| Glioma is one of the most common primary brain tumors,accounting for 74.6%of all malignant tumors of the brain and central nervous system(CNS).The treatment strategy for patients with glioma is mainly surgical resection combined with radiotherapy and adjuvant chemotherapy.At present,postoperative radiotherapy is widely recommended clinically,which can minimize the malignant progression of glioma and improve the survival of patients.Considering that the occurrence of drug resistance to radiotherapy and chemotherapy significantly weakens the therapeutic effect,the prognosis of glioma patients is poor,and the 5-year survival rate is about15%.Therefore,it is extremely urgent to explore the mechanism of glioma radiotherapy/chemotherapy resistance in order to relieve the pain of glioma patients and improve their quality of life.Transcriptional DNA synthesis binding template switching is a process of DNA replication repair after radiotherapy or chemotherapy,which protects cells from the adverse consequences caused by the continuous stagnation of replication fork.DNA damage induced proliferative nuclear antigen(PCNA)monoubiquitination recruits transcriptional synthesis polymerase to the stalled replication fork for pathological replication and plays an important role in post-replication repair.RAD18 is an E3 ubiquitin protein ligase,which can promote PCNA monobiquitin by forming e2-E3 complexes with RAD6 or HHR6A/HHR6 B.RAD18 deficiency can lead to hypersensitivity to a variety of DNA damage factors.It has been reported that RAD18 is significantly overexpressed in glioma cells,which reduces the radiation sensitivity of cells to ionization to a certain extent.However,the mechanism of RAD18 in glioma development and radiation resistance is still unclear.Based on this,this study explored the mechanism by which RAD18 regulates the occurrence of gliomas and participates in the body's resistance to radiation.Methods:1)Analysis of changes in the expression levels of RAD18 gene and protein in tumor tissues and cells: A total of 25 glioma tissues that received radiation therapy,10 glioma tissues that did not receive radiation therapy,and 15 normal brain tissues were included in the study.The study of human samples involved in the study was approved by the Ethics Committee of the China-Japan Union Hospital of Jilin University,and each subject of the study signed an informed consent.The obtained tumor tissues were detected by real-time quantitative polymerase chain reaction(RT-q PCR),western blot and immunohistochemical staining to detect the changes in the expression levels of RAD18 gene and protein in tumor tissues.Meanwhile,the expression changes of RAD18 gene and protein were verified in glioma cell lines(U251,U-87,A172,U-118,SHG-44).2)The effect of RAD18 on the survival of glioma cells: The human glioma cell U-118 and A172 were selected as the research object in the cell experiment,and the Lenti-RAD18 group,Lenti-NC group,sh-RAD18 group,For the sh-NC group model,the successful transfection was verified by western blot,and the transfection efficiency was detected.Then,the changes of cell proliferation ability after transfection were analyzed by CCK-8 method and clone formation assay;the changes of cell apoptosis were detected by flow cytometry.3)The effect of RAD18 on the function of glioma cells after radiotherapy:The above glioma cell lines were treated with 2,4,8 gy6MV-X photon beam irradiation respectively,and then the expression level of RAD18 protein in the cells was detected by western blot..Subsequently,CCK-8 kit and clone formation were used to detect the changes of cell proliferation ability after reflex therapy,and flow cytometry was used to detect the level of apoptosis.4)Analysis of the downstream regulatory mechanism of RAD18: RT-q PCR and western blot were used to detect the changes in the expression levels of p53 gene and protein in cells treated with the above.Meanwhile,the expression levels of cyclin(cyclin D1)and apoptosis-related protein(cleaved caspase-3)were detected by western blot.5)Nude mouse xenograft model to verify the effect of RAD18 overexpression on the proliferation of glioma after radiotherapy: To verify the effect of RAD18 on the malignant proliferation of xenograft tumors in nude mice,sh-RAD18 and sh-NC were infected to A172 glioma cells,respectively.Then,A172 cells were transplanted into the right armpit of nude mice,and the control group and the 2 Gy dose radiation treatment group were set.After 7 days of constructing the subcutaneously transplanted tumor model in nude mice,corresponding treatments were performed according to groups,2 Gy/time,3 times in total,and completed within one week.After 4 weeks,the animals were sacrificed,and the tumor tissues were isolated,and the long and short diameters of the tumor tissues were observed and measured.The changes of tumor volume and weight were observed,and the expression levels of RAD18 and p53 were detected by immunohistochemical staining.The expression levels of RAD18 gene and protein in tumor tissues were detected by RT-q PCR and western blot.In addition,the expression levels of proliferation and apoptosis-related proteins in tissues were detected by western blot.Results:1)The expression level of RAD18 is significantly up-regulated in gliomaThe gene and protein expression