基于RNA-seq的HBSS诱导细胞自噬的组学分析及实验验证

Omics analysis of HBSS-induced autophagy based on RNA-seq and experimental verification

为寻找Hank’s平衡盐溶液(HBSS)诱导细胞自噬的调控蛋白,对HBSS诱导大鼠肾上皮(NRK)细胞株的细胞自噬现象进行转录组分析和实验验证.用HBSS分别处理细胞不同时长,结合LC3B-Ⅱ蛋白表达量和细胞存活率确定最佳诱导时间.根据最佳诱导时间制作细胞样品,进行转录组测序(RNA-seq),原始数据质控过滤后进行注释和分析.选取表达差异变化排序靠前的基因进行敲降实验,筛选具有潜在调控功能的蛋白质,接着用荧光定量PCR实验对该蛋白质的上游基因表达情况进行辅助验证.结果发现,HBSS诱导细胞自噬的最佳诱导时间为3 h;在转录组结果中共鉴定到32305个基因,477个差异表达基因,差异基因的GO和KEGG富集分析结果显示这些基因主要参与代谢底物分解、氨基酸转运以及蛋白质修饰调控等信号调控通路;免疫印迹结果表明干扰基因Sat1的表达可以降低细胞内LC3B-Ⅱ的表达水平.本研究围绕HBSS诱导细胞自噬进行转录组学测序分析,并通过实验发现了影响LC3B-Ⅱ表达的潜在功能调控基因Sat1.综合考虑所有实验结果,Sat1可能通过GO和KEGG显著富集的信号通路影响HBSS诱导的细胞自噬,这些发现可以为细胞自噬相关机制的深入挖掘提供参考.

In order to find the regulatory proteins of the autogophy induced by Hank's balanced salt solution(HBSS),the normal rat kidney(NRK)cell lines were induced autophagy by HBSS,the transcriptome analysis and experimental verification were performed.The cells were treated with HBSS for different time,and the optimal induction time was determined by combining the protein expression levels of LC3B-Ⅱand cell survival rates.The transcriptome samples were prepared under the optimal induction conditions.After RNA Sequencing(RNA-Seq),the raw data was analyzed after quality examination.The knockdown experiment was performed on the genes with the highest ranking of differential expressed changes.The proteins with potential regulatory functions were screened out.At the same time,the upstream gene expression of the protein was verified by fluorescence quantitative PCR.The results found that the best induction time of HBSS induced autophagy was 3 h.A total of 32305 genes were identified in the transcriptome results,and 477 differentially expressed genes were found.GO and KEGG enrichment analysis of differentially expressed genes showed that these genes were mainly involved in signal regulation pathways such as metabolic substrate decomposition,amino acid transport,and the regulation of protein modification.Western blotting results showed that interference with the expression of Sat1 could reduce the protein level of LC3B-Ⅱ.In this study,the omics sequencing is used on HBSS-induced autophagy.A potential functional regulatory gene Sat1 is found,which affects the expression of LC3B-Ⅱ.Taking all the experimental results into consideration,Sat1 may affect the HBSS-induced autophagy through GO and KEGG significantly enriched signaling pathways.These findings can provide reference for further exploration on the mechanisms of autophagy.