Wu Xing1, Peng-Cheng Zhou2,3, Hao-Ye Zhang2, Li-Min Chen2, Yang-Mei Zhou2, Xue-Fei Cui1, Zhen-Guo Liu2,3. 1. Department of Radiology, Xiangya Hospital, Central South University, Changsha, China. 2. Department of Infectious Disease, The Third Xiangya Hospital, Central South University, Changsha, China. 3. Hunan Key Laboratory of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, China.
Abstract
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the leading causes of tumour-related death. Here, we investigated the molecular mechanism of HCC by studying the function of circ_GLIS2. METHODS: Human HCC specimens and cell lines were used. Sanger sequencing, actinomycin D and RNase R treatment were performed to validate circular RNA features of circ_GLIS2. qRT-PCR, western blotting, immunostaining, and IHC were employed to examine levels of circ_GLIS2, GLIS2 mRNA, and EMT-related markers. CCK-8, colony formation, flow cytometry, wound healing assay, and transwell assays were performed to evaluate cancer cell proliferation, apoptosis, migration, and invasion. RIP and RNA pull-down assay were used to validate EIF4A3/GLIS2 mRNA interaction. MSP was performed to measure the methylation status of GLIS2 promoter. Nude mouse xenograft model was used to examine tumour growth and metastasis in vivo. RESULTS: Circ_GLIS2 and linear GLIS2 mRNA were reduced in human HCC tissues and cells. Their low levels correlated with a poor survival rate of HCC patients. Overexpression of circ_GLIS2 and GLIS2 suppressed HCC cell proliferation, migration, and invasion but promoted cell apoptosis. GLIS2 promoter region was hypermethylated in HCC cells. EIF4A3 was directly bound with GLIS2 mRNA and promoted circ_GLIS2/GLIS2 expression. Moreover, overexpression of circ_GLIS2 restrained HCC tumour growth and metastasis in vivo. CONCLUSION: Circ_GLIS2 suppresses HCC growth and metastasis by inhibiting cell proliferation, migration, and invasion, but promoting cell apoptosis. These findings provide molecular insights into the mechanism of HCC and indicate that circ_GLIS2 could serve as a diagnosis marker or therapeutic target for HCC.
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the leading causes of tumour-related death. Here, we investigated the molecular mechanism of HCC by studying the function of circ_GLIS2. METHODS: Human HCC specimens and cell lines were used. Sanger sequencing, actinomycin D and RNase R treatment were performed to validate circular RNA features of circ_GLIS2. qRT-PCR, western blotting, immunostaining, and IHC were employed to examine levels of circ_GLIS2, GLIS2 mRNA, and EMT-related markers. CCK-8, colony formation, flow cytometry, wound healing assay, and transwell assays were performed to evaluate cancer cell proliferation, apoptosis, migration, and invasion. RIP and RNA pull-down assay were used to validate EIF4A3/GLIS2 mRNA interaction. MSP was performed to measure the methylation status of GLIS2 promoter. Nude mouse xenograft model was used to examine tumour growth and metastasis in vivo. RESULTS: Circ_GLIS2 and linear GLIS2 mRNA were reduced in human HCC tissues and cells. Their low levels correlated with a poor survival rate of HCC patients. Overexpression of circ_GLIS2 and GLIS2 suppressed HCC cell proliferation, migration, and invasion but promoted cell apoptosis. GLIS2 promoter region was hypermethylated in HCC cells. EIF4A3 was directly bound with GLIS2 mRNA and promoted circ_GLIS2/GLIS2 expression. Moreover, overexpression of circ_GLIS2 restrained HCC tumour growth and metastasis in vivo. CONCLUSION: Circ_GLIS2 suppresses HCC growth and metastasis by inhibiting cell proliferation, migration, and invasion, but promoting cell apoptosis. These findings provide molecular insights into the mechanism of HCC and indicate that circ_GLIS2 could serve as a diagnosis marker or therapeutic target for HCC.