基于IPA分析SF3B4作为胃癌潜在生物标志物的分子机制

Molecular mechanism of SF3B4 as a potential biomarker of gastric cancer based on IPA analysis

目的基于IPA(Ingenuity Pathway Analysis)进行生物信息学分析,探讨剪接因子3b亚基4(SF3B4)作为胃癌潜在生物标志物对人胃癌细胞的分子调控机制。方法采用免疫组化法分析SF3B4蛋白在人胃癌组织和癌旁正常组织中的表达情况,荧光定量PCR(RT-qPCR)法检测SF3B4 mRNA在人胃癌细胞系AGS、HGC-27、KATOⅢ、NCI-N87和MKN-74的表达水平。采用短发夹RNA(shRNA)技术沉默SF3B4基因,RNA测序(RNA-seq)检测SF3B4沉默后AGS细胞基因表达谱变化,输入IPA系统进行经典通路、疾病与功能、互作网络等分析,并筛选出关键基因进行RT-qPCR验证。结果SF3B4蛋白在胃癌组织中的表达显著高于癌旁正常组织(P<0.05),SF3B4 mRNA在人胃癌细胞系AGS、HGC-27、KATOⅢ和MKN-74中呈高表达,在NCI-N87中表达水平一般。RNA-seq显示,与对照组比较,SF3B4沉默组中105个基因为差异表达基因,其中上调基因64个,下调基因41个。IPA经典通路分析显示,SF3B4沉默后,坏死性凋亡信号通路、衰老途径显著抑制。疾病功能和互作网络分析显示,SF3B4在胃癌中发挥重要作用,主要影响自噬、细胞周期等功能。RT-qPCR验证发现,DNAJA3、PPP3CB、VDAC1、FOXO3、EI24和DRAM2基因在SF3B4沉默后表达下调。结论SF3B4在胃癌组织和胃癌细胞系中高表达,可能是潜在的胃癌生物标志物,可通过调控凋亡信号通路、衰老途径、自噬等通路参与胃癌的发生发展,并可能与DNAJA3、PPP3CB、VDAC1、FOXO3、EI24和DRAM2等基因相关。

Objective To explore the molecular mechanism of splicing factor 3b subunit 4(SF3B4)as a potential biomarker of human gastric cancer cells by bioinformatics analysis based on the Ingenuity Pathway Analysis(IPA).Methods SF3B4 protein expression in human gastric cancer tissues and adjacent normal tissues was analyzed by immunohistochemical method.The expression levels of SF3B4 mRNA in human gastric cancer cell lines AGS,HGC-27,KATOⅢ,NCI-N87 and MKN-74 were detected by RT-qPCR.SF3B4 gene was silenced by shRNA technique,and the changes in gene expression profile of AGS cells were detected by RNA sequencing(RNA-seq)after SF3B4 silencing.The classical pathway,disease and function,and interaction network were analyzed by IPA system.The expression levels of selected differential RNAs were verified by RT-qPCR.Results The expression of SF3B4 protein in gastric cancer tissues was significantly higher than that in adjacent normal tissues(P<0.05).SF3B4 mRNA was highly expressed in four gastric cancer cell lines(AGS,HGC-27,KATOⅢ,and MKN-74)and was moderately expressed in NCI-N87 cells.RNA-seq showed that the expression of 105 genes in SF3B4 silenced group was significantly different from that in the control group,including 64 up-regulated genes and 41 down-regulated genes.IPA classical pathway analysis showed that the necrotic apoptosis signaling pathway and senescence pathway were significantly inhibited after SF3B4 silencing.The analysis of disease function and interaction network showed that SF3B4 played an important role in gastric cancer,mainly affecting autophagy,cell cycle and other functions.RT-qPCR results further confirmed that DNAJA3,PPP3CB,VDAC1,FOXO3,EI24 and DRAM2 genes were down-regulated after SF3B4 silencing.Conclusion SF3B4 is highly expressed in gastric cancer tissues and gastric cancer cell lines,which may be a potential biomarker of gastric cancer.SF3B4 may be involved in the occurrence and development of gastric cancer by regulating apoptosis signaling pathway,aging pathway,autophagy and other pathways,and related to genes such as DNAJA3,PPP3CB,VDAC1,FOXO3,EI24 and DRAM2.