System biological and experimental validation of miRNAs target genes involved in colorectal cancer radiation response

Abstract

Background and objectivesPrediction of response to radiotherapy in colorectal cancer (CRC) patients is of great interest that protects radiation-resistant patients from imposing to high doses of X radiation. miRNAs and their target genes are potential candidates as biomarkers of cancer. Microarray provides a high-throughput analysis of cell transcriptome; however, this very complex data needs to be interpreted via network-based approaches. In this study, miRNAs target genes involved in the CRC radiation response were validated through a system biological and experimental manner. Material and methodsThe microarray expression data was obtained from NCBI GEO datasets. The miRNAs target genes were identified using miRNA-mRNA interaction prediction databases. Gene enrichment analysis performed using DAVID database to identify genes involved in the tumor radiation response. For selected miRNAs and targets, the miRNA-regulated protein–protein interactions network (PPIN) was constructed and analyzed using Cytoscape software. Survival analysis was done by Xena Browse. Radioresistant cell lines were established using fractional X irradiations and confirmed by clonogenic assay. After RNA isolation and cDNA synthesis, by Real-Time PCR, gene expression measurements were performed. SPSS V.22 was used for data analysis. ResultsFinally, the selected genes and miRNAs based on network analysis were comprises: mTOR, MAPK1, CDC42, EGFR, MAPK8, CCND1, BCL2, PIK3CG, hsa-miR-7-2, hsa-miR-17, hsa-miR-106a and hsa-miR-32. Among them, MAPK8 and CDC42 genes were significantly correlated with CRC patient's survival. Established radioresistant cell lines showed a morphological change and a significantly higher survival fraction compared to parental cell line. Real-Time PCR showed higher expression of MAPK8, CDC42 and CCND1 in radioresistant cell lines compared to parental cell line. ConclusionIn this study, via several enrichment stages, 2,069,706 target genes were highly enriched to 3 genes which have crucial roles in tumor radiation response and can be used as potential predictive biomarkers.