| Objective: To investigate the expression differences of miR-210-3p in invasive nonfunctional pituitary adenoma and non-invasive pituitary adenoma and the effects on the proliferation and migration of MMQ and GH3 pituitary adenoma cells to predict and verify the target genes of miR-210-3p,and provide new ways for the ultra-early diagnosis and related treatment of invasive NFPA.Methods: 1.In 20 cases of invasive NFPA and 20 cases of non-invasive NFPA,The expression of miR-210-3p was detected by RT-PCR and to confirm the difference in the expression of miR-210-3p in invasive NFPA and non-invasive NFPA.2.Cultured rat pituitary tumor MMQ and GH3 cell lines were divided into low-expression miR-210-3p group and Negative control vector(NC)group.Mi R-210-3p inhibitor and miR-210-3p NC were transfected,respectively,and the expression of miR-210-3p in the experimental group and the control group was detected by fluorescence quantitative PCR.3.CCK-8 method was used to detect proliferation at 24 h,48h and 72 h after transfection.4.Transwell method was used to detect the migration ability of pituitary tumor cells in rats.5.The downstream target gene of miR-210-3p was predicted to be FGFRL-1 using Target Scan 7.2.6.The target genes of miR-210-3p were verified by dual luciferase reporter gene assay.7.Westernboltting technique was used to detect the protein expression of FGFRL-1 in pituitary tumor cells of the low-expression miR-210-3p group and the negative control group.Results: 1.Compared with the invasive NFPA tissue,the expression of miR-210-3p was decreased in the non-invasive NFPA tissue(P < 0.05).2.In the MMQ and GH3 cell lines of pituitary tumor cells,the interference vector(Inhibitor)and meaningless sequence negative control vector(NC)of miR-210-3p were constructed.The results of RT-PCR analysis showed that the expression of miR-210-3p in MMQ cell lines was significantly decreased in MMQ-INH group compared with MMQ-NC group(P <0.01).In GH3 cell lines,compared with the GH3-NC group,the expression of miR-210-3p was also decreased in GH3-INH group(P<0.05).3.CCK-8 results indicated that the proliferation ability of MMQ and GH3 pituitary tumor cell lines after transfection with low expression of miR-210-3p virus was lower than that of negative control group(P < 0.05).4.Transwell results indicated that MMQ and GH3 pituitary tumor cell lines with low expression of miR-210-3p were less capable of cell migration than the negative control group(P < 0.05).5.The prediction results of Target Scan online prediction software showed that FGFRL-1 was the direct target of miR-210-3p in pituitary tumor cells.6.The results of double luciferase report experiment showed that the luciferase activity in pituitary tumor cells was significantly decreased after co-transfection of FGFRL-1 m RNA 3’UTR vector and miR-210-3p mimic(P<0.01),while there was no difference in luciferase activity after cotransfection of FGFRL-1 m RNA 3’UTR-mutr vector and miR-210-3p mimic(P>0.05).7.Western blotting results showed that the expression level of FGFRL-1 protein was low in MMQ and GH3 cells,while the expression level of FGFRL-1 protein was significantly increased after the low expression of miR-210-3p(P<0.0001,P<0.01).Conclusion: 1.The expression of miR-210-3p in non-invasive NFPA was lower than that in invasive NFPA.2.Mi R-210-3p can promote the proliferation and migration of MMQ and GH3 pituitary tumor cells,which provides a potential idea for us to further study the molecular mechanism of invasive NFPA in diagnosis and treatment.3.Through software prediction and experimental verification,we obtained that the target gene of miR-210-3p is FGFRL-1,and the expression of FGFRL-1 protein in MMQ and GH3 pituitary tumor cells with low expression of miR-210-3p is increased,which can inhibit the proliferation of pituitary tumor cells and promote the differentiation of pituitary tumor cells,providing clues for our further research. |
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