The Molecular Mechanism Of PTEN/AKT/fOXO1 Signal Pathway Regulated By MiR-21 In Multiple Myeloma

[Objective]Multiple myeloma?MM?is a genetically complex hematologic malignancy.It is the second most common hematological malignancy.Despite many therapeutic options,MM remains incurable,with a 5-year survival rate of 40%.The gene copy number changes,chromosomal translocations,mutations,transcriptional changes,and epigenetic changes that occurduring MM development result in the profound deregulation of micro-RNA?miRNA?transcription.This leads to aberrant messenger RNA?mRNA?translation and cell signaling.MicroRNA-21?miR-21?is always over-expressed in multiple myeloma and is implicated in cell proliferation,apoptosis,invasion,and drug resistance of MM cells.Our previous studies have established FOXO1 plays important role of proliferation and apoptosis in multiple myeloma.In this syudy,the FOXO1 interacting proteins were analyzed and the survival time of patients with multiple myeloma was detected by data mining.To explore the molecular mechanism of PTEN/AKT/FOXO1 signal pathway regulated by miR-21 in human multiple U266 cell lines.[Methods]1.The expression of FOXO1 in multiple myeloma was mined in Oncomine database.Thedifferent expressions of FOXO1 between the normal tissue and myeloma were analyzedby BIOGPS database.2.The survival time of patients with multiple myeloma was detected by GEPIA.TheFOXO1 interacting proteins were analyzed by String-DB database.3.miR-21 was detected on multiple myeloma cell lines by qRT-PCR.4.U266 cell line was transfected with miR-21inhibitor and its negative control.qRT-PCRwas used to detect the miR-21expression changes.5.Cell proliferation and apoptosis were determined by using CCK-8 and flow cytometry.Western blot was used to detect the protein expression of PTEN and FOXO1.6.The effect of gambogic acid with different concentrations on the proliferation of U266cells was detected by CCK-8 assay,while the effect of gambogic acid on apoptosis wasanalyzed by Annexin V/PI assay in cells expressing different mRNA levels of miR-21.[Results]1.The expression of FOXO1 was lower in multiple myeloma than in normal tissue byOncomine database and BIOGPS database.2.GEPIA analyzed that the expression level was not notable correlated with the prognosis ofmature B-cell neoplasms?LogrankP=0.39?.FOXO1 related proteins are PTEN?AKT byString-DB database.PTEN maybe regulate FOXO1 through AKT signal pathway.3.The related expression of miR-21 was detected in 5 different multiple myeloma cell linesby qRT-PCR.The U266 cell line was as research object.4.The related expression of miR-21 was lower in in experimental group than in negativecontrol group after transfection by qRT-PCR?0.30 0.005 vs0.97 0.06,P<0.001?.5.The proliferation of U266 cells in experimental group was?23.15 0.77?%aftertransfection,which was lower than negative control group?95.89 0.07?%?P<0.05?.The cellular apoptotic rate was significantly different,the experimental group was higherthan negative control group[?10.36 1.23?%vs?3.39 0.63?%,P<0.05].Western blotanalysis revealed up-regulation of PTEN and FOXO1protein expression in experimentalgroup by inhibiting miR-21 expression?P<0.05?.6.Gambogic acid inhibited the proliferation of U266 cells in a concentration and timedependent manner.The U266 cells transfected with miR-21 inhibitor were more sensitiveto gambogic acid and apoptotic cells were elevated significantly[?16.64 0.63?%vs?10.36 1.23?%,P<0.05].Gambogic acid effectively inhibited the phosphorylation ofAKT,down-regulated the expression of p-AKT^S308,and increased the expression oFOXO1 and Bim as revealed by Western blot assay?P<0.05?.[Conclusion]1.FOXO1 was closely related to multiple myeloma by bioinformatics network technology.PTEN maybe regulate FOXO1 through AKT signal pathway,it would be an scientific andtheoretical support for the further research.2.Inhibiting miR-21expression had distinct effects on apoptosis and proliferation inhibition,which were mediated by up-regulated levels of PTEN,down-regulated phosphorylationlevels of AKT and promoting the expression of FOXO1.PTEN/AKT/FOXO1 signalingpathway plays important role in the regulation of biological behavior of multiple myelomaby miR-21.3.Gambogic acid can induce the proliferation and apoptosis of U266 cells with higherexpression of miR-21 through mediation of AKT/FOXO1/BIM signaling.InhibitingmiR-21expression combined with gambogic acid can promote the apoptosis of U266 cells,both of which can play a synergistic role in anti-tumor.