Experimental Study Of The Regulation Of MiR-25 Target RAGE Biological Behavior In Nasopharyngeal Carcinoma

ObjectiveIn the present study,miR-25 expression was detected in tumor tissues of patients with nasopharyngeal carcinoma,and to explore its correlation with clinicopathological parameters in patients with nasopharyngeal carcinoma,and analyze the impact of miR-25 expression in nasopharyngeal carcinoma cells growth,proliferation,invasion,explore miR-25 signal activation on RAGE expression in NPC cells,invasion mechanism of nasopharyngeal carcinoma cells,aims to understand the mechanism of occurrence and development of NPC,as well as nasopharyngeal carcinoma clinical diagnostics and targeted therapy provide a reliable basis.Methods(1)60 cases of nasopharyngeal carcinoma tissues and corresponding normal tissue samples were collected from Anhui Medical University Affiliated Provincial Hospital and Tongling Municipal People's Hospital ENT laboratory,sample collection time was in May 2013-August 2016.All samples are nasopharyngeal biopsies diagnosed with nasopharyngeal carcinoma by pathological examination,histological type poorly differentiated or undifferentiated carcinoma.Sources patients with nasopharyngeal carcinoma tissue samples were selected,37 male,female 23 cases;aged <45 years 29 cases of patients,?45 years were 31 cases.All patients according to TNM clinical stage to be divided,where I of 11 cases,II of 18 cases,III of 14 cases,IV of 17 cases;according to T divided stage,where T1 ~ T2 of 41 cases,T3 ~ T4 of 19 cases;divided according to N staging,which is 39 cases of N0,N1 ~ 3 which is 21 cases.All patients with nasopharyngeal carcinoma pathological tissue sources were not receiving chemotherapy treatment.Fluorescence quantitative PCR miR-25 next to the NPC tissues and corresponding normal tissues analyzed with clinicopathological parameters in patients with nasopharyngeal carcinoma correlation.(2)miR-25 mimic,miR-25 inhibitor and miR-25 control was designed and synthesized,then three are separately transfected human NPC 5-8F cells,building miR-25 over-expression or inhibit the expression of 5-8F cells cell lines,then expression of miR-25 in the above-mentioned cells after transfection.Then after transfection proliferative activity of the above cells;clonogenic assay growth;invasion of assay cells.(3)MiR-25 target genes were screened using bioinformatics software.Western blot to detect transfected with miR-25 control,miR-25 mimic or miR-25 inhibitor of 5-8F cells RAGE expression of miRNA and protein.RAGE 3'UTR using site-directed mutagenesis RAGE 3'UTR Mut was constructed containing the luciferase reporter gene(mutant)plasmid vector,while the key structure RAGE 3'UTR WT(wild type)plasmid vector,respectively,the two miR-25 mimic co-transfected 5-8F cells.Using luciferase reporter system for detecting the expression of luciferase cells.Use si-RAGE RAGE expression construct inhibition 5-8F cell lines and expression of RAGE mRNA and protein in cells.The 5-8F cells were divided into 3 groups were detected by Transwell chamber Control group,miR-25 mimic groups and si-RAGE + miR-25 mimic cells invasive ability.Results(1)Fluorescence results showed that the nasopharyngeal carcinoma miR-25 expression was significantly higher than in the corresponding normal tissues adjacent cancering(P<0.05).miR-25 in patients with nasopharyngeal carcinoma clinicopathological parameters analysis showed that,TNM clinical stage miR-25 expression in patients with NPC's,T stage,N stage,but not regardless of gender and age of the patients.(2)Fluorescence results showed that the miR-25 mimic cells expression level was significantly higher(P <0.05)with the control group,miR-25 inhibitor cells expression was significantly reduced(P <0.05),showed that over-expression of miR-25 or inhibit the expression of 5-8F cells was constructed successfully.Test results showed that the transfected miR-25 mimic of 5-8F cells two days,three days,four days when the growth rate was significantly higher(P <0.05);transfected with miR-25 inhibitor 5-8F cells two days,three days,four days when the growth rate was significantly lower than the control group(P <0.05).The results show that the number of colony forming miR-25 mimic group 5-8F cells was significantly higher(P <0.05);miR-25 inhibitor group 5-8F cell colony formation was significantly lower than the control group(P <0.05).The results showed that,miR-25 mimic invasive group cells 5-8F cells were significantly higher(P <0.05).(3)The results show,RAGE is miR-25 target genes.Were transfected with miR-25 mimic after,5-8F cells,the expression of RAGE miRNA and protein was significantly increased(P <0.05);transfected with miR-25 inhibitor after,5-8F cells,the expression of RAGE mRNA and protein was significantly reduced(P <0.05).The results showed that comparing with the control group,miR-25 mimic RAGE 3'UTR WT co-transfected was significantly higher(P <0.05);but miR-25 mimic and RAGE 3' UTR Mut co-transfected cells is no significant difference(P> 0.05)compared with the control group.After expression 5-8F cells RAGE mRNA and protein was significantly decreased.Transwell chamber assay results showed that compared group,the invasion of cells miR-25 mimic cells increased significantly(P <0.05),while si-RAGE + invade cells miR-25 inhibitor group cells was significantly reduced(P <0.05).ConclusionsmiR-25 was highly expressed in nasopharyngeal carcinoma,and its expression level associated with TNM clinical stage,T stage,N stage in NPC patients.miR-25 to facilitate the invasion of nasopharyngeal carcinoma cells directly regulated by expression of RAGE,thereby contributing to the occurrence and development of NPC.