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.

中文翻译：

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通过 lncRNA CDKN2B-AS1/miR-324-5p/ROCK1 轴调节喉鳞状细胞癌中的细胞周期进程。

目的评价长链非编码RNA ANRIL（CDKN2B-AS1）在喉鳞状细胞癌（LSCC）中的作用，并探讨其作用机制。方法采用逆转录定量PCR（RT-qPCR）检测CDKN2B-AS1在LSCC组织和细胞系（Tu177、HN4、AMC-HN-8和NP69）中的表达水平。然后用 CDKN2B-AS1 的 siRNA 转染 AMC-HN-8 细胞。CDKN2B-AS1对细胞增殖、细胞周期和凋亡蛋白的影响分别通过CCK-8测定、流式细胞术分析和蛋白质印迹测定。采用双荧光素酶报告基因测定和 RNA 免疫沉淀 (RIP) 测定来验证 CDKN2B-AS1 的目标。还将 miR-324-5p 模拟物或 miR-324-5p 抑制剂和 ROCK1 过表达质粒转染到 AMC-HN-8 细胞中以进行进一步分析。结果CDKN2B-AS1在LSCC组织中表达上调，CDKN2B-AS1的上调与总生存期、晚期临床分期和淋巴结转移相关。在 AMC-HN-8 细胞中，siRNA 敲低 CDKN2B-AS1 可抑制细胞活力，阻断 G1 期细胞周期，并增加细胞周期蛋白依赖性激酶抑制剂 1A (p21)、切割的 caspase3 和切割的 PPoly 的表达水平。 ADP-核糖) 聚合酶 1. 双荧光素酶报告基因检测结果表明，miR-324-5p 可以与 CDKN2B-AS1 或含有蛋白激酶 1 (ROCK1) 的 Rho 相关卷曲螺旋结合。最后，ROCK1 在 AMC-HN-8 细胞中的过表达修正了 CDKN2B-AS1 siRNA 对细胞生长的抑制作用。讨论 CDKN2B-AS1 的上调与总生存期、晚期临床分期、