Effects And Mechanisms Of MiR-9-FOXP2 Signaling In Inhibition Of Human Glioma Cells By Resveratrol

PartⅠ The Expression and Clinical Prognosis of miR-9-FOXP2 Signal in Human GlioblastomaObjective:To examine the expression of miR-9 and FOXP2 gene in the glioma tissue,and explore the relationship between the clinical characteristics and the survival time of FOXP2 and miR-9 of the glioblastoma.Methods:From January 2015 to December 2016 period,a total of 70 cases of glioma patients were selected from the Department of Neurosurgery of People’s Hospital of Wuhan University.The immunohistochemistry and qPCR test were used to detect FOXP2 and miR-9 in glioma expression.According to the expression level of FOXP2 in GBM,the first 20 cases were low expression group and 20 cases were high expression group.The relationship between GBM and FOXP2 and miR-9 expression was analyzed and compared.To summarize the clinical data and follow-up results of glioma patients,Kaplan-Meier method was used to analyze the survival time and prognosis of FOXP2 and miR-9 expression levels.Results:1 The expression of FOXP2 in glioma tissues was higher than 90%in high grade(WHOⅢ,Ⅳ grade)glioma tissues.The expression site is mainly in the nucleus,followed by the cytoplasm.2 The expression of FOXP2 in different pathological types of gliomas was different,low expression in low grade gliomas,high expression in high grade gliomas,P<0.05.3 There was no significant difference in sex,age,KPS score and clinical asymptomatic symptoms between FOXP2 expression and glioma patients,P>0.05.4 The expression of FOXP2 in GBM was negatively correlated with clinical prognosis,and the survival time of FOXP2 was shorter than that of low expression.There was significant difference(P<0.05).5 The expression level of miR-9 in glioma specimens was positively correlated with the prognosis,and negatively correlated with the expression level of FOXP2(P<0.05).Conclusion:1.FOXP2 protein was expressed in different grades of gliomas.2.The expression level of FOXP2 protein was positively correlated with the pathological grade and the malignancy of glioma.3.The level of FOXP2 protein is closely related to the prognosis of glioma,which affects the quality of life and prognosis of patients.4.miR-9 was expressed in GBM and negatively correlated with FOXP2.Part Ⅱ Res Regulated the miR-9-FOXP2 Signaling in Glioma Cells and its MechanismObjective:To study the expression and the biological function of miR-9-FOXP2 signaling pathway,and to explore the mechanism of resveratrol on the glioma cellsMethods:The miR-9 regulatory transcription factor and target protein were screened by bioinformatics software Pictar,miRBase and Targetscan.CCK8,EdU,cell cycle assay Cell viability and proliferation,Annexin V-FITC and flow cytometry were used to detect apoptosis.Resveratrol-treated glioma cells were transfected with miR-9 mimic and miR-9 The expression and significance of miR-9 and FOXP2 in U251 cell line were detected by Western blot and qPCR.The effect of miR-9-FOXP2 on the inhibition of glioblastoma was confirmed by reversing the experiment.The effect of niR-9-FOXP2 on the expression of FOXP2 was detected by double luciferase.Results:1.miRNA database successfully screened FOXP2 as hsa-miR-9 target gene.2.RES inhibited the proliferation and proliferation of glioblastoma cells,and the cell viability was significantly lower than that of the control group(P<0.05).Cell cycle arrest in G1 phase,ras protein and cyclinD1 protein expression decreased.3.RES promoted the apoptosis of glioblastoma cell line U251,increased the apoptosis rate,cleaved-caspase3,Bax protein expression,Bcl-2 protein expression decreased,P<0.05.4.The expression of miR-9 and FOXP2 protein in RES treatment group was significantly lower than that in control group(P<0.05).The expression of miR-9 in RES group was higher than that in control group(P<0.05).5.miR-9 regulates FOXP2 and downstream protein expression in glioblastoma U251 cells.After expression of miR-9,the expression of FOXP2 decreased,the expression of ras and cyclinD1 decreased,the expression of cleaved-caspase3 and Bax increased,-2 protein expression decreased,P<0.05.6.miR-9 directly regulates FOXP2 protein expression,double luciferase reporter gene expression analysis,hsa-miR-9-5p mimics FOXP2 wild type and mutant vector reported fluorescence has a significant down-regulation effect.7.miR-9 reversal experiments showed that RES on the regulation of FOXP2 disappeared.8.Compared the EGFR expression level in glioblastoma U251 cells,RES group were decreased lower than the control group,P<0.05.Conclusion:1.Successfully screened FOXP2 as the target gene for miR-9.2.RES can make the glioblastoma U251 cell arrested G1 phase,thereby inhibiting cell proliferation.By promoting bax protein expression,bcl-2 decreased,cleaved-caspase3 protein activity increased,promote glioblastoma cell U251 apoptosis.3.RES increased the expression of mir-9 in glioblastoma U251 cells and decreased the expression of FOXP2.Mir9 had the same regulation on FOXP2 and cleaved-caspase3,bcl-2 bax,ras and cyclindl proteins.4.FOXP2 is the target gene of mir9,RES inhibits glioblastoma growth by regulating mir-9-foxp2 signal.MiR-9 regulates FOXP2 and downstream protein expression in glioblastoma U251 cells.5.We can found that the new target of resveratrol and FOXP2 protein function,promote tumor inhibition,induced apoptosis.MiR-9 can down-regulate the expression of FOXP,EGFR mRNA and protein,and hsa-miR-9-5p may regulate the expression of 3’UTR gene with FOXP2 gene.Part III RES In Vivo Regulation of miR-9-FOXP2 Signaling Inhibits Gliomas GrowObjective:To observe the effect of resveratrol on tumor growth in nude mice,to analyze the expression of FOXP2 protein in nude mice and explore the anti-tumor effect of miR-9-FOXP2 signal in resveratrol.Methods:U251 glioma cells were transplanted subcutaneously in nude mice of BALB/C to establish nude mice model,resveratrol 50mmol/L L every three days of gavage,dynamic recording of tumor volume and weight changes were observed in transplanted tunor growth.The expressions of miR-9 and FOXP2 protein were detected by flow cytometry,HE staining,qPCR and Western blot trail.The histopalthological changes of tumor were observed.Results:1.The tumor growth of nude mice was successfully established.The tumor volume was about 6mm3 and the rate of tumor formation was 100%after 5 days.2.The effect of resveratrol on the growth of nude mice was observed.The tumor growth rate and volume of RES group were slower than that of DMSO group.The difference between the two groups was statistically significant at day 21 and day 24 21 days P = 0.0231,24 days P ＝ 0.009,(P<0.05).3.RES on tumor inhibition,and RES group nude mice obvious skin ulceration and tumor hemorrhage necrosis was significantly less than DMSO group.4.The expression of FOXP2 in RES and DMSO group was significantly higher than that in the control group(P<0.001).The expression of FOXP2 in the RES and DMSO groups was significantly higher than that in the control group(P<0.001).The changes of miR-9 in RES group and DMSO group after resveratrol were significantly different between the two groups,the difference was statistically significant(P = 0.0038,P<0.01).The correlation coefficient between FOXP25.and mIR-9 r2 = 0.4928,P = 0.0236.Conclusion:1.a certain dose of resveratrol can play an anti-tumor effect in vivo,resveratrol inhibit tumor growth.2.resveratrol can play an anti-tumor effect in nude mice,with the duration of anti-tumor effect.3.resveratrol can reduce the expression of FOXP2 in glioma cells.4.resveratrol 1 play an anti-tumor effect by miR-9-FOXP2 signa.