Evaluation The Role Of MiR-486-5p In Non-small Cell Lung Cancer Cells Through Proteomic Approach

Objective: Our previous work found that miR-486-5p was one of the most significantly down-regulated miRNAs in lung cancer.It was a useful marker for early diagnosis of lung cancer both in sputum and peripheral blood.Furthermore,miR-486-5p was involved in lung cancer growth,invasion and metastasis by targeting ARHGAP5 and Pim-1 expression.Considering the complexity of miRNA regulation on its targeted genes,we determined to further screen the targeted proteins by miR-486-5p through an iTRAQ-based quantitative proteomic approach.Our results may provide more data on the mechanism of miR-486-5p in NSCLC.Methods: H1299 and H157 cells were cultured in vitro and transfected with miR-486 plasmid or empty vector,respectively.The level of mi R-486-5p was confirmed by Taqman micro RNA assay in obtained stable cell lines.iTRAQ-based quantitative proteomic approach was used to detect the dysregulated proteins in H1299 and H157 cells further.SPSS Statistics 17.0 software was used to analyze the statistical significance.Data are presented as mean ± SD.Statistical comparisons between experimental groups were analyzed by t-test.P<0.05 was taken to indicate statistical significance.Results:1.Proteomics analysis the protein targets for miR-486-5p in H1299 cells using iTRAQ approachCompared with the control groups,25 proteins were indentified to be upregulated,and 27 proteins to be down-regulated in H1299-miR-486-5p cells.Further analysis showed that the up-regulated proteins were mainly associated with the aminoacyl-tRNA biosynthesis process,while the down-regulated proteins were mainly related to the ribosome pathway.The interrelationship between dysregulated proteins mainly included SLIT1(down-regulation)-POTEJ(up-regulation)-PDP2(up-regulation);AP4S1(up-regulation)-RAB6B(down-regulation)-TB22B(up-regulation);DD19B(up-regulation)-XPO6(down-regulation).2.Proteomics analysis the protein targets of miR-486-5p in H157 cells using iTRAQ approachFollowing miR-486-5p overexpression in H157 cells,70 proteins showed a significant increase in expression,and 135 proteins showed a significant decrease in expression,respectively.The relevant signaling pathways of the up-regulation proteins involvement included ribosome biogenesis,spliceosome,as well as RNA transport.The down-regulated proteins were mainly involved in protein processing in endoplasmic reticulum pathway.Furthermore,interactions between dysregulated proteins were more complicated in H157 cells than that in H1299 cells.For examples,SLIT1(down-regulation)-EPHA2(down-regulation)-RHOA(down-regulation)-DOCK1(up-regulation);TYY1(down-regulation)-BIRC5(up-regulation)-CTCF(down-regulation)-GRB10(up-regulation)and so on.3.Selection the common co-regulated proteins of miR-486-5p in H1299 and H157 cellsAfter getting the intersection of the dysregulated protein lists from H1299-miR-486-5p cells and H157-miR-486-5p cells,it was found that SLIT1 was the only one protein which was down-regulated in both of cells.Conclusions:1.Stable overexpression miR-486-5p cells were generated both in H1299 and H157 cells.2.Twenty-five up-regulated proteins in H1299-miR-486-5p cells were mainly associated with aminoacyl-tRNA biosynthetic pathways,while the twenty-seven down-regulated proteins were mainly associated with ribosome pathways.3.The related signal pathways in the seventy up-regulated proteins of H157-miR-486-5p cells were involved in the pathway of spliceosome,ribosome biogenesis,RNA transport,etc.while the 137 down-regulated proteins was most significantly involved in the pathway of protein processing in endoplasmic reticulum.4.SLIT1 gene was down-regulated both in H1299 and H157 cells overexpressing miR-486-5p,which suggested that SLIT1 gene might be one of the target genes regulated by miR-486-5p.