Effect Of MicroRNA-34a On Radiotherapy Sensitivity Of Nasopharyngeal Carcinoma By Targeted Regulation Of Raf1

Background:Radiotherapy is the main treatment of Nasopharyngeal carcinoma(NPC)and prolonging patient survival,but the sensitivity of radiotherapy and the prognosis of radiotherapy exist individual differences.mi R-34a,as a tumor suppressor gene,has been shown to be closely related to the occurrence and development of a variety of malignant tumors,and affects tumor chemoradiotherapy sensitivity by regulating downstream genes involved in the proliferation,invasion and apoptosis of cancer cells.At present,there is no clear conclusion on the role of mi R-34a in NPC and the correlation of radiotherapy sensitivity.Raf1 is overexpressed in a variety of tumor tissues and is thought to be closely related to radiosensitivity of tumors.The bioinformatics software predicted that Raf1 might be one of a target genes of mi R-34a.Therefore,this study aims to explore the correlation between mi R-34a and radiotherapy sensitivity of NPC patients from clinical and cellular experiments,and whether mi R-34a targeting Raf1 is a molecular mechanism involved in the regulation of radiotherapy sensitivity,so as to provide certain reference for the formulation of personalized radiotherapy regiments and the study of prognostic molecular markers for NPC patients.Objective:To detect the expression of miR-34a in NPC patients with different radiosensitivity and analyze the correlation between mi R-34a and recurrence.To explore the effect of mi R-34a targeting Raf1 on the radiosensitivity of NPC cells CNE-2 at the cellular level.Methods:Blood samples were collected from 84 NPC patients treated in the department of radiotherapy of author’s hospital.The expression of mi R-34a in patients with radiotherapy sensitivity and radiotherapy tolerance was detected by q RT-PCR.Kaplan-Meier method was used to analyze the survival of NPC patients with different mi R-34a levels,and Cox regression analysis was used to analyze the correlation between mi R-34a and prognosis.mi R-34a expression was up-regulated in NPC cell lines CNE-2 after transfection with mi R-34a mimic,and the cells were radiated by2Gy radiation.Changes in proliferation,apoptosis,cell cycle and invasion ability of CNE-2 cells after treatment were detected by CCK8,flow cytometry and Transwell assay,and expressions of apoptotic proteins were detected by Western blot.The dual luciferase reporter gene was used to verify the targeting relationship between mi R-34a and Raf1.mi R-34a mimic and pc DNA3.1-Raf1 were co-transfected into CNE-2 cells to analyze the effects of up-regulation of mi R-34a and Raf1 on cell proliferation,apoptosis,migration ability and radiation sensitivity under 2Gyγradiation.Results:The expression of miR-34a was significantly higher in NPC patients with radiosensitive than that in patients with radiation tolerance.The relapse free survival time of patients with high mi R-34a was significantly lower than that of patients with low mi R-34a.The high expression of mi R-34a might be an independent protective factor for the prognosis and recurrence of NPC.Up-regulation of mi R-34a could inhibit the cell cycle process,blocked cells at G0/G1phase,inhibit the proliferation and invasion,promote apoptosis,and enhance the radiosensitivity of CNE-2 cells toγ-ray.mi R-34a negatively regulated Raf1.Overexpression of Raf1 could promote the cell at G0/G1phase transition to S phase,promote the proliferation and invasion,suppress the apoptosis in CNE-2 cells transfected with mi R-34a mimic,reduce the radiosensitivity of cells to a certain extent.The expression level of mi R-34a in radiotherapy sensitive NPC patients was significantly higher than that in radiotherapy tolerant patients(P<0.05).The relapsion-free survival of patients in the high mi R-34a group was significantly higher than that in the low mi R-34a group(χ~2=6.336,P=0.012).The high expression of mi R-34a is an independent protective factor for the recurrence of NPC.After the expression level of mi R-34a mimic was up-regulated in CNE-2 cells,the proliferation and cell cycle process of CNE-2 cells could be inhibited,cells were blocked at G0/G1phase,apoptosis was promoted and migration was inhibited.After up-regulation of mi R-34a on the basis of 2Gy radiation,the proliferation activity,cell cycle progression and migration ability of the cells were further inhibited,and the apoptosis of the cells was increased,the radiotherapy sensitivity of the cells was increased.Dual-luciferase reporter gene assay showed that mi R-34a can target and negatively regulate Raf1.Raf1 expression decreased after mi R-34a was up-regulated.Meanwhile,the proliferation,cycle process and migration ability of up-regulated mi R-34a and overexpressed Raf1 cells were higher,while the apoptosis rate was lower than that of CNE-2 cells up-regulated mi R-34a alone,which reversed the effect of up-regulation of mi R-34a on cells,that is,the radiotherapy sensitivity of CNE-2cells was reduced to a certain extent.Conclusion:miR-34a is closely related to radiotherapy sensitivity and prognosis of NPC patients.mi R-34a plays an anticancer role in NPC cells.Up-regulation of mi R-34a can inhibit the proliferation,cell cycle progression and invasion,promote apoptosis and enhance the sensitivity of radiotherapy of CNE-2 cells.The tumor suppressive effect of mi R-34a on NPC cells and the radiotherapy sensitization effect may be achieved by targeting negative regulation of Raf1.