The Role And Mechanism Of Mir-6858-5p In Inhibiting The Malignant Biological Behavior Of Glioma By Melatonin

Glioblastoma multiforme(GBM)is one of the most common and lethal primary malignant brain tumor in the central nervous system.Despite advances in surgery and comprehensive treatment,the life expectancy of patients with glioblastoma after diagnosis is only 12 to 18 months.Therefore,it is urgent to find new treatment strategies to improve and prolong the survival time of patients.MicroRNAs(miRNAs)is the endogenous,short ribonucleic acid molecules containing only 19-25 nucleotides,non-coding,and highly conservative,which can directly react with 3'UTR sequences to perform related functions.They are mainly involved in the regulation of various biological processes and they can also be used as a kind of cancer-promoting factor or anti-cancer factor to participate in the regulation of cancer progression.It has been reported that a variety of miRNAs can regulate most of the malignant biological behavior processes of GBM cells such as stemness maintenance,invasion and angiogenesis.It was reported that GBM cells can acquired the resistance to temozolomide though the c-Myc/Mir-29c/REV3L signaling pathway,so as to regulate the chemotherapy effect.However,miR-93 can regulate the therapeutic effect to temozolomide of GBM cells by inhibiting a variety of autophagy regulatory factors.Therefore,there is very important value to study the role of miRNAs in glioma has important value.Melatonin(MEL),the amine hormone secreted by the glands in the brain,has the function of aging resistance and immune regulation.MEL plays the antitumor role in many types of cancer,including gastric cancer,lung cancer,and GBM.MEL can alter the biological characteristics of GSC and inhibit the proliferation of GSC to reduce the growth capacity of GBM.In addition to directly targeting GBM,MEL has also been shown to modulate the micro-environment of GBM cells by targeting SIRT1.Our works also demonstrates that MEL exerts anti-migratory and anti-invasive activities in GBM cells under both normoxia and hypoxic conditions.However,whether MEL can exert its anti-GBM effect by regulating the expression of miRNA is not fully understood.In this study,we investigated the anti-GBM potential of MEL both in vitro and in vivo.We found that MEL possesses profound antitumor effects in U251,U87,A172 and GBM#P3(primary GBM)cells.Using miRNA sequencing,we identified candidate miRNAs induced by MEL in GBM cell lines.It was found that MEL could inhibit the growth of GBM by increasing the expression level of Mir-6858-5p,and then inhibit the SIRT3/AKT signaling pathway.These results offer new opportunities for therapeutic strategies by targeting GBM cells.Part I Melatonin inhibits migration and invasion of glioma cells by up-regulating miR-6858-5pObjectsTo explore the effect of melatonin on GBM cells migration and invasion in vitro.Methods1.The effects of MEL on U87,U251,A172 and GBM#P3 cells were detected by CCK-8 kit.2.The effect of MEL on the proliferation of U87 and GBM#P3 cells was determined by using EdU staining.3.Wound healing assay was used to detect the effect of MEL on migration ability of U87 cells.4.To detect the effect of MEL on the invasion ability of U87 cells by Transwell.5.To screen target miRNA of MEL by miRNA gene sequencing.6.qRT-PCR was used to further detect and analyze the expression of miR-6858-5p in U87 and GBM#P3 cells treated by MEL.Results1.MEL inhibits proliferation of GBM cells in vitro(1)MEL reduced the cell viability of U87,U251,A172 and GBM#P3 cells in a doseand time-dependent manner.(2)After MEL treatment,the proliferation of U87 and GBM#P3 cells were weakened obviously in a dose-dependent manner.2.MEL inhibits migration and invasion of GBM cells in vitro(1)The wound closure of U87 cells after MEL treatment was significantly weaker than that of the control group(2)The invasion ability of U87 weakened with the increase of melatonin concentration.3.MEL induces miR-6858-5p level of GBM cells(1)The results of miRNA sequencing show that miR-6858-5p is obviously increased in GBM cells after exposure to MEL.