| Pituitary adenomas(PAs)are the third most common cranial tumors after meningioma and glioma,accounting for 10?15%of all cranial tumors.Although most pituitary adenomas are benign adenomas,many patients with pituitary adenomas have significant endocrine disorders or neurological compressions caused by pituitary adenomas.The major challenges of PAs are due to a lack of diagnostic markers and a poor response to available therapies.Abnormal expression of miRNAs are known to be associated with various human diseases.In pituitary adenoma,the abnormal expression of multiple miRNAs is associated with the development of pituitary adenomas.MiRNAs may play an important role in the pathogenesis of pituitary adenomas and may provide new molecular targets for the diagnosis and treatment of pituitary adenomas.Therefore,it has important clinical significance to study new pituitary adenoma-associated miRNAs.In this study,we explored the expression of miR-378 in tumor tissues of pituitary adenoma patients and found that miR-378 is down-regulated in pituitary adenoma tissues.Furthermore,we explored the cell biological function and related molecular mechanisms of miR-378 in pituitary adenomas,providing a new theoretical basis for understanding the mechanism of pituitary adenomas.Part I Expression and clinical significance of microRNA-378 in pituitary adenomaObjective:To investigate the expression changes of miR-378 in pituitary adenomas and adjacent normal tissues,and to analyze the relationship between the expression of miR-378 and the clinical features of pituitary adenomas.Methods:In this chapter,25 paired pituitary adenomas and the corresponding adjacent normal tissues were collected.The expression of miR-378 in pituitary adenomas and adjacent normal tissues was detected by real-time PCR.Clinical information such as age,gender,tumor size,tumor invasiveness and tumor function were collected from 25 patients with pituitary adenoma.The relationships between miR-378 expression in pituitary adenomas tissues and age,sex,tumor size,tumor invasiveness,or tumor function were analyzed.Results:1.The qRT-PCR method detected the expression level of miR-378 in human pituitary adenoma tissues and adjacent normal adjacent tissues.The results showed that the expression of miR-378 in human pituitary adenoma tissues was significantly lower than that of adjacent normal tissues(P<0.05).2.There was no significant correlation between the expression level of miR-378 and the patient's age,gender and tumor function(P>0.05).3.The expression level of miR-378 was significantly correlated with tumor size and tumor invasiveness(P<0.05).Conclusions:MiR-378 exhibits a low expression level in human pituitary adenomas.The expression level of miR-378 in pituitary adenomas is correlated with tumor size and tumor invasiveness.MiR-378 may have a role in inhibiting tumorigenesis and development in pituitary adenomas.Part ? Effects of MicroRNA-378 on the Biological Behavior of Pituitary Adenoma CellsObjective:To investigate the effects of miR-378 on proliferation and migration on pituitary adenoma cells in vitro.Methods:MiR-378 mimics and miR-378 inhibitors were designed to construct gain-and loss-of-function cell models.The effect of miR-378 overexpression or interference on the proliferation of GH3 pituitary adenoma cells was studied by CCK-8 assay.The effect of miR-378 overexpression or interference on the migration ability of GH3 pituitary adenoma cells was studied by cell scratch assay.Results:1.After GH3 cells were transfected with miR-378 mimics,the relative expression of miR-378 was up-regulated by 26-fold(P<0.05).After GH3 cells were transfected with miR-378 inhibitors,the relative expression of miR-378 in cells was down-regulated by 78%(P<0.05).2.miR-378 mimics significantly inhibited the proliferation of GH3 cells at 48 and 72 hours after transfection(P<0.05).Conversely,compared with the control,the proliferation of GH3 cells transfected with miR-378 inhibitors was significantly increased after 48 hours and 72 hours(P<0.05).3.In the miR-378 overexpression group,the migration ability of GH3 cells was inhibited,which was statistically different from the control group(P<0.05).Conversely,in the miR-378 interference group,the migration ability of GH3 cells was statistically increased compared with the control group(P<0.05).Conclusions:Overexpression of MiR-378 can inhibit the proliferation and migration of pituitary adenoma cells.Conversely,inhibition of miR-378 expression promotes the proliferation and migration of pituitary adenoma cells.MiR-378 functions as a"cancer suppressor".gene in pituitary adenomas.Part ? Molecular mechanisms of microRNA-378 on the biological effects of pituitary adenoma cellsObjective:To explore the target genes of miR-378 in pituitary adenoma cells,and to study the possible mechanisms of miR-378 in pituitary adenoma cells.Methods:Bioinformatics analyse were used to predict possible target genes for miR-378.Dual luciferase assays were used to confirm the binding of miR-378 and RNF31 genes.The effect of miR-378 on the expression of RNF31 gene was studied by real-time PCR and immunoblotting.The effects of RNF31 gene on proliferation and migration of pituitary adenoma cells were studied by CCK-8 assay and cell scratch assay.In addition,cell rescue experiments were performed to investigate whether miR-378 exerts biological functions in pituitary adenomas through RNF31 gene.Results:1.The dual luciferase reporter gene results showed that miR-378 mimics significantly inhibited the dual luciferase activity of the pGL3-WT-RNF31-3'-UTR vector(P<0.05),while miR-378 mimics could not inhibite the dual luciferase activity of the pGL3-MUT-RNF31-3'-UTR vector(P>0.05).2.Overexpression of miR-378 can inhibit the level of RNF31 mRNA in GH3 cells,which is statistically different from the control group(P<0.05).Conversely,interference with miR-378 expression in GH3 cells can statistically promote RNF31 mRNA expression levels(P<0.05).3.Overexpression of miR-378 in GH3 cells can inhibit the expression of RNF31 gene protein,which is statistically different from the control group(P<0.05).Conversely,interference with miR-378 expression in GH3 cells can promote RNF31 protein expression levels(P<0.05).4.CCK-8 experiments showed that siRNAs-RNF31 significantly inhibited the proliferation of GH3 cells at 48 and 72 hours after transfection(P<0.05).Cell scratch assay showed siRNAs-RNF31 significantly inhibited the migration of GH3 cells after transfection(P<0.05).5.Cell rescue experiments showed that siRNAs-RNF31 could rescue the proliferation promotion effect of miR-378 inhibitors on GH3 cells(P<0.05).In addition,siRNAs-RNF31 could rescue the migration promotion effect of miR-378 inhibitors on GH3 cells(P<0.05).Conclusions:MiR-378 can target RNF31 gene and play a biological role as "tumor suppressor" gene by inhibiting RNF31 gene in pituitary adenomas. |
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