Study Of The CREB1/miR-433 Reciprocal Feedback Loop In Colorectal Cancer Proliferation And Metastasis

Objectives:(1)To detect the expression of miR-433 in 35 paired colorectal cancer and adjacent normal mucosa specimens and 9 CRC cell lines.And to conduct KEGG pathway analysis.(2)To investigate the biological impact after alteration of miR-433.(3)To explore and determine the downstream targets of miR-433 in CRC.(4)To explore and predicate the upstream regulon of miR-433.(5)To assess the expression of markers associated with cell cycle progression and epithelial-mesenchymal transition.Methods:(1)Real-time PCR was used to detect the expression of miR-433 in CRC tissues and cell lines.The miRWalk dataset was applied to perform KEGG pathway analysis.The LinkedOmics database was used to plot a survival curve.(2)MTT,colony formation,transwell,subcutaneous tumor formation assays and an intra-splenic injection liver metastasis model were performed to investigate the impact on CRC cells after over-expression or inhibition of miR-433.(3)The TargetScan allied with micoRNA.org and miRDB datasets were adopted to predict the targets of miR-433.Western Blotting and dual-luciferase assay were carried out to determine the targeting relationship.(4)The UCSC combined with LASAGNA,JASPAR,CONSITE and DBTSS databases were conducted to predict upstream regulon of miR-433.ChIP and dual-luciferase assays were conducted to determine the interaction.(5)Western Blotting was utilized to detect the downstream markers expression after alteration of miR-433.Results:(1)miR-433 is down-expressed in CRC tissues and cell lines,and associated with Wnt、Ras、MAPK、cAMP pathways and cell adhesion molecules.miR-433 may serve as a protective factor in CRC prognosis.(2)gain-of-function or knockdown approach significantly repressed or reinforced CRC cells proliferation,colony formation,migration,invasion,oncogenesis and metastasis activity.(3)Bioinformatics aligned with Western Blotting and dual-luciferase assays demonstrated that miR-433 targeted CREB1,CCAR1 and JNK1.(4)Bioinformatics joint with ChIP and dualluciferase assays illustrated that CREB1 could bound to miR-433 promotor.(5)A downregulation of phospho-Smad2,phospho-c-Jun,CDK2,snail and slug and an accompanying upregulation of p21,p27,E-cadherin and β-catenin were present after overexpression of miR-433.Conclusions:(1)miR-433 is downregulated in CRC tissues.(2)Overexpression or inhibition of miR-433 suppressed or enhanced CRC cells propagation,colony formation,migration,invasion,tumorigenicity and metastasis property.(3)CREB1,CCAR1 and JNK1 simultaneously served as miR-433’s targets;meanwhile,CREB1 transactivated the expression of miR-433.Which in other words,CREB1/miR-433 composed a reciprocal feedback loop in CRC.(4)miR-433 abrogated cell cycle progression and epithelial-mesenchymal transition.