| PurposeThe purpose of the study is to investigatethe molecule mechanism of the downregulation of long non-coding RNA(lncRNA)PVT1(plasmacytoma variant translocation 1)inhibiting the cell proliferation in Burkitt`s lymphoma(BL)cell lines(Raji and Namalwa).MethodsAt first,differential expression levels including lncRNA-PVT1 and miRNAs were obtained through using analysis tools of bioinformatics,and two databases of Starbase and Jefferson were used to scan and screen the miRNA(miR-29b)which was significantly related with PVT1for next researches.Meanwhile,the correlation of expression of PVT1 and miR-29b in different tumor cells was analyzed by CCLE database.Secondly,expression of miR-29b was detected by qRT-PCR after the expression of PVT1 decreased in Raji cells,and through luciferase reporter assay,it would be illustrated that miR-29b combined with PVT1.Thirdly,after the expression of PVT1 and miR-29b by siRNA and mimics changed,the cell proliferation was observed in Raji and Namalwa cells.CCK8 assay was used to monitor cell proliferation,cell cycle and apoptosis were detectedby flow cytometry assay.qRT-PCR and western bloting(WB)were utilized to assay the changes of expression of proteins CDK(CDK4)and CKI(CDKN1A,CDKN1B,and CDKN2A).Finally,the activity of signal pathway of AKT/FOXO1 was analyzed through measuring the expression of AKT/p-AKT and FOXO1/p-FOXO1.Especially,inhibitor of FOXO1,AS1842856,was utilized to treat BL cells,similarly,the cell growth and cell cycle were assayed,and expression of proteins of CDK/CKI were examined by WB.ResultsThe analysis of Gene Expression Omnibus(GEO)database showed that there is obvious variation of expression of PVT1 and miR-29b in the tissues of BL.In contrast with normal group,up-regulation on levels of PVT1 was more than 1.7 times in BL group(p<0.001),and the expression of miR-29b decreased approximately four-fold(p=0.003).Moreover,the correlation analysis illustrated that levels of PVT1 were negative correlative with levels of miR-29b in the majorities of tumors(r~2=-0.359,p=0.031).By two databases of Starbase and Jefferson,the binding site between PVT1 and miR-29b was found in 3`-UTR of PVT1 RNA(chr8:128952169-128952190[+]).The study of down-regulation of PVT1 by RNA interference in Raji cells succeeded to induce the increasement of expression of miR-29b.In addition,the luciferase reporter assay supported that miR-29b combined with 3`-UTR of PVT1 RNA(p<0.05).The function experiments of PVT1 and miR-29b in BL cells declared that growth ability of cells was prohibited with the decrease of PVT1 or increase of miR-29b,and it was significant for adding inhibitor of miR-29b to offset the limited growth.Meanwhile,the decrease of PVT1 or increase of miR-29b were remarkable to augment the percentage of G0/G1 phase of cell cycle in BL cells,and similarly with above situation,the the percentage of G0/G1 phase also was lowered through adding inhibitor of miR-29b.From the studies for cell cycle proteins,we discovered both mRNA and protein of CKI,such as CDKN1A,CDKN1B and CDKN2A,were elevated after PVT1 decreased,or after miR-29b increased in Raji cells.On the contrary,CDK4was reduced.In final,from the analysis of activity of AKT/FOXO1 pathway,it was clarified that AKT/FOXO1 pathway was negatively activated by both miR-29b and PVT1.Using inhibitor of FOXO1,AS1842856,markedly varied the proportion of G0/G1 phase,and induced the change of levels of two types of proteins involving CDK and CKI.Conclusions1)There is abnormal high expression of PVT1 and low expression of miR-29b in tissues of BL,and levels of PVT1 were negatively correlated with levels of miR-29b by Bioinformatics analysis.2)The downregulation of PVT1 and upregulation of miR-29b inhibit the proliferation in Raji and Namalwa cells.3)PVT1 targets miR-29b to affect the proliferation of BL cells(Raji and Namalwa cells),and to disturb G0/G1 phase of cell cycle,which is related with the change of AKT/FOXO1 pathway and its downstream cell cycle protein(CDK4 and CDKN1A/CDKN1B/CDKN2A)in Raji cells. |
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