A Positive Feedback Loop of lncRNA HOXD-AS2 and SMYD3 Facilitates Hepatocellular Carcinoma Progression via the MEK/ERK Pathway

Abstract

Jin Sun,1– 3 Yingnan Li,1– 3 Mengjiao Shi,1,2 Hongwei Tian,1– 3 Jun Li,1– 3 Kai Zhu,1– 3 Ying Guo,1– 3 Yanhua Mu,1– 3 Jing Geng,1– 3 Zongfang Li1– 4 1National-Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China; 2Shaanxi Provincial Clinical Research Center for Hepatic & Splenic Diseases, the Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China; 3Center for Tumor and Immunology, the Precision Medical Institute, Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China; 4Department of Geriatric General Surgery, the Second Affiliated Hospital of Xi’ an Jiaotong University, Xi’an, Shaanxi, People’s Republic of ChinaCorrespondence: Jing Geng; Zongfang Li, 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, Tel [phone] ; Tel/Fax [phone], Email [email protected]; [email protected]: HOX cluster-embedded long noncoding RNAs (HOX-lncRNAs) have been shown to be tightly related to hepatocellular carcinoma (HCC). However, the potential biological roles and underlying molecular mechanism of HOX-lncRNAs in HCC largely remains to be elucidated.Methods: The expression signature of eighteen HOX-lncRNAs in HCC cell lines were measured by qRT-PCR. HOXD-AS2 expression and its clinical significance in HCC was investigated by bioinformatics analysis utilizing the TCGA data. Subcellular localization of HOXD‐AS2 in HCC cells was observed by RNA-FISH. Loss‑of‑function experiments in vitro and in vivo were conducted to probe the roles of HOXD-AS2 in HCC. Potential HOXD-AS2-controlled genes and signaling pathways were revealed by RNA-seq. Rescue experiments were performed to validate that SMYD3 mediates HOXD-AS2 promoting HCC progression. The positive feedback loop of HOXD-AS2 and SMYD3 was identified by luciferase reporter assay and ChIP-qPCR.Results: HOXD-AS2 was dramatically elevated in HCC, and its up-regulation exhibited a positive association with aggressive clinical features (T stage, pathologic stage, histologic grade, AFP level, and vascular invasion) and unfavorable prognosis of HCC patients. HOXD-AS2 was distributed both in the nucleus and the cytoplasm of HCC cells. Knockdown of HOXD-AS2 restrained the proliferation, migration, invasion of HCC cells in vitro, as well as tumor growth in subcutaneous mouse model. Transcriptome analysis demonstrated that SMYD3 expression and activity of MEK/ERK pathway were impaired by silencing HOXD-AS2 in HCC cells. Rescue experiments revealed that SMYD3 as downstream target mediated oncogenic functions of HOXD-AS2 in HCC cells through altering the expression of cyclin B1, cyclin E1, MMP2 as well as the activity of MEK/ERK pathway. Additionally, HOXD-AS2 was uncovered to be positively regulated at transcriptional level by its downstream gene of SMYD3.Conclusion: HOXD-AS2, a novel oncogenic HOX-lncRNA, facilitates HCC progression by forming a positive feedback loop with SMYD3 and activating the MEK/ERK pathway.Keywords: hepatocellular carcinoma, HOXD-AS2, SMYD3, MEK/ERK signaling

Keywords

• hepatocellular carcinoma
• hoxd-as2
• smyd3
• mek/erk signaling