Molecular Mechanism Of Proliferation And Invasion Mediated By MiR-200c And EP300 Target Genes In Nephroblastoma

This research aims to reveal a new regulating mechanism of miR-200c,approaching the role of miR-200c/EP300/P27 in the development of nephroblastoma,and provide a new miRNA and target gene for clinical therapy of nephroblastom.Iin this study,we screened differently expressed miRNAs in nephroblastoma tissues and cells,and determined miR-200c,which was associated with proliferation and invasion of neplhroblastoma as our research candidated by means of miRNA microarray.We demonstrat ed the role of miR-200c in proliferation and invasion of nephroblastoma.The main contens of this research were as the following:(1)Screening and identifying miRNA associated with proliferation and invasion of nephroblastoma.(2)Predicting and identifying target genes of miR-200c,and demonstrating their roles in proliferatioand invasion of nephroblastoma.(3)To identify the signal pathway of EP300 target gene of miR-200c,and to explore the role of PI3K/AKT/FOXO pathway in proliferation and invasion of nephroblastoma.Methods1.Screening and identifying differently expressed miRNAs in tissues and cells of nephroblastoma.2.Prediction and identification of miR-200c targets.3.Effects of miR-200c on biological behaviors of nephroblastoma.4.The relational nephroblastoma signal pathway of miR-200c was detected.Results1.Screening and identifying of miRNA associated with proliferation and invasion of nephronblastoma.(1)Six miRNA microarray results showed that 27 miRNAs were downregulated in primary nephroblastoma tissues in comparison witlh normal renal tissues,including miR-200c,which was significantly downregulated in nephroblastoma tissues.(2)Detecting the expression of miR-200c in 24 matched fresh nephroblastoma tissues and two cell lines by real-time PCR.The statistical analysis showed that the expression of miR-200c in nephroblastoma tissues were significantly lower than normal renal tissues.Similarly,the expression of miR-200c in SK-NEP-1 and G401 cell were lower than that of 293T cell line.2.Predicting and identifying target genes of miR-200c(1)Results showed that EP300(predicted by four common databases)was associated with proliferation and invasion.So we chosed EP300 as miR-200c target gene.(2)The double luciferase reporter system showed that the luciferase activity was decreased significantly respectively in wt EP300 group compared to mt EP300 group.The above results indicated that miR-200c inhibited the activity of luciferase throuth the binding sites of EP300 3'UTR region.(3)The results of RT-PCR and Western blot showed that the expression of miR-200c and target gene EP300 was negatively correlated.3.Effects of miR-200c on biological behaviors of nephroblastoma.(1)MiR-200c lentivirus-expressing vector was successfully designed and established.After viral packaging,it was transfected into SK-NEP-1 and G401 cells lines.The stable miRNA-expressing nephroblastoma cell lines were detected the expression of miR-200c by real-time PCR.(2)The CCK-8 cell proliferation assays,soft agar assays,transwell migration and invasion assays,apoptosis and cell cycle by flow cytometry were carried out to detect SK-NEP-1 and G401 cells in cell proliferation,plate clone formation,migration and invasion in vitro,apoptosis and cell cycle after transfection of miR-200c and miR-200c inhibitor.The results showed that miR-200c inhibited the biological functions of SK-NEP-1 and G401 cells.(3)Xenograft tumors were generated by subcutaneous injection to assess the effect of miR-200c and miR-200c inhibitor on tumors growth in vivo.A orthotopic transplantation assay was employed to evaluate the effect of miR-200c and miR-200c inhibitor on the proliferation of cell lines.The above results indicated that miR-200c could partially inhibited tumorigenicity and proliferation.4.Predicting and confirming the transcription factor of EP300,and demonstrating the signaling pathway in nephroblastoma.(1)Chromatin immunoprecipitation test(CHIP)was adopted to detect the binding sites between target gene EP300 and transcription factor P27,and results showed that there was a direct binding site between EP300 and P27.(2)Western blot detects the upstream and downstream signaling pathways of EP300.Conclusions:1.Downregulation of miR-200c was associated with proliferation and invasion of nephroblastoma.2.miR-200c/EP300/P27 axis inhibits proliferation and invasion of nephroblastoma.3.miR-200c is a PI3K/AKT/FOXO signalling pathway regulator in nephroblastoma.The PI3K/AKT/FOXO pathway has been considered relevant to the proliferation and invasion of nephroblastoma.Innovation points of this paper:1.We have clarified the key role of miR-200c/EP300/P27 axis in the proliferation and invasion of nephroblastoma,which provides new ideas and therapeutic targets for cancer diagnosis and therapy.2.We have provided evidence for the potential predictive value of miR-200c in proliferation and invasion of nephroblastoma.