(2)QRT-PCR verified that the level of miR-6858-5p significantly was increased in U87 and GBM#P3 cells by MEL treatment.ConclusionMelatonin can significantly inhibit the proliferation of glioma cells and effectively inhibit the migration and invasion of GBM cells by up-regulating the level of miR-6858-5p.Part II Melatonin inhibits the malignant progression of glioma through the miR-6858-5p/SIRT3/AKT signaling pathwayObjectsTo explore the mechanism of melatonin's influence on glioma cell growth and its influence on glioblastoma malignant progression in vivoMethods1.The effects of miR-6858-5p mimics on the activity of U87 and GBM#P3 cells were tested by CCK-8 kit.2.To detect the effect of miR-6858-5p mimics on the invasion of U87 cells by Transwell.3.After the miR-6858-5p inhibitor co-transfected with MEL,the level of miR-6858-5p was tested by CCK-8 kit.4.EdU staining was used to detect the effect of miR-6858-5p inhibitor on the proliferation of MEL treated U87 cells.5.U87 cells were pretreated with miR-6858-5p inhibitor,and then the effects of MEL on migration and invasion were examined by wound healing and Transwell assay.6.The direct target protein of miR-6858-5p was predicted by online Targetscan and miRDB database.7.To search the related studies of predicted proteins in glioma though PubMed.8.mRNA expressions of BCAT1,S1PR1,FNDC3B,GFRA1,POU3F3,HBP1,SCAI,WNT5A,RUNX3 and SIRT3 were detected by qRT-PCR.9.The expression levels of p-AKT,p-mTOR,MMP-2,CDK-4,cyclin B and cyclin D were detected by western blot in U87 and GBM#P3 cells treated by MEL,miR-6858-5p inhibitor or miR-6858-5p mimics,respectively.10.The interaction between miR-6858-5p and SIRT3 was tested by miRNA luciferase assay.11.The effects of MEL on glioma growth were verified by using a mouse intracranial xenotransplantation model.12.Using qRT-PCR to analyze the level of miR-6858-5p.13.The level of p-AKT,SIRT3 and miR-6858-5p was analyzed by western blot in vivo.Results1.MEL inhibits GBM progression by up-regulating miR-6858-5p(1)miR-6858-5p mimic can inhibit the proliferation of U87 and GBM#P3 cells in a time-dependent manner.(2)miR-6858-5p inhibitor can increase the activity of U87 and GBM#P3 cells after MEL treatment.(3)The results of EdU staining showed that MEL inhibited the proliferation of U87 cells,and the proliferation was improved after the addition of miR-6858-5p inhibitor.(4)Wound healing and Transwell results showed that miR-6858-5p inhibitors could inhibit MEL-induced decreased U87 cell migration and invasion energy retention.2.MEL inhibits GBM progression through the miR-6858-5p/SIRT3/AKT signaling pathway(1)Target genes of miR-6858-5p were predicted by Targetscan and miRDB databases.2862 and 168(target score>70)target genes,138 of them were included in both databases.(2)After searched 138 genes in PubMed one by one,10 most likely genes related with glioma were obtained,including BCAT1,S1PR1,FNDC3B,GFRA1,POU3F3,HBP1,SCAI,WNT5A,RUNX3 and SIRT3.(3)QRT-PCR results showed that mRNA levels of BCAT1,SNPR1 and SIRT3 were significantly reduced in U87 cells treated by MEL,while the remaining seven genes showed no significant changes.(4)Western blot results showed that the expression levels of p-mTOR,MMP-2,p-AKT,CDK-4,cyclin B and cyclin D decreased in U87 and GBM#P3 cells treated by MEL.However,there was no significant change in the two cells co-treated by miR-6858-5p inhibitor and MEL.(5)After transfected with miR-6858-5p mimics,the expression levels of these proteins in U87 and GBM#P3 cells also decreased significantly.(6)Dual luciferase reporter assay results showed that miR-6858-5p could act on the 3'UTR of SIRT3.3.MEL inhibits the growth of GBM cells in vivo(1)In mice implanted with GBM#P3 cells,the number of intracranial tumors in mice treated with MEL was significantly smaller than that in the non-treatment group.(2)The total survival rate of mice treated with MEL was obviously higher than that of the non-treatment group.(3)miR-6858-5p was up-regulated by MEL in vivo.(4)The expression levels of p-AKT,SIRT3 were decreased in vivo.ConclusionMEL inhibits SIRT/AKT signaling pathway by up-regulating miR-6858-5p,and inhibiting the malignant behavior of GBM.At the same time,MEL inhibits the growth of GBM cells in vivo.