| Pituitary Adenomas(PAs)invade the surrounding tissue structure,resulting in low cure rate of surgical resection,which leads to disease progression in many patients after surgical failure.To limit morbidity and mortality in such patients,effective and safe treatment regimens are needed.Minimally invasive liquid biopsy has the potential to improve the efficiency of early screening and to monitor prognosis.Minimally invasive liquid biopsy may improve the efficiency of early screening and monitor prognosis by facilitating diagnostic procedures.Different biomarkers obtained from PA tissues may have different potential to evaluate PA.PA usually develops from the cumulative effect of many small genetic or epigenetic changes.Many long non-coding RNAs(LncRNAs)are involved in the development of PA,which may be the key regulatory nodes representing effective markers for diagnostic testing.Targeted drugs may be more beneficial to the therapeutic effect.The purpose of this study was to investigate the role and mechanism of MEG3 in PA.PartⅠ The expression of lncRN A MEG3 in PA tissuesObjective:To detect the expression of lncRNA MEG3 in PA tissues.Methods:1.GSE77517 was downloaded from GEO database.2.The expression of lncRNA MEG3 was detected by qRT-PCR.3.Pearson’s χ2 was used to analyze the relationship between the expression of MEG3 and the clinical features of PA.Results:1.The lncRNA MEG3 expression in PA tumor tissues was significantly decreased when compared to that in normal tissues.2.The expression of LncRNA MEG3 in the tissues of patients with stage Ⅲ-Ⅳ PA was significantly lower than that of patients with stage Ⅰ-Ⅱ PA.3.The expression of lncRNA MEG3was significantly correlated with the invasiveness and clinical grades of PA.Conclusion:The low expression of lncRNA MEG3 in PA is associated with aggression and clinical grade of pituitary tumors.Part Ⅱ The effect of lncRNA MEG3 on the cell activities of rat PA cellsObjective:To explore the effect of lncRNA MEG3 on the activities of rat PA cell lines.Methods:1.The over-expression sequence of MEG3 was designed to up-regulate the expression of MEG3 in GH3 and MMQ cells.2.CCK-8 was performed to assess cell proliferation.3.Cell apoptosis was detected by flow cytometry and western blot.4.The PA cell invasion and migration were both analyzed using transwell assay.5.The expressions of E-cadherin,Twist,Slug,MMP-7,which were associated with EMT,were tested using western blot.Results:1.Up-regulation of MEG3 expression inhibited the proliferation of GH3 and MMQ cells.2.Up-regulation of MEG3 expression promoted the apoptosis of GH3 and MMQ cells.3.Up-regulation of MEG3 expression reduced the expression of Survivin and Bcl-2 in GH3 and MMQ cells,and increased the expression of Cleaved-caspase-3 and Bax.4.Up-regulation of MEG3 expression inhibited the proliferation,migration and invasion of GH3 and MMQ cells.5.Up-regulation of MEG3 expression increased the expression of E-cadherin in GH3 and MMQ cells,and inhibited the expression of Twist,Slug and MMP-7.Conclusion:Up-regulation of IncRNA MEG3 inhibited the proliferation,migration,invasion and EMT of rat PA cells GH3 and MMQ,and promoted their apoptosis.Part Ⅲ LncRNA MEG3 targeting miR-23b-3p/FOXO4Objective:To investigate the molecular mechanism of IncRNA MEG3 regulating PA.Methods:1.Online tools were used to predict the target miRNA of IncRNA MEG3.2.Luciferase report assay was carried out to verify the prediction.3.RNA pull down assay was used to verify the prediction,too.4.QRT-PCR was used to detect the expression of the miRNA in PA tissue samples.5.Spearman’s correlation analysis was used to calculate the correlation between miRNA expression and MEG3 expression.6.Online biological tools were performed to predict the downstream mRNA of the above miRNA.7.Luciferase report assay was carried out to verify the prediction.8.RNA pull down assay was used to verify the prediction,too.9.QRT-PCR and Western blot were both used to detect the mRNA expression in GH3 and MMQ cells transfected with miR-23b-3p mimic/miR-NC.10.QRT-PCR and Western blot were both used to detect the mRNA expression in GH3 and MMQ cells transfected with MEG3/NC.11.QRT-PCR was used to detect the mRNA expression in PA tissues.12.Spearman’s correlation analysis was used to calculate the correlation between MEG3/miR-23b-3p and predicted mRNA expression.Results:1.LncRNA MEG3 targeted miR-23b-3p.2.Up-regulation of MEG3 expression inhibited the expression of miR-23b-3p in GH3 and MMQ cells.3.MiR-23b-3p was highly expressed in PA tissues.4.The expression of MEG3 was negatively correlated with the expression of miR-23b-3p in PA tissues.5.LncRNA MEG3/miR-23b-3p targeted FOXO4.6.Up-regulation of miR-23b-3p inhibited FOXO4 expression in GH3 and MMQ cells.7.Up-regulation of IncRNA MEG3 increased FOXO4 expression in GH3 and MMQ cells.8.The expression of FOXO4 was significantly lower in PA tissues compared to that in normal tissues.9.In PA tissues,the expression of FOXO4 was positively correlated with MEG3 expression and negatively correlated with miR-23b-3p expression.Conclusion:LncRNA MEG3 targeted miR-23b-3p/FOXO4 in PA.Part Ⅳ LncRNA MEG3 regulates PA cell activities and EMT by targeting miR-23b-3p.Objective:To investigate the mechanism of lncRNA MEG3 on PA.Methods:1.According to different treatments,GH3 cells were divided into three groups:shNC+miR-NC,shMEG3+miR-NC and shMEG3+miR-23b-3p inhibitor.2.CCK-8 was performed to assess cell proliferation.3.Cell apoptosis was detected by flow cytometry and western blot.4.The PA cell invasion and migration were both analyzed using transwell assay.5.The expressions of E-cadherin,Twist,Slug,MMP-7,which were associated with EMT,were tested using western blot.Results:1.Down-regulation of MEG3 expression in GH3 cells significantly promoted cell proliferation,while down-regulation the expression of miR-23b-3p in GH3 cells largely restored this effect.2.Down-regulation of MEG3 expression in GH3 cells inhibited cell apoptosis,while down-regulation of miR-23b-3p expression in GH3 cells restored this effect.3.In GH3 cells,down-regulation of MEG3 expression increased cell migration and invasion,while down-regulation of miR-23b-3p expression counteracted the above effects.4.Down-regulation of MEG3 expression promoted EMT of GH3 cells,while down-regulation of miR-23b-3p expresison eliminated this effect.Conclusion:LncRNA MEG3 regulated cell proliferation,apoptosis,migration,invasion and EMT of PA cells by targeting miR-23b-3p. |
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