Hsa_circ_0035796 promotes non-small cell lung cancer cell progression via the miR-142-3p-HMGB1 axis and interaction with FOXO1
Authors: Cui Yumeng , Wu Xiaojie , Jin Jie , Man Weiling , Li Jie , Li Xiang , Li Yanghua , Yao He , Liu Mengsi , Zhong Rongbin , Zhao Jin , Wu Jiahui , Zhu Tianhao , Lin Yanli , Xu Junjie , Wang Youliang 
Published: September 2022

Abstract
Background
Noncoding RNAs such as circular RNAs (circRNAs) are abundant in the human body and influence the occurrence and development of various diseases. Non-small cell lung cancer (NSCLC) is one of the most common malignant cancers. Information on the functions and mechanism of circRNAs in lung cancer is limited; thus, the topic needs more exploration. The purpose of this study was to identify aberrantly expressed circRNAs in lung cancer, unravel their roles in NSCLC progression, and provide new targets for lung cancer diagnosis and therapy.
Methods
High-throughput sequencing was used to analyze differential circRNA expression in patients with lung cancer. qRT‒PCR was used to determine the level of circ_0035796 in lung cancer tissues and plasma samples. Gain- and loss-of-function experiments were implemented to observe the impacts of circ_0035796 on the growth, invasion, and metastasis of lung cancer cells in vitro and in vivo. Mechanistically, dual luciferase reporter assays, fluorescent in situ hybridization (FISH), RNA immunoprecipitation (RIP) and RNA pull-down experiments were performed to confirm the underlying mechanisms of circ_0035796. Nucleoplasmic localization of FOXO1 was determined by nucleocytoplasmic isolation and immunofluorescence. The interaction of circ_0035796 with FOXO1 was verified by RNA pull-down, RNA immunoprecipitation (RIP) and western blot assays. The proliferation and migration of circ_0035796 in vivo were verified by subcutaneous and tail vein injection in nude mice.
Results
Circ_0035796 was significantly upregulated in lung cancer tissues and cells, and its expression was correlated with the clinical stage of patients with lung cancer. Ectopic expression of circ_0035796 strikingly facilitated the proliferation, invasion and metastasis, and inhibited the apoptosis of lung cancer cells in vitro and in vivo. However, knockdown of circ_0035796 exerted the opposite effects. Circ_0035796 was mainly distributed in the cytoplasm. Further mechanistic research indicated that circ_0035796 acted as a competing endogenous RNA of miR-142-3p to relieve the repressive effect of miR-142-3p on its target HMGB1, activating the MAPK/ERK and NF-κB pathways and promoting cell migration and invasion. More importantly, we found that circ_0035796 could bind FOXO1 and sequester it in the cytoplasm, adjusting the feedback AKT pathway. The accumulation of FOXO1 in the cytosol and nuclear exclusion promoted cell proliferation and inhibited apoptosis.
Conclusions
Circ_0035796 is a new circRNA that promotes tumor function in NSCLC and may serve as a potential prognostic biomarker and therapeutic target for NSCLC. Our findings indicate that circ_0035796 facilitates the invasion and metastasis of NSCLC cells by regulating the miR-142-3p/HMGB1 axis and activating the MAPK/ERK and NF-κB pathways. In addition, circ_0035796 can promote cell proliferation and inhibit apoptosis by sequestering FOXO1 in the cytoplasm to regulate AKT activity and BIM transcription.