RANBP9调控结直肠癌细胞凋亡的信号通路分析  

Signaling Pathway Analysis of RANBP9 in Regulating Apoptosis of Colorectal Cancer Cells

作　　者：秦春枝[1] 武广彬[1] 李吉[1] QIN Chunzhi;WU Guangbin;LI Ji(Jinshan Hospital of Fudan University,Shanghai 201508,China)

机构地区：[1]复旦大学附属金山医院,上海201508

出　　处：《中国细胞生物学学报》2020年第7期1177-1186,共10页Chinese Journal of Cell Biology

基　　金：上海市金山区卫生健康委基金(批准号:JSKJ-KTMS-2017-01)资助的课题。

摘　　要：RANBP9是RAS超家族成员RAN的一种结合蛋白,参与多种肿瘤的发生发展。为探索它对结直肠癌细胞凋亡的调控作用,该研究利用慢病毒感染结直肠癌HCT116、HT29细胞的方法,建立RANBP9-shRNA稳转细胞系,经氟尿嘧啶诱导凋亡后利用流式细胞仪和caspase-2酶活力实验检测细胞凋亡;抽提实验组和对照组HCT116细胞总RNA,经质检合格后进行基因表达谱芯片实验,筛选出RANBP9敲减前后结直肠癌细胞的差异表达基因并进行定量PCR验证,基于Gene Ontology数据库进行分子功能注释,基于Ingenuity Pathway Analysis数据库进行通路分析。流式细胞分析显示,在HCT116和HT29细胞中RANBP9-shRNA均促进氟尿嘧啶诱导的细胞凋亡;基因芯片数据分析得到差异表达mRNAs 857个(|Fold Change|>1.5且FDR<0.05),其中上调表达677个,下调表达180个,涉及的分子功能主要包括γ-谷氨酰转移酶活性、钙离子结合、胰岛素受体结合、病毒受体活性、GTP酶活性、细胞外基质结合、β-连环蛋白结合、SMAD结合、转录调节、AMP活化蛋白激酶活性、蛋白质转运蛋白活性、细胞骨架结合等,其显著参与的信号通路主要涉及癌症的TGF-β、BMP、IL-8和RhoA等。实时定量PCR证实上述通路中的SMAD3、SMAD7、BMP6、BMP7、CXCL8、RAPGEF6的mRNA表达水平在RANBP9-shRNA组中显著高于对照组。综上,RANBP9可能通过多个信号通路来调控结直肠癌细胞的凋亡,该研究为阐明其中的分子机制提供了新的思路。RANBP9 is a binding protein of RAN,a member of RAS superfamily.It is involved in the development of various tumors.To explore its effect on the apoptosis of colorectal cancer cells,RANBP9-shRNA and control cell lines were established by infection with lentivirus in HCT116 and HT29 cells.Flow cytometry and caspase-2 activity assay were used to detect the apoptotic cells treated with fluorouracil.Total RNAs of RANBP9-shRNA and Control cells were extracted.Microarray hybridization was performed after quality evaluation.Differentially expressed genes before and after knockdown of RANBP9 were screened and some of them were verified by real-time quantitative PCR.Molecular function annotation of the differentially expressed genes was performed based on Gene Ontology database,and pathway analysis was performed based on Ingenuity Pathway Analysis database.In HCT116 and HT29 cells,RANBP9-shRNA promotes apoptosis induced by fluorouracil.Eight hundred and fifty-seven differentially expressed genes(|Fold Change|>1.5 and FDR<0.05)were obtained by microarray analysis,of which 677 were up-regulated and 180 were down-regulated.The molecular functions mainly included gamma-glutamyltransferase activity,calcium binding,insulin receptor binding,viral receptor activity,GTPase activity,extracellular matrix binding,β-catenin binding,SMAD binding,transcriptional regulation,AMP-activated protein kinase activity,protein transporter activity,cytoskeleton binding and so on.The signaling pathways involved in cancer included TGF-β,BMP,IL-8,RhoA and so on.Real-time quantitative PCR confirmed that the mRNA levels of SMAD3,SMAD7,BMP6,BMP7,CXCL8 and RAPGEF6 in the above pathways were significantly higher in RANBP9-shRNA group than in Control group.Altogether,RANBP9 may regulate the apoptosis of colorectal cancer cells through multiple signaling pathways.This study provides new sights to elucidate the molecular mechanism of RANBP9 in CRC.

关 键 词：RANBP9 凋亡 基因芯片 结直肠癌 

分 类 号：R735.37[医药卫生—肿瘤]