The Mechanism Of MiR-20b-5p Targeting AEN To Regulate Apoptosis Of Nasopharyngeal Carcinoma Cells

Objective: To clarify the effect of mi R-20b-5p on proliferation and apoptosis of nasopharyngeal carcinoma cells,and explore the mechanism of mi R-20b-5p targeting AEN to regulate the proliferation and apoptosis of nasopharyngeal carcinoma cells.Method: QRT-PCR was used to detect the expression of mi R-20b-5p in nasopharyngeal carcinoma patients' serum,and nasopharyngeal carcinoma cell lines CNE1,HNE2,HONE1,and immortalized normal nasopharyngeal epithelial cells NP69;Using nasopharyngeal carcinoma cells CNE1 and HONE1 as the research subjects,transfected NC control,mi R-20b-5p mimics and mi R-20b-5p inhibitor to up-regulate or down-regulate the expression of mi R-20b-5p,respectively,and verified by q RT-PCR detection.Plate clone formation experiment,CCK8,and tumor formation in nude mouse were used to analyze the effects of mi R-20b-5p expression changes on cell colony formation,cell proliferation,and nude mouse tumorigenesis from in vivo and in vitro;Flow cytometry was used to detect the apoptosis of CNE1 and HONE1,after transfect mi R-20b-5p mimics and mi R-20b-5p inhibitor 48 h later;bioinformatics analysis was used to predict the target gene of mi R-20b-5p and a luciferase reporter gene was used to verified;Western blot was used to detect the effects of mi R-20b-5p expression changes on the expression of AEN and apoptosis proteins such as Caspase3,Parp,Bax,Bcl-2,Bcl-xl.In addition,analysis the effects of transfected mi R-20b-5p mimics simultaneously overexpressing AEN,or transfection of mi R-20b-5p inhibitor simultaneously with knockdown of AEN expression,were detected by CCK-8,flow cytometry,and Western blot,to detect the proliferation,apoptosis,and the expression changes of AEN,Caspase3,Parp,Bax,Bcl-2,Bcl-xl and other proteins.Results: The expression of mi R-20b-5p in nasopharyngeal carcinoma cells was significantly higher than that of normal nasopharyngeal epithelial cells(P<0.001),and the expression of mi R-20b-5p in the serum of patients with nasopharyngeal carcinoma was also significantly higher than that of healthy people(P<0.0001),clinical significance analysis showed that the high expression of mi R-20b-5p was significantly positively correlated with the large mass of nasopharyngeal carcinoma patients and the later clinical stage(P<0.01).Transfection with mi R-20b-5p mimics significantly increased the expression of mi R-20b-5p in nasopharyngeal carcinoma cells,while transfection with mi R-20b-5p inhibitor significantly decreased the expression of mi R-20b-5p(P<0.001).After transfection of mi R-20b-5p inhibitor,the expression of mi R-20b-5p significantly down-regulated.Meanwhile,the cloning ability of cells was significantly weakened,cell proliferation was significantly inhibited,apoptosis was obvious,and the growth of subcutaneous implanted tumors in nude mice was slow;mi R-20b-5p mimics upregulated mi R-20b-5p,the cloning ability and cell proliferation ability of cells were significantly enhanced,the apoptosis was significantly reduced,and the growth of subcutaneous implanted tumors in nude mice was significantly increased.Bioinformatics predictions show that AEN is the downstream target gene of mi R-20b-5p,and luciferase reporter gene detection shows that mi R-20b-5p can indeed target the transcriptional activity of AEN.At the same time,our research found that over-surface AEN can significantly inhibit the proliferation of nasopharyngeal cancer cells and induce apoptosis of nasopharyngeal cancer cells,while silencing the expression of AEN significantly promotes the proliferation of nasopharyngeal cancer cells and inhibits their apoptosis;when transfected with mi R-20b-5p mimics overexpressing AEN at the same time can significantly weaken the promotion of mi R-20b-5p on the proliferation and anti-apoptotic effect of nasopharyngeal carcinoma fine cells,while transfecting mi R-20b-5p inhibitor and knocking down AEN expression at the same time can significantly resist mi R-20b-5p knockdown inhibits the proliferation and anti-apoptosis of nasopharyngeal carcinoma cells.Further research found that mi R-20b-5p inhibitor can significantly up-regulate the expression of AEN,Caspase3,Parp,Bax and other proteins,and down-regulate the expression of Bcl-2 and Bcl-xl proteins(P<0.05);mi R-20b-5p mimics obviously down-regulate the expression of AEN,Caspase3,Parp,Bax and other proteins,and up-regulate the expression of Bcl-2 and Bcl-xl proteins(P<0.05).Conclusion:1.Mi R-20b-5p is highly expressed in NPC,which is positively related to the clinical stage of NPC,and may be a potential biomarker of NPC.2.Mi R-20b-5p promotes the proliferation of NPC cells,and its mechanism is related to the inhibition of apoptosis of NPC cells by targeted down-regulation of AEN expression.