levels of RAD18 were significantly up-regulated in both glioma tissues and glioma cell lines.Meanwhile,the protein expression of RAD18 was significantly up-regulated in glioma cell lines after radiotherapy.The above results suggest that the significant up-regulation of RAD18 may be involved in the carcinogenesis of gliomas and in the radiation resistance of gliomas.2)The effect of RAD18 on the proliferation,apoptosis and radiosensitivity of glioma cellsBy constructing silencing and overexpression models,the effect of RAD18 on the proliferation and apoptosis of glioma cells was first investigated.The results of CCK-8 showed that compared with the control group,the cell proliferation ability in the overexpression RAD18 group(Lenti-RAD18)was significantly enhanced,at the same time,the cell proliferation ability in the silent group(sh-RAD18)was significantly weakened;flow cytometry results showed that compared with the control group,the apoptosis level in the Lenti-RAD18 group was significantly decreased,and the apoptosis level in the sh-RAD18 group was up-regulated;The results of the clone formation experiment showed that compared with the control group,the clone formation ability of the Lenti-RAD18 group was significantly increased,and the clone formation ability of the cells in the sh-RAD18 group was lower than that of the sh-NC group.After radiotherapy,the results of CCK-8 method and clone formation assay showed that when the dose of radiation therapy was 4 Gy,overexpression of RAD18 could promote the proliferation of glioma cells after radiotherapy.The results of flow cytometry showed that RAD18 could inhibit the glioma cells after radiotherapy.tumor cell apoptosis.It is suggested that RAD18 may be associated with the radiosensitivity of glioma cells.3)RAD18 down-regulates p53 expression and inhibits nuclear accumulationRT-q PCR and Western blot analysis of silenced/overexpressed RAD18 cells showed that the results of p53 expression showed that overexpression of RAD18down-regulated the expression of p53,while silencing of RAD18 significantly up-regulated the expression of p53.The effect of RAD18 on the expression of p53 in the cytoplasm and nucleus was further explored.The results showed that the expression level of p53 protein in the nucleus of the overexpression group of RAD18 was significantly down-regulated,while the expression level of p53 protein in the cytoplasm was significantly up-regulated.It is suggested that RAD18 may be involved in promoting the nuclear export of p53 and down-regulating the expression of p53.In addition,overexpression of RAD18 reduced the expression of cleaved caspase-3 and increased the expression levels of Bcl-2 and Cyclin D1.4)RAD18 regulates the expression of p53 and affects the malignant progression of xenografted tumors in nude miceThe results of the tumor-bearing experiment in nude mice showed that: compared with the control group,the tumor tissue in the sh-RAD18 group was significantly reduced,and the tumor tissue was also reduced to a certain extent after radiotherapy;little.It is suggested that sh-RAD18 can significantly reduce the weight and volume of tumor tissue.Immunohistochemical results showed that compared with the control group,the positive signal of RAD18 in the sh-RAD18 group was significantly reduced;compared with the radiotherapy group,the positive signal of RAD18 in the radiotherapy+sh-RAD18 group was significantly reduced.Proteins were extracted from tumor tissues of nude mice,and the expression levels of RAD18 and p53 in each histone were detected by western blot.The results showed that compared with the control group,the protein expression level of the sh-RAD18 group was significantly down-regulated;compared with the radiotherapy group,the protein expression of RAD18 in the radiotherapy+sh-RAD18 group was also significantly decreased.At the same time,the expression level of p53 protein in the sh-RAD18 group was significantly up-regulated compared with the control group;the protein expression level of p53 in the radiotherapy+sh-RAD18 group was also significantly up-regulated compared with the radiotherapy group.The above results suggest that RAD18 may affect the expression of p53 and affect the sensitivity of radiotherapy.It is suggested that RAD18 can regulate the expression of p53 and further play the role of signal transmission.Silencing RAD18 can up-regulate the expression of p53,promote the expression of cleaved caspase-3,inhibit the expression of Cyclin D1,and enhance the radiosensitivity of glioma.Conclusion:Our study found that RAD18 was highly expressed in glioma tissues and cell lines,and RAD18 was significantly upregulated after radiotherapy.Further studies have confirmed that RAD18 can regulate the expression of p53,promote the proliferation and inhibit apoptosis of glioma cell lines after radiotherapy.In vivo experiments have also confirmed that overexpression of RAD18 can significantly enhance the radiotherapy sensitivity of glioma,and the mechanism of action is also closely related to down-regulation of p53 expression.Based on the above results,we speculate that RAD18 plays a key role in glioma radiation resistance,which may provide a potential target for drug development to reduce glioma radiation resistance. |
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