Wang Shanshan, Feng Rilu, Shi Ying, Chen Dexi, Weng Honglei, Ding Huiguo, Zhang Chenguang
Beijing Institute of Hepatology, Beijing You' An Hospital, Capital Medical University, Beijing, 100069, China.
Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, 68167, Mannheim, Germany.
Sci Rep. 2021 Jan 25;11(1):2146. doi: 10.1038/s41598-021-81678-7.
Retinoic acid and retinoid acid receptor (RA-RAR) signaling exhibits suppressive functions in the progression of hepatocellular carcinoma (HCC) through multiple mechanisms. However, whether RA-RAR signaling induces autophagy that contributes its anti-tumor activity in HCC remains elusive. In the current study, the effects of RA-RAR pathway on autophagy were investigated in two HCC cell lines: alpha-fetoprotein (AFP) positive PLC/PRF/5 and AFP negative HLE cells. Cell autophagy was analyzed with western blot for detection of LC3 conversion and p62/SQSTM1 degradation while autophagy flux was assayed using the mRFP-GFP-LC3 reporter. Cell apoptosis and viability were analyzed by caspase-3 activity, TdT-mediated dUTP nick end labeling (TUNEL) assay, and Cell Counting Kit (CCK)-8, respectively. Chromatin immunoprecipitation (ChIP) was employed to detect the binding of RAR onto the promoter of autophagy-relevant 7 (ATG7), and co-immunoprecipitation (CoIP) was used to analyze the interaction of AFP and RAR. The results showed that ATRA dosage and time-dependently induced high levels of cell autophagy in both the PLC/PRF/5 and HLE cells, which was accompanied with up-regulation of ATG7. ChIP assay showed that RAR was able to bind to its responsive elements on ATG7 promoter. Impairment of ATG7 induction or blockade of autophagy with chloroquine aggravated ATRA induced apoptosis of HCC cells. Furthermore, intracellular AFP was able to complex with RAR in PLC/PRF/5 cells. Knockdown of AFP in PLC/PRF/5 cells augmented the up-regulation of ATG7 by ATRA while overexpression of AFP in HLE cells attenuated ATRA induced ATG7 expression and autophagy. Thus, ATRA induced ATG7 and autophagy participated in its cytotoxicity on HCC cells and AFP interfere with the induction of ATG7 and autophagy through forming complex with RAR.
维甲酸和维甲酸受体（RA-RAR）信号通路通过多种机制在肝细胞癌（HCC）进展中发挥抑制作用。然而，RA-RAR信号通路是否诱导自噬从而在HCC中发挥其抗肿瘤活性仍不清楚。在本研究中，在两种肝癌细胞系中研究了RA-RAR通路对自噬的影响：甲胎蛋白（AFP）阳性的PLC/PRF/5细胞和AFP阴性的HLE细胞。用蛋白质免疫印迹法分析细胞自噬以检测LC3转化和p62/SQSTM1降解，同时使用mRFP-GFP-LC3报告基因检测自噬通量。分别通过半胱天冬酶-3活性、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）试验和细胞计数试剂盒（CCK）-8分析细胞凋亡和活力。采用染色质免疫沉淀（ChIP）检测RAR与自噬相关蛋白7（ATG7）启动子的结合，并采用免疫共沉淀（CoIP）分析AFP与RAR的相互作用。结果表明，全反式维甲酸（ATRA）剂量和时间依赖性地在PLC/PRF/5和HLE细胞中诱导高水平的细胞自噬，同时伴有ATG7的上调。ChIP试验表明，RAR能够结合到ATG7启动子上的反应元件。用氯喹抑制ATG7的诱导或阻断自噬会加重ATRA诱导的肝癌细胞凋亡。此外，细胞内AFP能够与PLC/PRF/5细胞中的RAR形成复合物。敲低PLC/PRF/5细胞中的AFP可增强ATRA对ATG7的上调作用，而在HLE细胞中过表达AFP则减弱ATRA诱导的ATG7表达和自噬。因此，ATRA诱导的ATG7和自噬参与了其对肝癌细胞的细胞毒性作用，而AFP通过与RAR形成复合物干扰了ATG7的诱导和自噬。
