RIPK3基因转染的 SH-SY5Y 细胞中 HIF-1α基因及其信号通路相关基因表达变化

摘要

Objective To observe the expression changes of hypoxia-inducible factor 1α( HIF-1α) and its signaling pathway related gene in the neuroblastoma cell line SH-SY5Y transfected by receptor interacting serine/threonine kinase 3 (RIPK3) gene.Methods The pCMV6-AC-GFP plasmid expressing RIPK3 gene (recombinant plasmid) was construc-ted.SH-SY5Y cells were cultured and then were transfected with the recombinant plasmid and empty vector plasmid as the experimental group and control group respectively.Expression of RIPK3 protein was detected by Western blotting, and the OD value was detected by MTT assay at 8, 14, 20, 26, 32 and 38 h.RNA transcriptome sequencing (RNAseq) and Inge-nuity Pathway Analysis ( IPA) was used to detect and screen the key genes in the downstream signaling pathway of RIPK3-HIF-1α.Droplet Digital PCR ( ddPCR) was used to detect the HIF-1αmRNA in the two groups.Results The RIPK3 ex-pression in the experimental group (0.806 ±0.097 5) was higher than that of the control group (0.455 ±0.088 6), P<0.05.The proliferation of SH-SY5Y was inhibited as the cell incubation time was prolonged.The expression of HIF-1αmRNA in the experimental group (0.01543 ±0.00347) was strongly down-regulated as compared with that of the control group (0.04628 ±0.01026) ( P<0.05) .In the interaction network of HIF-1αwhich was taken as the core, we screened the key molecules such as ubiquitin-conjugating enzyme ( UBC) , von Hippel-Lindau ( VHL) , transcription elongation fac-tor B polypeptide 1 (TCEB1) and vascular endothelial growth factor A (VEGFA).Conclusion After the SY5Y cells were trasfected by RIPK3, the HIF-1αmRNA expression was down-regulated in SH, and the expression levels of HIF-1αsignaling pathway-related genes ( UBC, VHL, TCEB1 and VEGFA) were affected.%目的：观察受体相互作用蛋白激酶3（RIPK3）基因转染的神经母细胞瘤细胞系SH-SY5Y中低氧诱导因子1α（ HIF-1α） mRNA及其信号通路相关基因表达变化。方法构建表达RIPK3基因的pCMV6-AC-GFP质粒（重组质粒），培养SH-SY5Y细胞，分为实验组及对照组，分别转染重组质粒和空载质粒。采用Western blotting法检测细胞中的RIPK3蛋白，分别于培养8、14、20、26、32、38 h后，通过MTT实验检测细胞增殖情况（ OD值）。采用转录组测序技术（ RNAseq）及Ingenuity Pathway Analysis（ IPA）软件检测并筛选RIPK3-HIF1α下游信号通路中的关键基因。采用微滴式数字PCR（ ddPCR）检测两组细胞中的HIF-1αmRNA。结果实验组细胞中RIPK3蛋白相对表达量（0．806±0．0975）高于对照组（0．455±0．0886），P＜0．05。随培养时间延长，实验组细胞增殖受到抑制。实验组细胞中HIF-1αmRNA相对表达量（0．01543±0．00347）低于对照组（0．04628±0．01026），P＜0．05。在HIF-1α为核心的相互作用关系网络中，筛选出关键分子泛素缀合酶样蛋白（ UBC）、希佩尔－林道蛋白（ VHL）、转录延伸因子B多肽1（ TCEB1）、血管内皮生长因子A（ VEGFA）。结论 RIPK3基因转染SH-SY5Y后，细胞中HIF-1αmRNA表达下调，同时HIF-1α信号通路相关基因（ UBC、VHL、TCEB1、VEGFA）的表达水平受到影响。