基于RNA-seq的脂代谢相关基因Tmem176a功能分析

Functional verification of candidate lipid meTableolism gene Tmem176a based on transcriptome sequencing

探讨在Hepa1-6细胞中过表达、敲低Tmem176a基因对脂质代谢途径的影响。将构建成功的Tmem176a慢病毒过表达载体、siRNA分别转染进Hepa1-6细胞中,将过表达或敲低Tmem176a后的Hepa1-6细胞进行RNA-seq、生物信息学分析、Real-Time PCR验证。与对照组相比,Tmem176a在Hepa1-6细胞中过表达182倍(P<0.05)和敲低0.2倍(P<0.05),OE-Tmem176a相较于OE-Vector差异表达基因共计424个,其中下调基因192个,上调基因共233个;KD-Tmem176a相较于KD-Scramble差异表达基因共计405个,其中下调基因248个,上调基因共157个,部分差异基因富集在脂肪的消化吸收、胆固醇代谢、甘油酯代谢、脂肪酸合成等代谢途径,Real-Time PCR验证部分差异表达的基因,差异基因表达水平的变化趋势与RNAseq一致。RNAseq结果提示Tmem176a基因会影响脂质代谢基因响应。Tmem176a表达水平改变会促进小鼠肝癌Hepa1-6细胞中脂质代谢基因表达水平发生显著变化。

The effects of overexpression and knockdown of Tmem176a on lipid metabolic pathways in Hepa1-6 cells were investigated.The constructed Tmem176a overexpression lentivirus vector and siRNA were transfected into Hepa1-6 cells.The overexpressed or knocked down with Tmem176a of Hepa1-6 cells were subjected to RNA-seq,bioinformatics analysis,and Real-Time PCR verification.Compared with the control group,Tmem176a was overexpressed 182 times(P<0.05)and knocked down 0.2 times(P<0.05)in He-pa1-6 cells.There were 424 differentially expressed genes(DEGs)in OE-Tmem176a compared with that in OE-Vector,of which 233 genes were upregulated and 192 genes were downregulated.There were 405 DEGs of KD-Tmem176a compared with that of KD-Scram-ble,of which 157 genes were upregulated and 248 genes were downregulated.Some of the DEGs were enriched in metabolic pathways such as fat digestion and absorption,cholesterol metabolism,glyceride metabolism,and fatty acid synthesis.The Real-Time PCR was used to investigate some of the DEGs,and the change trend of the expression level of some investigated DEGs was consistent with that of RNAseq.RNAseq results indicated that Tmem176a affected gene response to lipid metabolism,and the changed expression level of Tmem176a promoted significant changes of gene expression level of lipid metabolism in mouse hepatoma Hepa1-6 cells.