TMEM206基因敲除小鼠小脑组织中差异表达基因的筛选及生物学功能分析

Screening of differentially expressed genes in cerebellar tissues of TMEM206-knockout mice and analysis of their biological functions

目的筛选TMEM206基因敲除小鼠小脑组织中的差异表达基因并进行生物学功能分析,探讨TMEM206基因的功能。方法采用CRISPR/Cas9基因编辑技术制备TMEM206基因敲除小鼠,并通过配殖获得TMEM206基因敲除纯合小鼠。选取3只8周龄雌性TMEM206基因敲除纯合小鼠和3只同窝雌性野生小鼠,分离小脑组织并提取总RNA,转录组测序后,使用Cuffdiff(v2.2.1)软件的EBseq算法筛选差异表达基因,并使用DAVID数据库对差异表达基因进行基因属性(GO)和京都基因和基因组百科全书(KEGG)通路富集分析。结果共筛选出474个差异表达基因,与正常小脑组织相比,TMEM206基因敲除小鼠小脑组织中上调的基因265个、下调的基因209个。GO功能分析结果显示,差异表达基因的表达产物定位于细胞外膜和主要组织相容性复合体Ⅰ类蛋白(MHCⅠ)较多,功能属性主要归类于结合转录因子、脂类分子及抗原分子等;差异表达基因参与的生物学过程众多,如DNA损伤的细胞反应、细胞分化选择、骨质矿化等。KEGG通路富集分析结果显示,差异表达基因在分子属性上多为细胞黏附分子和肿瘤坏死因子路径的分子,功能上主要与细胞吞噬和衰老有关,此外还与人乳头瘤病毒、Ⅰ型人类T淋巴细胞白血病病毒、单纯疱疹病毒、Ⅷ型人疱疹病毒及EB病毒等感染有关。结论TMEM206基因敲除后,小鼠小脑组织中筛选得到474个差异表达基因,差异表达基因与维持细胞外膜结构稳定、细胞黏附、病毒感染有关。

Objective To screen the differentially expressed genes in the cerebellar tissues of TMEM206-knockout mice and to analyze their biological functions to explore the function of the TMEM206 gene.Methods TMEM206-knock-out mice were generated via the CRISPR-Cas9 system.The homozygous TMEM206-knockout mice were obtained by mat-ing.Total RNAs were extracted from cerebellum in three 30-week-old homozygous TMEM206-knockout female mice and three wild-type female littermates.Transcriptome sequencing was performed in cerebellum,the differentially expressed genes were screened using Cuffdiff(v2.2.1)software with EBSeq algorithm.GO functional enrichment analysis and KEGG pathway analysis were performed using the DAVID database.Results Totally 474 differentially expressed genes were screened out,including 265 up-regulated genes and 209 down-regulated genes in the cerebellar tissues of TMEM206-knockout mice.Gene ontology analysis showed that these differentially expressed genes were mainly focused on the exter-nal side of plasma membrane and major histocompatibility complex I;the molecular function involved in sequence-specific DNA binding transcription factor activity,lipid binding,and antigen binding.Differentially expressed genes were involved in many biological processes,such as cell response to DNA damage,cell differentiation selection,bone mineralization,etc.KEGG pathway enrichment analysis showed that the differential expression genes were mainly cell adhesion molecules and tumor necrosis factor pathway molecules in terms of molecular properties,and were mainly related to cytophagocytosis and senescence in function,and were related to the infection of human papillomavirus,human T-lymphocytic leukemia vi-rus type I,herpes simplex virus,human herpesvirus typeⅤⅢand EB virus.Conclusion After TMEM206 gene knock-out,474 differentially expressed genes are screened out in the mouse cerebellar tissues,and these genes are related to the stability of extracellular membrane structure,cell adhesion and virus infection.