Regulating of cell cycle progression by the lncRNA CDKN2B-AS1/miR-324-5p/ROCK1 axis in laryngeal squamous cell cancer.

OBJECTIVE:To evaluate the function of long non-coding RNA ANRIL (CDKN2B-AS1) in laryngeal squamous cell cancer (LSCC), and to explore the underlying mechanism. METHODS:The expression levels of CDKN2B-AS1 in LSCC tissues and cell lines (Tu177, HN4, AMC-HN-8 and NP69) were determined by reverse transcription quantitative PCR (RT-qPCR). AMC-HN-8 cells were then transfected with siRNAs of CDKN2B-AS1. The effects of CDKN2B-AS1 on cell proliferation, cell cycle, and apoptotic protein were determined by CCK-8 assay, flow cytometry analysis, and western blot, respectively. Dual luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to verify the targets of CDKN2B-AS1. The miR-324-5p mimics or miR-324-5p inhibitor and ROCK1 over-expression plasmids were also transfected into AMC-HN-8 cells for further analysis. RESULTS:CDKN2B-AS1 was upregulated in LSCC tissues, and the upregulation of CDKN2B-AS1 was correlated with overall survival, advanced clinical stage, and lymph node metastasis. In AMC-HN-8 cells, the knockdown of CDKN2B-AS1 by siRNA inhibited cell viability, blocked cell cycle in G1 phase, and increased the expression levels of cyclin-dependent kinase inhibitor 1A (p21), cleaved caspase3, and cleaved PPoly (ADP-Ribose) polymerase 1. Results of dual luciferase reporter assay showed that miR-324-5p could bind to CDKN2B-AS1 or Rho-associated coiled-coil containing protein kinase 1 (ROCK1). Finally, over-expression of ROCK1 in AMC-HN-8 cells revised the inhibitory effect of CDKN2B-AS1 siRNA on cell growth. DISCUSSION:The upregulation of CDKN2B-AS1 was correlated with overall survival, advanced clinical stage, and lymph node metastasis and promoted LSCC cell growth via miR-324-5p/ROCK1 axis.

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