The Role Of DNMT3B In The Acquired Radioresistance Of Nasopharyngeal Carcinoma

Part I The correlation between the expression of DNMT3 B and the clinicopathological characteristics and prognosis of nasopharyngeal carcinoma Objective: To explore the relationship between the expression of DNMT3 B and the clinicopathological characteristics of nasopharyngeal carcinoma and its effect on the prognosis of patients with nasopharyngeal carcinoma.Methods: Analyze the difference of DNMT3 B expression between nasopharyngeal carcinoma tissues and normal nasopharyngeal tissues in the Gene Expression Omnibus(GEO)public database;Considering the limitations of the small sample size of nasopharyngeal carcinoma in the GEO database and lack of specific NPC samples in The Cancer Genome Atlas(TCGA),we further analyze the TCGA database to explore the difference of DNMT3 B expression between head and neck squamous cell carcinoma(HNSCC)tissues and normal head and neck tissues.Using tissue microarray to analyze the correlation between the expression of DNMT3 B and clinicopathological characteristics;Kaplan-Meier plot was used to analyze the effect of DNMT3 B expression on overall survival(OS)of patients with nasopharyngeal carcinoma.Results: We first analyzed the data from the GEO database(GSE12452 and GSE40290)and found that DNMT3 B m RNA expression was upregulated in nasopharyngeal carcinoma samples compared to normal nasopharyngeal epithelial samples.TCGA database analysis showed that DNMT3 B m RNA expression was higher in head and neck squamous cell carcinoma samples compared with normal tissues.We further isolated 40 paired samples from HNSCC,including tumors and adjacent non-tumor tissues with detailed patient information,showing the similar results.To corroborate these results above,then we conducted tissue microarray analysis to investigate the expression of DNMT3 B protein in nasopharyngeal carcinoma.The results showed that the expression of DNMT3 B was mainly located in the nuclei of nasopharyngeal carcinoma cells.High expression of DNMT3 B was observed in 92/132(69.7%)nasopharyngeal carcinoma samples,while the low expression of DNMT3 B was observed in 40/132(30.3%).The expression of DNMT3 B was not related to age,histological classification or lymph node stage,but was significantly related to gender(P = 0.029),distant metastasis(P = 0.004)and clinical stage(P = 0.006).In addition,Kaplan-Meier analysis showed that the expression of DNMT3 B was significantly correlated with the overall survival of patients with nasopharyngeal carcinoma.The overall survival of patients with high expression of DNMT3 B in nasopharyngeal carcinoma was shorter than that in patients with low expression of DNMT3B(P = 0.0205),indicating that high expression of DNMT3 B predicted a poor prognosis in patients with nasopharyngeal carcinoma.The expression level of DNMT3 B has nothing to do with the disease-free survival rate of patients with nasopharyngeal carcinoma.In addition,from the TCGA data,we found that although there was no significant relationship between DNMT3 B and overall survival in patients with head and neck squamous cell carcinoma,the 5-year overall survival rate of patients with low levels of DNMT3 B was significantly higher than that of patients with high levels of DNMT3B(51.9% vs 42.6%).Conclusion: DNMT3 B is highly expressed in patients with nasopharyngeal carcinoma and is closely related to the poor prognosis of patients with nasopharyngeal carcinoma.Part II The effect of DNMT3 B on the radiosensitivity of nasopharyngeal carcinoma cells.Objective: To detect the effect of silencing of DNMT3 B on the radiosensitivity of nasopharyngeal carcinoma cells.Methods: Clone formation assay was used to detected the difference in radiosensitivity between NPC cell line CNE2 and NPC radioresistance cell line CNE2-R,and verified the successful construction of CNE2-R cell line.Immunoblotting experiment was used to detect the difference of DNMT3 B