The Function Of MIIP Gene To Enhance The Radiosensitivity Of Nasopharyngeal Carcinoma

Objective:To explore the expression of MIIP and its clinical significance in nasopharyngeal carcinoma.To detect the biological behavior,expression of apoptosis-related genes and changes in ? H2 AX focus of in overexpressed MIIP cells in nasopharyngeal carcinoma after irradiation,and to explore the possible mechanism of MIIP gene in radiation enhancement of nasopharyngeal carcinoma.Methods: 1.MIIP gene and expression data were retrieved from the bioinformation databases and the relationship between MIIP and prognosis of head and neck cancer patients was retrieved from an online survival analysis tool “Kaplan-Meier Plotter” database.2.Using immunohistochemistry to detect MIIP expression of NPC(n=87,year 2014-2018)paraffin specimens,and to analyze its relationship with clinical TNM stages.3.Adenovims-MIIP was used to overexpress MIIP in 5-8F and CNE2 cells.The changes in radiosensitivity of the cells were analyzed by colony formation assay.The changes in cell apoptosis and cycle distribution following irradiation were detected by flow cytometry.4.5-8F and CNE2 cells were treated with gradient irradiation..The changes in radiosensitivity of the cells were analyzed by colony formation assay.The changes in cell apoptosis and cycle distribution following irradiation were detected by flow cytometry.5.The expression of BCL2 associated X,apoptosis regulator/B-cell lymphoma 2 was evaluated using western blot.The expression levels of related proteins at different radiation doses and time points were analyzed.6.DNA damage was analyzed by counting ?-H2 AX foci.The expression levels of ?-H2 AX were evaluated by immunofluorescence and western blot.The expression levels of at different radiation doses and time points were analyzed?-H2 AX.Results: 1.MIIP was moderately expressed in normal nasopharyngeal tissues in the HPA database,while MIIP was negatively expressed in tumor tissues of nasopharyngeal carcinoma.Only one study(41samples)were involved MIIP in NPC tissues and normal tissues.MIIP expression levels in NPC was lower than that in normal tissues(P<0.05).Kaplan-Meier Plotter of survival analysis indicated that low expression of MIIP was not associated with survival in head and neck cancer,including overall survival(HR=0.79,95%CI:0.6-1.06,P=0.11),relapse-free survival(HR=1.87,95%CI:0.71-4.93,P<0.2).Low expression of MIIP was only found in female patients with head and neck cancer,and the difference between total survival and high expression was significant(HR=0.48,95%CI: 0.26-0.87,P=0.013).2.Among 87 nasopharyngeal carcinoma specimens,23 cases of MIIP were weakly positive(positive rate: 26.44%).MIIP gene expression was correlated with T stage(P=0.004),N stage(P=0.010)and TNM stage(P=0.015),but not with age(P=0.659)and gender(P=0.544).3.A dose-dependent decrease in survival occurred in 5-8F and CNE2 cells following irradiation(0,2,4,6 and 8 Gy).These results confirmed that the overexpression of MIIP was able to suppress the growth of NPC cells following irradiation.4.The MIIP gene overexpression groups exhibited markedly higher apoptotic rate compared with the negative control groups in the absence of IR.Following radiation with 6 Gy,the apoptotic rates of the MIIP gene overexpression groups were also significantly higher compared with that of the negative control groups(P<0.05).5.In the absence of IR,no significant difference was observed between the cell cycle profiles of cells in the MIIP gene overexpression and control groups.By contrast,the percentage of cells in the MIIP gene overexpression group was markedly higher in the G2/M phase compared with the control cells at 24 h following 6 Gy irradiation(P<0.01).The overexpression of MIIP gene enhanced the G2/M cell cycle arrest that was induced by IR.6.The 5-8F OE and CNE2 OE cells exhibited notably higher Bax expression and considerably lower Bcl-2 expression compared with 5-8F NC and CNE2 NC cells.The upregulation of Bax expression is more pronounced(P<0.01)when irradiated as well as the downgrade of Bcl-2 expression(P<0.01).7.The 5-8F OE and CNE2 OE cells exhibited slower decay of ?-H2 AX foci following irradiation compared with 5-8F NC and CNE2 NC cells.In addition,5-8F OE and CNE2 OE groups exhibited higher levels of ?-H2 AX compared with 5-8F NC and CNE2 NC,respectively.MIIP gene was able to increase the induction and persistence of IR-induced ?-H2 AX foci.Conclusion: 1.The bioinformation databases found that the expression of MIIP in nasopharyngeal carcinoma was lower than that in normal tissues,and the low expression of MIIP in head and neck tumors may be related to tumor staging and prognosis.2.The low expression level of MIIP in NPC tissues was correlated with T staging,N staging and TNM staging of NPC,which may be an independent staging factor of NPC.3.Overexpression of MIIP gene inhibits the proliferation of nasopharyngeal carcinoma cells,promotes apoptosis,and causes cell arrest in G1 phase and reduces the proportion of cells in S phase.MIIP gene is a tumor suppressor gene,and its deletion will induce tumorigenesis.4.MIIP gene and its products can increase radiosensitivity in nasopharyngeal carcinoma cells.Most cells are blocked in G2/M phase,and MIIP may be an important candidate gene for improving curative effect of radiotherapy.5.The overexpression of MIIP and irradiation increased cell apoptosis by activating the Bax/Bcl-2 signaling pathway in NPC cells,which is one of the potential underlying mechanisms of radiosensitization.6.The overexpression of MIIP may increase the number of ?-H2 AX foci and slow down its decay.It was indicated that MIIP expression is associated with the radiosensitivity of NPC cells and has a significant role in regulating cell radiosensitivity.