Long noncoding RNA HClnc1 promotes proliferation and migration of liver cancer cells by targeting RBBP5/KAT2B complex to enhance ODC1 transcription

doi:10.12122/j.issn.1673-4254.2025.09.11

Abstract

Abstract:

Objective To investigate the role of long noncoding RNA (lncRNA) HClnc1 in regulating proliferation, invasion, and migration of hepatocellular carcinoma (HCC) cells and the regulatory mechanism. Methods HClnc1 expression levels in liver cancer tissues were analyzed using data from the TCGA database. BrdU incorporation, plate cloning, and transwell assays were employed to examine the effects of HClnc1 silencing/overexpression and/or ODC1 silencing on proliferation, invasion, and migration of liver cancer cells. The effects of HClnc1 silencing on ODC1 protein and mRNA expression in the liver cancer cells were analyzed using qRT-PCR and Western blotting. The activity of ODC1 promoter was analyzed using a dual luciferase reporter gene assay. Pull-down experiment, mass spectrometry analysis, and chromatin immunoprecipitation (ChIP) assay were used for identification of HClnc1-binding proteins and their interactions. Protein interactions with the ODC1 promoter region and their binding efficiencies were investigated using RNA interference and ChIP analysis. Results HClnc1 was significantly overexpressed in HCC tissues. In liver cancer cells, HClnc1 silencing significantly inhibited cell proliferation, invasion, and migration, while HClnc1 overexpression promoted these behaviors. ODC1 silencing also suppressed malignant behaviors of liver cancer cells, and counteracted the effects of HClnc1 overexpression. Interference of HClnc1 obviously inhibited ODC1 promoter activity. RBBP5 and KAT2B proteins were identified to bind simultaneously with HClnc1. HClnc1 overexpression upregulated ODC1 protein expression, while interference of RBBP5 or KAT2B downregulated ODC1 protein expression and blocked HClnc1-induced upregulation of ODC1 protein. Both RBBP5 and KAT2B could directly bind to ODC1 promoter region; knocking out KAT2B or RBBP5 reduced the binding efficiency, while knocking out HClnc1 reduced the binding of both RBBP5 and KAT2B to ODC1 promoter region. Conclusion By targeting the RBBP5/KAT2B epigenetic modification complex, HClnc1 increases ODC1 promoter activity to enhance ODC1 transcription and promote the proliferation and migration of liver cancer cells.

Key words: long noncoding RNA, hepatocellular carcinoma, RBBP5, KAT2B, ODC1, HClnc1

Zhihui FENG, Wenyue LI, Mingxiu ZHANG, Peipei WANG, Yangyang SHUAI, Hong ZHANG. Long noncoding RNA HClnc1 promotes proliferation and migration of liver cancer cells by targeting RBBP5/KAT2B complex to enhance ODC1 transcription[J]. Journal of Southern Medical University, 2025, 45(9): 1919-1926.

Figures/Tables 6

Fig.1 Effects of HClnc1 silencing on proliferation, invasion, and migration of liver cancer cells. A: Expression of HClnc1 in HCC (T) and adjacent regular tissues (N) from TCGA database. B: Proliferation of HepG2 cells assessed using BrdUrd assay. C, D: Colony formation of HepG2 cells (Original magnification:×1). E-G: Migration and invasion of HepG2 cells assessed using Transwell assay. **P<0.01, ***P<0.001 vs control group.

Fig.2 Effects of HClnc1 overexpression on proliferation, invasion, and migration of liver cancer cells. A: Proliferation of Huh7 cells assessed using BrdUrd assay. B, C: Colony formation of Huh7 cells. D-F: Migration and invasion of Huh7 cells assessed using Transwell assay. *P<0.05, **P<0.01, ***P<0.001 vs control group.

Fig.3 ODC1 knockdown reverses HClnc1-induced proliferation, invasion, and migration of hepatocellular carcinoma (HCC) cells. A, B: Gene expression levels of ODC1 in HCC and normal tissues from TCGA clinical samples and their correlation with survival. C, D: Relative mRNA and protein expression levels of ODC1 in HepG2 cells transfected with control, HClnc1 shRNA1/2, or ODC1 shRNA. E: ODC1 promoter activity assessed by luciferase activity assay. F: Proliferation of HepG2 cells assessed using BrdUrd assay. G-H: Migration and invasion of HepG2 cells assessed using Transwell assay (×20). I-K: HepG2 cells were co-transfected with control or HClnc1 plasmid plus control or ODC1 shRNA. I: Proliferation of HepG2 cells assessed using BrdUrd assay. J-K: Migration and invasion of HepG2 cells assessed using Transwell assay (×20). *P<0.05, **P<0.01, ***P<0.001 vs control or control plasmid +control shRNA. #P<0.05, ##P<0.01, ###P<0.001 vs HClnc1 plasmid+control shRNA.

Fig.4 HClin1 binds to RBBP5 and KAT2B proteins. A: Silver staining of biotinylated HClnc1-associated proteins. The proteins were excised from the two HClnc1-specific bands (red arrows) for mass spectrometry analysis. B: 55 identified proteins. C: Western blotting of proteins from antisense HClnc1 and HClnc1 pull-down assay. D: RNA immunoprecipitation with anti-RBBP5 or anti-KAT2B antibody. E, F: Relative RNA levels of HClnc1 and GAPDH detected using real-time PCR assay. The nuclear fraction of HepG2 cells was immunoprecipitated with anti-RBBP5, anti-KAT2B, or IgG control antibodies. ***P<0.001 vs IgG.

Fig.5 Knockdown of HClnc1 disrupts the protein-protein interaction between RBBP5 and KAT2B. A: Co-immunoprecipitation of RBBP5 and KAT2B in HepG2 cells. B, C: Interaction of RBBP5 and KAT2B after HClnc1 shRNA transfection (B) or RNase treatment (C).

Fig.6 HClnc1 regulates the transcription of ODC1 by connecting RBBP5 and KAT2B. A: ODC1 protein levels in HepG2 cells transfected with control shRNA or RBBP5/KAT2B shRNA detected using Western blotting (with GAPDH as the internal control). B: Detection of ODC1 DNA in anti-RBBP5 or anti-KAT2B antibody-immunoprecipitated chromatin of HepG2 cells. C: ODC1 gene promoter levels in chromatin immunoprecipitation samples detected using real-time PCR analysis. ***P<0.001 vs control shNC or control plasmid.

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