expression between CNE2 and CNE2-R.Immunoblotting and q RT-PCR experiment were used to detect the expression changes of DNMT3 B,DNMT3A and DNMT1 protein and m RNA levels in various NPC cell lines(CNE2,HNE1,CNE1,and CNE1-LMP1)after 6Gy irradiation at different time points(0,6,12,24 and 48h).The basic expression levels of DNMT3 B in the four NPC cell lines were tested by Immunoblotting.Four lentiviruses,Vector,sh DNMT3B#1,sh DNMT3B#2,and sh DNMT3B#3 were transfected into CNE2 and HNE1,respectively.Immunoblotting and q RT-PCR were used to detect their transfection efficiency.The effect of silencing of DNMT3 B on the proliferation and radiosensitivity of NPC cells was detected by clone formation experiment.The scratch and Transwell assays were applied to detect the effect of silencing DNMT3 B on the migration and invasion ability of NPC cells.Immunoblotting and q RT-PCR experiments were also applied to detect the protein and m RNA expressions of E-cadherin,N-cadherin and Vimentin after silencing of DNMT3 B.Results: Based on the principle of dose gradient increasing,the survival rate of CNE2-R constructed was significantly higher than that of CNE2,indicating that CNE2-R was successfully constructed,and DNMT3 B was highly expressed in CNE2-R.Immunoblotting and q RT-PCR found that after 6Gy irradiation,the m RNA and protein levels of DNMT3 B in each NPC cell line gradually increased with time,while the responses of DNMT1 and DNMT3 A to radiation at the m RNA and protein levels were inconsistent in the different cell lines.It was speculated that DNMT3 B may be related to the radioresistance of NPC.Compared with CNE1 cell line,DNMT3 B was highly expressed in CNE2,HNE1 andCNE1-LMP1.CNE2 and HNE1 were selected for lentivirus transfection to silencing DNMT3 B to investigate the role of DNMT3 B in NPC.Immunoblotting and q RT-PCR showed that sh DNMT3B#3 showed the best transfection efficiency,so we chose CNE2-sh DNMT3B#3 and CNE2-Vector,and HNE1-sh DNMT3B#3 and HNE1-Vector cell lines for the next experiment.The clone formation experiment found that after silencing of DNMT3 B,the proliferation of CNE2-sh DNMT3B#3 and HNE1-sh DNMT3B#3 was inhibited compared to the control group.Combining different doses of radiation(0,2,4,6,8,10Gy),silencing of DNMT3 B increased the radiosensitivity of NPC cells.The scratch experiment and Transwell experiment found that silencing of DNMT3 B inhibited the migration and invasion ability of NPC cells.Immunoblotting and q RT-PCR were used to detect the expression of EMT-related markers and found that after silencing of DNMT3 B,the epithelial marker protein E-cadherin increased,while the mesenchymal marker proteins N-cadherin and Vimentin decreased,especially in HNE1 cell line.The m RNA level of EMT markers showed the same tendency.The results indicated that silencing of DNMT3 B could inhibit EMT of NPC cells.Conclusion: Radiation can induce the expression of DNMT3 B in NPC,leading to the acquired radioresistance of NPC.Silencing of DNMT3 B can enhance the radiosensitivity of NPC cells.In addition,after silencing of DNMT3 B,E-cadherin increased,while N-cadherin and Vimentin decreased,especially in HNE1,thereby inhibiting the occurrence of EMT of NPC,which in turn inhibited migration and invasion.Part III The study on the mechanism of regulation of DNMT3 B in the radiosensitivityof nasopharyngeal carcinoma cellsObjective: To study the mechanism of DNMT3 B regulating the radiosensitivity of nasopharyngeal carcinoma cellsMethods: Flow cytometry was used to detect the cell cycle distribution of CNE2 and HNE1 cell lines after silencing DNMT3 B.Immunoblotting was used to detect the expression of cell cycle-related proteins p53,p21 and Cyclin D1 after silencing DNMT3B;q RT-PCR was also applied to detect the m RNA expression of p53 and p21 after silencing DNMT3 B.The apoptosis of CNE2 and HNE1 cell lines was detected by Flow cytometry after silencing DNMT3B;Immunoblotting was used to detect the expression of