Regulatory Effect And Mechanism Of TTC9C In Radiosensitivity Of Glioma

BackgroundGlioma was one of the major malignant tumors that threaten human health that occur in the neuroectodermal layer,which had highly invasion and proliferation.It was characterized by high morbidity,high postoperative recurrence rate,high mortality,and low cure rate.In addition,malignant glioma was difficult to be cured by surgery and was generally insensitive to radiotherapy and chemotherapy.At the same time,the existing radiosensitizers to malignant glioma had the disadvantages of poor sensitization effect and large toxic and side effects,and their radiosensitization effect was still controversial,so it cannot completely meet the needs of clinical glioma radiosensitization.Due to the difficulty of complete resection,insensitivity to chemotherapeutic drugs,significant radiation resistance,and lack of sensitizing drugs,the prognosis of patients with malignant glioma was still poor,and most tumors will still relapse and progress in the radiotherapy field.Therefore,it was of great clinical significance to find a new target for malignant glioma radiosensitization and further improve the radiotherapy effect.In the field of tumor radiotherapy,the mechanism of radiotherapy resistance had not been fully understood,the needs for new and more efficient radiosensitization genes was still urgent.In recent years,with the development of tumor biology and radiation oncology,targeted therapy for tumor radiotherapy sensitivity-related genes had become a research hotspot,and genome screening libraries were expected to screen more efficient tumor radiosensitization targets from the human genome.CRISPR Cas9 technology is a gene-targeting modification technology that has developed rapidly in recent years.The CRISPR Cas9 human genomic library constructed on this technology provided a new method for screening new targets for tumor radiosensitization.In this study,we used CRISPR Cas9 human genomic library to infect malignant human glioma cells,and through a high-throughput sequencing negative screening method,we discovered a new target for radiotherapy sensitization TTC9 C.The radiotherapy sensitization effect of TTC9 C was further verified at the cytology,zoology and clinical level,and the molecular mechanism was further explored.Contents and Method1.High-throughput screening of new targets for tumor radiosensitization by CRISPR Cas9 whole human genome library(1)Construction of candidate gene pool by negative screening of high-throughput second-generation sequencing:In this study,the optimal MOI value of human glioma cell U87 MG infected with the gene library was first determined,then U87 MG cells were infected with the library,and then treated with 6Gy gamma rays.Then high-throughput second-generation sequencing was performed on the two groups of cells,and candidate gene pools were constructed by negative selection.(2)Target gene determined:Next,we used different tumor-derived cell lines U87 MG,Hela,and A549,constructed knockdown cell lines of candidate genes,and further detected the radiotherapy sensitization effect of the candidate target gene by means of clone formation rate,apoptosis,and DNA damage repair pathway detection.The TTC9 C was selected as the target gene with the best radiosensitization effect.2.Research on the regulation effect of TTC9 C on the radiation sensitivity of glioma cellsWe used different glioma cell lines U87 MG and U251 MG from different backgrounds to construct TTC9 C knockout and over-expressing stably transformed cells,and further clarify its radiosensitization effect by tumor cell proliferation,migration,clone formation rate,apoptosis,cell cycle,immunofluorescence,DDR pathway detection,furthermore we explored the effect of TTC9 C on DNA damage repair.3.Research on the regulatory mechanism of TTC9 C on glioma radiation sensitivity(1)TTC9C recruitment and modification after irradiation:First,the DSBs foci method was used to detect the recruitment of TTC9 C at the DSBs spot after irradiation,and to determine whether it directly recruited to the DSBs spot to play a role.Next,the IP and Western Blot were used to test whether the TTC9 C protein had been correspondingly modified after irradiation.(2)Finding TTC9 C interaction molecules and clarifying their interaction sites:We determined whether the interaction proteins of TTC9 C were related to DNA damage repair by IP mass spectrometry,and then used Co-IP and Western Blot to determine its direct acting molecule is ATR.Next,by constructing different domain of ATR,we determined TTC9 C and ATR specific binding targets.4.Effect of TTC9 C on radiosensitivity of intracranial gliomas and clinical research(1)Regulation effect of TTC9 C on radiation sensitivity of intracranial gliomas:A radiotherapy model of intracranial glioma in nude mice was constructed,and the radiosensitization effect of TTC9 C knockout in intracranial gliomas was further clarified by means of survival rate observation,brain tissue anatomy,pathological section,and immunohistochemistry.Next,we determined the inhibitory effect of TTC9 C on HR repair pathway.(2)Clinical samples bioinformatics of TTC9C:By means of bioinformatics analysis,the expression characteristics and co-expression relationship of TTC9 C in tumor cells were further explored,and the biological functions of TTC9 C were further clarified through clinical sample information.ResultsThe main results of this study were as follows:1.Within the human genome,a new target for glioma radiosensitization,TTC9 C,was successfully screened;2.In vitro experiments,TTC9 C knockout played a significant radiosensitization effect in glioma cells:In this study we found that TTC9 C knockout can significantly inhibit the proliferation and migration of human glioma cells,reduce the cell viability and clone formation,promote apoptosis of human glioma cells after irradiation,and significantly reduce the human brain HR repair of DNA damage in glioma cells after irradiation;3.TTC9 C was combined with ATR and directly participates in HR repair of DNA damage after irradiation:We found that TTC9 C undergoes phosphorylation modification after irradiation and directly recruits to DNA strand break spots and participate in DNA repair.At the same time,TTC9 C can directly combine with ATR to participate in HR repair,and its binding site was located at N side of ATR;4.In vivo experiments,TTC9 C knockdown played a significant role in radiosensitivity of intracranial glioma radiotherapy,and we further confirmed that TTC9 C played an important role in the occurrence and development of glioma:In vivo experiments,we found that TTC9 C knockout significantly prolonged the survival time of glioma radiotherapy mice,delayed the proliferation,promoted apoptosis and inhibited HR repair pathway activation of intracranial gliomas after irradiation.In addition,through bioinformatics analysis,we found that the expression level of TTC9 C significantly affected the prognosis of tumor patients,and TTC9 C has significant co-expression relationship with related molecules of DNA damage repair.ConclusionIn this study,we discovered a new target for glioma radiosensitization TTC9 C,it can directly bind to ATR,inhibit HR repair of DNA damage caused by ionizing radiation,and play a significant radiosensitization effect in human glioma cells.Therefore,we believe that TTC9 C has great potential application value in human glioma radiotherapy.