旋毛虫抗原诱导的MCF-7乳腺癌细胞差异表达基因分析

Analysis of genes differentially expressed by MCF-7 cells challenged with Trichinella spiralis larval antigen

目的分析旋毛虫抗原刺激MCF-7乳腺癌细胞前后的差异表达基因,为乳腺癌的治疗提供新的靶点及思路。方法利用Illumina HiSeq对旋毛虫幼虫抗原刺激前、后的MCF-7细胞进行转录组测序,通过cutadapt对原始数据进行过滤,用RPKM(Reads Per Kilo bases per Million reads)方法计算基因表达量,以差异基因log2fold change≥1且FDR≤0.05筛选差异表达基因(differentially expressed genes,DEGs),通过Gene Ontology(GO)数据库、KEGG pathway数据库对差异表达基因的功能和参与的信号通路进行分析,利用qRT-PCR对差异表达基因进行验证。 结果转录组测序数据过滤后分别得到51 617 374和44 494 010条有效序列,从中筛选出21个差异表达基因,与对照MCF-7细胞(CON组)相比,旋毛虫抗原刺激的MCF-7细胞(AG组)有13个上调基因,8个下调基因。差异表达基因主要富集于分子结合、催化活性、细胞组分、胞膜组分、生物调节、细胞过程等,主要参与TGF-β、Jak-STAT、ErbB、Hippo、mTOR信号通路调节及碳代谢、氨基酸生物合成、丙氨酸-天冬氨酸-谷氨酸代谢、视黄醇代谢、甘氨酸-丝氨酸-苏氨酸代谢及血管平滑肌收缩等。利用qRT-PCR对显著下调基因受体活性修饰蛋白3(Receptor activity modifying protein 3,RAMP3)的表达进行验证,结果与转录组测序一致。 结论旋毛虫抗原刺激导致MCF-7乳腺癌细胞RAMP3表达降低,RAMP3可能参与旋毛虫抗原抗肿瘤过程。

Objective To examine the genes differentially expressed by MCF-7breast cancer cells with or without stimulation fromTrichinella spiralis larval antigen in an attempt to provide new targets for breast cancer therapy. Methods Illumina HiSeq was performed to obtain the transcriptome of MCF-7cells with or without treatment of T.spiralis larval antigen.The raw data were filtered using cutadapt,and the abundance of gene expression was calculated using the reads per kilobase of transcript per million mapped reads(RPKM).Differentially expressed genes(DEGs)were identified based on the standard of a log2-fold change≥1and FDR≤0.05.Gene function and pathway analysis were performed by comparing DEGs to the Gene Ontology(GO)and KEGG pathway databases,and DEGs were verified using qRT-PCR. Results A total of 51 617 374and 44 494 010genes were obtained after cutadapt filtering,and screening yielded 21DEGs for analysis.Of the 21DEGs,13genes were upregulated and 8genes were downregulated in MCF-7 cells treated with the T.spiralis larval antigen(Ag group)compared to controls treated with PBS(Con group).The DEGs mainly had enriched binding,catalytic activity,cell components,membrane components,biological regulation,and cellular processes,and DEGs were involved in the TGF-β,Jak-STAT,ErbB,Hippo,and mTOR signaling pathways and the processes of carbon metabolism,biosynthesis of amino acids,alanine,aspartate,and glutamate metabolism,retinol metabolism,glycine,serine,and threonine metabolism,and vascular smooth muscle contraction.The level of receptor activity modifying protein 3(RAMP3)mRNA was evaluated using qRT-PCR and was consistent with the data from transcriptome sequencing. Conclusion The expression of RAMP3is downregulated after treatment with T.spiralis larval antigen,which indicates that RAMP3may be involved in the antitumor action of T.spiralis larval antigen.