apoptotic and anti-apoptotic proteins Bax,Bcl-2 and Cleared-caspase3 after silencing DNMT3 B.The STING database was applied to analyze whether there was a direct(physical)or indirect(functional)relationship between DNMT3 B and p53 and p21.Methylation-specific PCR(MSP)and Bisulfite sequencing PCR(BSP)were used to detect the changes of methylation levels of p53 and p21 promoter regions after silencing DNMT3 B.After treatment with 6Gy,flow cytometry was used to detect the apoptosis rate together with silencing of DNMT3 B,further proving the relationship between DNMT3 B and radiosensitivityResults: Flow cytometry showed that silencing of DNMT3 B could block cell cycle of NPC cells in G1 phase.Immunoblotting detection found that cell cycle-related proteins p53 and p21 were significantly up-regulated while Cyclin D1 was significantly down-regulated;P53 and p21 m RNA expressions were also significantly up-regulated.While silencing DNMT3 B,the apoptosis rate increased significantly,and the apoptotic protein Bax was up-regulated,the anti-apoptotic protein Bcl-2 was significantly down-regulated.In addition,the expression of Cleaved-caspase3 also increased.STING database analysis further found that DNMT3 B was expected to regulate p53 and p21 through epigenetic modification.We designed multiple pairs of primers for MSP and BSP in the p53 and p21 promoter regions.MSP and BSP detection showed that after silencing DNMT3 B,the p53 and p21 promoter regions were demethylated,resulting in increased expression of p53 and p21,thus restoring and activating p53/p21 pathway.Flow cytometry further confirmed that silencing of DNMT3 B could significantly increase the radiation-induced apoptosis,thereby increasing the radiosensitivity.Conclusion: Silencing of DNMT3 B can restore and activate the p53/p21 signaling pathway through DNA demethylation,leading to blockage of the G1 phase,which further promotes apoptosis and thereby improves radiosensitivity.Therefore,DNMT3 B can be a potential target for radiosensitization of NPC.Part IV The effect of DNMT3 B on the radiosensitivity of nasopharyngeal carcinoma cells in vivoObjective: To explore the effect of DNMT3 B on the radiosensitivity of nasopharyngeal carcinoma cells in vivo with nude mice transplanted with tumorsMethods: The nude mice xenograft model was established and randomly divided into 4 groups,namely CNE2-Vector group,CNE2-sh DNMT3B#3 group,CNE2-Vector + radiotherapy group and CNE2-sh DNMT3B#3 + radiotherapy group.There are 6 mice in each group.The irradiation group was irradiated with 8Gy radiation,and the time of tumor onset and tumor volume were recorded.After 18 days,nude mice were sacrificed,and the tumor was taken out and weighed.The expression of related proteins DNMT3 B,p53,p21 and Cleared-caspase3 were detected by immunohistochemistry.Results: All the nude mice in 4 groups formed tumors,and the average tumor formation time was 3 days.Considering that the NPC cell line is sensitive to radiation,the nude mice in the radiotherapy group were given local 8Gy radiation after 12 days.Compared with the CNE2-Vector group,the tumors in the CNE2-sh DNMT3B#3 group were smaller and the growth rate was lower,and the tumor weight showed the same trend.After 8 Gy irradiation,the tumor growth rate of the irradiated group was significantly lower than that of the non-irradiated group.It is worth mentioning that the tumor size in the irradiated CNE2-sh DNMT3B#3 group was significantly smaller than that in the irradiated CNE2-Vector group,suggesting that silencing of DNMT3 B could increase the radiosensitivity of NPC cells in vivo.Immunohistochemistry of tumor tissue showed that silencing of DNMT3 B increased p53 and p21 expression.And the apoptosis protein Cleaved-caspase3 was also significantly increased.Conclusion: After silencing of DNMT3 B,it can activate the p53/p21 signaling pathway at the animal level,promote cell apoptosis,and then increase the radiosensitivity of NPC cells in vivo.