RNA sequencing analysis of the CAL-27 cell response to over-expressed ZNF750 gene. RNA sequencing analysis of the CAL-27 cell response to over-expressed ZNF750 gene

Background: Zinc-finger protein 750 (ZNF750) is a potential tumor suppressor in oral squamous cell carcinoma (OSCC). However, the molecular mechanisms underlying its anti-tumor effect remain elusive in OSCC. We report the application of RNA sequencing to identify differentially expressed genes (DEGs) between vector groups and ZNF750 groups (over-expressed ZNF750 in CAL-27 cell), and to elucidate the genes and pathways involved in tumor suppression following the ZNF750 over-expression in OSCC cell line CAL-27 cell. Methods: The RNA sequence libraries were constructed, and the data were analyzed to identify DEGs between vector groups and ZNF750 groups. QPCR and western-blot was used to validate differential expression of candidate genes with cell cycle regulation. The cell cycle distribution was analyzed by BrdU staining. Results: By RNA sequencing profiling, 7,131 genes were differentially expressed in ZNF750 groups. Among the DEGs, 3,285 genes were upregulated, 3,846 genes were downregulated and 4,507 genes were identified in three main categories (cellular_component, biological process and molecular function) based on the gene ontology (GO) classification. The Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis defined the DEGs could be categorized into 280 pathways and identified the top two most significant pathways involved in spliceosome and cell cycle. Functional categorization and enrichment analysis revealed that most of DEGs involved in binding and catalytic activity, and the cell cycle associated genes was significantly enriched in response to ZNF750 over-expression. ZNF750 induced cell cycle arrest in G0/G1 phase of the cell cycle. Conclusion: Data from this study revealed that the cell cycle pathway was a key factor involved in the anti-tumor effect of ZNF750 in CAL-27 cells. Overall design: RNA sequence profiles of CAL-27 cell transduced with or without ZNF750 lentivirus were generated by deep sequencing, in duplicate, using Illumina Hiseq 2500 platform.

背景：锌指蛋白750（Zinc-finger protein 750, ZNF750）是口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）中潜在的抑癌因子。然而其在OSCC中发挥抗肿瘤作用的分子机制尚未明确。本研究利用RNA测序技术，鉴定转染空载体组与ZNF750过表达组（在CAL-27细胞中过表达ZNF750）之间的差异表达基因（differentially expressed genes, DEGs），并阐明OSCC细胞系CAL-27中ZNF750过表达后参与肿瘤抑制过程的基因与通路。

方法：构建RNA测序文库，对数据进行分析以鉴定空载体组与ZNF750过表达组之间的DEGs。采用实时荧光定量PCR（qPCR）与蛋白质印迹法（western-blot）验证与细胞周期调控相关的候选基因的差异表达。通过BrdU染色法分析细胞周期分布。

结果：通过RNA测序分析，ZNF750过表达组中共鉴定出7131个差异表达基因。在这些DEGs中，3285个基因呈上调表达，3846个基因呈下调表达；基于基因本体（gene ontology, GO）分类，共将4507个基因归入细胞组分（cellular_component）、生物过程（biological_process）和分子功能（molecular_function）三大功能类别。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分析显示，DEGs可被划分为280条通路，其中富集程度最高的两条通路为剪接体通路与细胞周期通路。功能分类与富集分析结果表明，大部分DEGs参与结合与催化活性过程，且与细胞周期相关的基因在ZNF750过表达后显著富集。ZNF750可诱导细胞周期阻滞于G0/G1期。

结论：本研究数据表明，细胞周期通路是ZNF750在CAL-27细胞中发挥抗肿瘤作用的关键调控通路。

整体实验设计：利用Illumina HiSeq 2500平台，对转染ZNF750慢病毒与空载慢病毒的CAL-27细胞进行深度测序，设置两次生物学重复，以获取RNA测序表达谱。