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

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 gammaglutamyltransferase 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.