NRF1-Induced lncRNA DDX11-AS1 Contributes to the Progression of Hepatocellular Carcinoma via Activating CA9 Expression and the MEK/ERK Pathway

Yingnan Li,1,2 Mengjiao Shi,1,3 Beibei Bie,4 Hongwei Tian,1,3 Jun Li,1,3 Zongfang Li,1– 3 Jin Sun1,3 1Department of General Surgery, National-Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710004, People’s Republic of China; 2Center for Tumor and Immunology, The Precision Medical Institute, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710115, People’s Republic of China; 3Shaanxi Provincial Clinical Research Center for Hepatic & Splenic Diseases, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710004, People’s Republic of China; 4Department of Pharmacy, Medical School, Xi’an Peihua University, Xi’an, 710125, People’s Republic of ChinaCorrespondence: Zongfang Li, Department of General Surgery, National & Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, The Second Affiliated Hospital, Xi’an Jiaotong University, No. 157 West 5th Road, Xi’an, 710004, People’s Republic of China, Email [email protected] Jin Sun, Department of General Surgery, National & Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, The Second Affiliated Hospital, Xi’an Jiaotong University, No. 157 West 5th Road, Xi’an, 710004, People’s Republic of China, Email [email protected]: DDX11 antisense RNA 1 (DDX11-AS1) has been recognized for its strong correlation with hepatocellular carcinoma (HCC). Nevertheless, the exact biological functions and fundamental molecular processes of DDX11-AS1 in HCC require further in-depth investigation.Methods: A comprehensive bioinformatics analysis was carried out to explore the expression of DDX11-AS1 and its clinical implication in HCC utilizing the TCGA data. qRT-PCR was employed to validate the expression of DDX11-AS1 in HCC tissues/cell lines. RNA fluorescence in situ hybridization (RNA-FISH) was used to observe the subcellular localization of DDX11-AS1 in HCC cells. Loss-of-function experiments, both in vitro and in vivo, were executed to elucidate the biological functions of DDX11-AS1 in HCC. RNA sequencing (RNA-seq) was employed to identify genes and signaling pathways potentially regulated by DDX11-AS1. Rescue experiments were conducted to validate that carbonic anhydrase IX (CA9) mediates DDX11-AS1 promoting HCC progression. The influence of nuclear respiratory factor 1 (NRF1) on the transcription of DDX11-AS1 was investigated through dual-luciferase reporter assays and ChIP-qPCR.Results: The increased expression of DDX11-AS1 is positively associated with several aggressive clinical characteristics (pathologic T stage, histologic grade, AFP level, and vascular invasion), and is closely linked to unfavorable outcomes in HCC patients, acting as a separate hazardous factor for overall survival. DDX11-AS1 is predominantly situated in the nucleus of HCC cells. DDX11-AS1 knockdown impeded the growth, migration, and invasion capabilities of HCC cells in vitro, and reduced the tumor enlargement in a subcutaneous mouse model. RNA-Seq unveiled that silencing DDX11-AS1 lessened the expression of CA9 and suppressed the activity of the MEK/ERK signaling cascade in HCC cells. Rescue experiments uncovered that CA9 acts as a downstream target facilitating the cancer-causing roles of DDX11-AS1 in HCC. Furthermore, DDX11-AS1 was revealed to be transcriptionally regulated by NRF1.Conclusion: DDX11-AS1, a NRF1-induced lncRNA, facilitates HCC development by upregulating CA9 expression and activating the MEK/ERK signaling cascade.Keywords: hepatocellular carcinoma, DDX11-AS1, CA9, MEK/ERK signaling, NRF1