The Role And Mechanism Of NF-?B-YY1-miR-103a Pathway In Radioresistance Of Glioblastoma

Background Gliomas are the most common malignant tumors in the central nervous system,glioblastoma(GBM,WHO IV)accounts for about 54% of gliomas.GBM has the characteristics of easy recurrence and strong invasiveness,and the median survival time is only 12-15 months.Due to the invasive growth of GBM,surgery is often difficult to completely remove the tumor.Radiotherapy plays an extremely important role in the treatment of GBM,postoperative radiotherapy can effectively kill residual tumor cells and prolong the progression-free survival and overall survival of patients.For GBM patients which can not be resected surgically,radiotherapy can also be used to inhibit the growth of tumor cells,improve the clinical symptoms and quality of life of patients.The damage of brain function caused by radiotherapy is also lower than that of surgery.However,compared with other types of glioma patients,GBM patients often have poor radiotherapy effects.A large number of literature studies have shown that GBM radioresistance may be related to DNA damage repair,glioma stem cells(GSCs),autophagy and tumor microenvironment.Therefore,systematic elucidation of the pathological mechanism involved in radiotherapy resistance of GBM has important clinical significance in improving radiotherapy sensitivity and inhibiting recurrence of GBM patients.Objective Searching for key molecules and related pathways involved in radiotherapy resistance of GBM and elucidate its mechanism,so as to provide potential drug targets for enhancing radiotherapy sensitivity and reversing radiotherapy resistance of GBM patients.Method 1.Firstly,a radioresistance model which can simulate clinicopathological characteristics of GBM patients was constructed by using Patient-Derived tumor Xenograft(PDX)model,and the radioresistance cell subset was isolated;2.The biological functions of radiation-tolerat persister(RTP)cells were identified by CCK-8,colony formation,3D invasion,scratch and RNA sequencing experiments;3.Differential screening of mi RNAs in radioresistant PDX model showed that mi R-103 a was significantly down-regulated,and the expression of mi R-103 a was verified in RTP cells and TCGA data;4.The functional and molecular mechanisms of mi R-103 a in RTP cells were elucidated by bioinformatics,Western Blot,dual luciferase reporter,single cell gel electrophoresis and stem cell sphere formation;5.The molecular mechanism of down-regulation of mi R-103 a in RTP cells was elucidated by bioinformatics,epigenetics,transcription factor microarray,chromatin immunoprecipitation and other techniques;6.The function of NF-?B-YY1-mi R-103 a pathway in RTP cells was confirmed by CCK-8,colony formation,immunofluorescence,single cell gel electrophoresis and stem cell sphere formation;7.The correlation of NF-?B-YY1-mi R-103 a pathway with radiotherapy sensitivity and recurrence was evaluated by immunohistochemistry and in situ hybridization of mi R-103 a in clinical GBM samples;8.Nanoparticles targeting GBM were prepared from mi R-103 a and their effects on radiosensitivity were validated in orthotopic GBM model.Result 1.We have successfully constructed a radioresistance model of GBM which can reflect the clinicopathological characteristics,and confirmed that radioresistance cell subsets have stronger malignant phenotypes;2.DNA damage repair and GSCs self-renewal pathway changed most significantly in RTP cells;3.Differential screening of mi RNAs revealed that mi R-103 a were significantly down-regulated in RTP cells,and we confirmed the accuracy of the screening results in RTP cells which formed by various cell lines.TCGA database also confirmed that mi R-103 a were low expressed in GBM;4.Overexpression of mi R-103 a can enhance the radiosensitivity of RTP cells.Further studies have found that mi R-103 a can directly regulate the expression of XRCC3 and FGF2.On the one hand,XRCC3 can enhance the DNA damage repair ability of GBM cells,on the other hand,FGF2 can promote the self-renewal of GSCs,thus,the dual regulation of mi R-103 a on DNA damage repair and self-renewal of GSCs in RTP cells is formed;5.NF-?B and YY1 are the transcription factors which have been significantly activated in RTP cells.Bioinformatics analysis showed that YY1 binding sites existed in the promoter region of mi R-103 a,and YY1 could inhibit the transcription of mi R-103 a,further studies have found that activated NF-?B can negatively regulate the expression of mi R-103 a by promoting transcriptional activation of YY1;6.NF-?B-YY1-mi R-103 a pathway mediates the function of RTP cells in radioresistance by affecting DNA damage repair and GSCs self-renewal;7.Immunohistochemical and in situ hybridization results of GBM samples showed that P65,YY1,FGF2 and XRCC3 were significantly increased in recurrent GBM patients,while mi R-103 a was significantly decreased in recurrent GBM patients.Spearman correlation analysis showed that the expression level of mi R-103 a was negatively correlated with FGF2,XRCC3,YY1 and p65,while p65 was positively correlated with YY1,FGF2 and XRCC3,and YY1 was positively correlated with FGF2 and XRCC3.The above correlation analysis further confirmed the existence of NF-?B-YY1-mi R-103 a pathway in GBM patients;8.Transferrin-modified mi R-103 a nanoparticles can target orthotopic GBM model through blood-brain barrier and enhance its radiosensitivity.Conclusion We successfully constructed a PDX radioresistance model that can simulate clinicopathological characteristics and confirmed the existence of NF-?B-YY1-mi R-103 a regulatory pathway in RTP cells.Radiotherapy can induce the continuous activation of NF-?B.Activated NF-?B can promote the transcriptional activation of YY1,YY1 up-regulates the expression levels of XRCC3 and FGF2 through the negative regulation of mi R-103 a,XRCC3 enhances the ability of DNA damage repair,and FGF2 enhances the self-renewal ability of GSCs,which ultimately leads to the radioresistance of RTP cells.The confirmation of this pathway and elucidation of its molecular mechanism provide potential drug targets for enhancing radiosensitivity of GBM and reversing radioresistance.