migration assay were applied to evaluate CRC cell proliferation and migration. The tumor growth indicators were quantified in nude mice xenografted with shRFWD3 and shCtrl RKO cells. The results indicated that RFWD3 knockdown restricted CRC development and . In exploring the downstream mechanism of RFWD3's action, we found that RFWD3 could transcriptionally activate BIRC5 by interacting with E2F transcription factor 1 (E2F1). Accordingly, we identified BIRC5 as a downstream gene of RFWD3 regulating CRC. Subsequent loss- and gain- of function experiments demonstrated that upon overexpressing BIRC5 in RKO cells with down-regulated RFWD3, the inhibitory effects of cell proliferation, migration and colony formation could be reversed, while the capacity of cell apoptosis was ameliorated, suggesting that the effects of RFWD3 depletion was mainly due to BIRC5 suppression. Taken together, this study revealed that RFWD3 participates in the occurrence and development of colorectal cancer E2F1 transcriptional regulation of BIRC5.
结直肠癌（CRC）是最常见的人类恶性肿瘤之一。据报道，DNA损伤反应（DDR）通路的改变正成为包括CRC在内的不同癌症类型治疗的新靶点。RFWD3在癌细胞中复制蛋白A（RPA）介导的DNA损伤中起关键作用。更重要的是，当G1细胞周期检查点被激活时，RFWD3可通过正向调节p53稳定性来响应DNA损伤。然而，现有文献尚未报道RFWD3在CRC中的功能意义。在此，我们通过免疫组化分析和癌症基因组图谱（TCGA）数据库揭示了RFWD3在CRC组织中的高表达。此外，qRT-PCR和蛋白质免疫印迹分析也证实了RFWD3在CRC细胞系中的过表达。采用Celigo细胞计数法和伤口愈合/Transwell迁移试验评估CRC细胞的增殖和迁移。对接种shRFWD3和shCtrl RKO细胞的裸鼠体内肿瘤生长指标进行定量分析。结果表明，敲低RFWD3可抑制CRC的发展。在探索RFWD3作用的下游机制时，我们发现RFWD3可通过与E2F转录因子1（E2F1）相互作用转录激活BIRC5。因此，我们确定BIRC5是RFWD3调节CRC的下游基因。随后的功能缺失和功能获得实验表明，在RFWD3下调的RKO细胞中过表达BIRC5后，细胞增殖、迁移和集落形成的抑制作用可被逆转，而细胞凋亡能力得到改善，这表明RFWD3缺失的影响主要是由于BIRC5受到抑制。综上所述，本研究揭示了RFWD3通过E2F1对BIRC5的转录调控参与结直肠癌的发生发展。
