Translated Long Non-Coding Ribonucleic Acid ZFAS1 Promotes Cancer Cell Migration by Elevating Reactive Oxygen Species Production in Hepatocellular Carcinoma

Micropeptides (≤100 amino acids) are essential regulators of physiological and pathological processes, which can be encoded by small open reading frames (smORFs) derived from long non-coding RNAs (lncRNAs). Recently, lncRNA-encoded micropeptides have been shown to have essential roles in tumorigenesis. Since translated smORF identification remains technically challenging, little is known of their pathological functions in cancer. Therefore, we created classifiers to identify translated smORFs derived from lncRNAs based on ribosome-protected fragment sequencing and machine learning methods. In total, 537 putative translated smORFs were identified and the coding potential of five smORFs was experimentally validated via green fluorescent protein-tagged protein generation and mass spectrometry. After analyzing 11 lncRNA expression profiles of seven cancer types, we identified one validated translated lncRNA, ZFAS1, which was significantly up-regulated in hepatocellular carcinoma (HCC). Functional studies revealed that ZFAS1 can promote cancer cell migration by elevating intracellular reactive oxygen species production by inhibiting nicotinamide adenine dinucleotide dehydrogenase expression, indicating that translated ZFAS1 may be an essential oncogene in the progression of HCC. In this study, we systematically identified translated smORFs derived from lncRNAs and explored their potential pathological functions in cancer to improve our comprehensive understanding of the building blocks of living systems Transcriptomeanalyses of ZFAS1 overexpression and si in SK-Hep1 human hepatoma cell lines by deep sequencing. In total transcriptome data set, include two PCDH control, two ZFAS ORF overexpression, two si GFP, and two si ZFAS1

微肽（Micropeptides，由≤100个氨基酸构成）是生理与病理过程的关键调控因子，可由源自长链非编码RNA（long non-coding RNAs, lncRNAs）的小开放阅读框（small open reading frames, smORFs）编码。近年来研究证实，由lncRNA编码的微肽在肿瘤发生进程中发挥核心调控作用。由于翻译型smORFs的鉴定仍存在技术难题，目前学界对其在癌症中的病理功能尚所知有限。为此，本研究基于核糖体保护片段测序（ribosome-protected fragment sequencing）与机器学习方法，构建了可鉴定lncRNA来源翻译型smORFs的分类模型。本研究共鉴定出537个推定的翻译型smORFs，并通过绿色荧光蛋白标记蛋白表达实验与质谱检测对其中5个smORFs的编码潜能完成了实验验证。在分析7种癌症类型的11套lncRNA表达谱后，我们鉴定出1个已验证的翻译型lncRNA ZFAS1，该分子在肝细胞癌（hepatocellular carcinoma, HCC）中显著上调。功能实验显示，ZFAS1可通过抑制烟酰胺腺嘌呤二核苷酸脱氢酶（nicotinamide adenine dinucleotide dehydrogenase）的表达，提升细胞内活性氧生成水平，进而促进癌细胞迁移，提示翻译型ZFAS1可能是肝细胞癌进展过程中的关键癌基因。本研究系统性鉴定了lncRNA来源的翻译型smORFs，并探索了其在癌症中潜在的病理功能，以期增进学界对生命系统基本组成单元的全面认知。此外，本研究通过深度测序对过表达ZFAS1及敲低ZFAS1的人肝癌SK-Hep1细胞系开展转录组分析，总转录组数据集包含2个PCDH对照样本、2个ZFAS开放阅读框过表达样本、2个si GFP样本与2个si ZFAS1样本。