The Role And Mechanism Of RPA3 In Promoting Proliferation,Migration And Invasion Of Glioma By Activating PI3K-AKT-mTOR Pathway

Glioma is the most common primary malignant brain tumor,which can occur in any parts of the central nervous system,with the characteristics of invasive growth,high mortality and disability,short survival and easy recurrence.Although surgery and postoperative radiotherapy and chemotherapy can prolong the survival time of patients,the prognosis of most glioma patients is still very poor,and the exact molecular mechanism of glioma formation is not clear.In recent years,as the molecular genetics of tumor has become a research hotspot,the specific gene targeting therapy of glioma provides a new direction for the treatment of glioma.Replication protein A(replication protein A,RPA)is a highly conserved multi-subunit singlestranded DNA binding protein complex,which consists of p70(RPA1),p34(RPA2)and p14(RPA3).It is mainly responsible for regulating DNA replication,cell cycle checkpoint and DNA recombination.Its defects can lead to genomic instability,gene mutation and chromosome mismatch,and induce cancer.The expression level of RPA3 protein in a variety of malignant tumors has become an important prognostic indicator,but the role of RPA3 in glioma is not clear.The purpose of this study is to study the effect of RPA3 on the proliferation,migration and invasion of glioma cells and its possible mechanism,and to provide a theoretical basis for the study of potential prognostic biomarkers and new therapeutic targets of glioma.Part One The expression of RPA3 in glioma tissues and cells and its correlation with survival time of patientsObjective: to analyze the expression of RPA3 in glioma tissues and cells,and detect the correlation between the expression of RPA3 and the survival time of glioma patients.Methods:1.The database of Hiplot platform was used to analyze the difference of RPA3 m RNA expression between glioma and normal tissues.2.The correlation between the expression level of RPA3 and the survival time of glioma patients was analyzed by using multiple databases of Gliovis website,and survival analysis and meta-analysis were carried out.3.The tumor samples of 30 glioma patients treated in Baoding First Central Hospital from January 2021 to November 2021 were analyzed,including 7 cases of WHO II grade,13 cases of WHO III grade and 10 cases of WHO IV grade.8 non-tumor brain tissue samples(taken from nonfunctional areas of patients undergoing decompression of craniocerebral injury)were selected.Glioma and non-tumor brain tissue samples were collected immediately after resection and preserved in liquid nitrogen.For later use.Two neuropathologists confirmed the histological diagnosis of each specimen according to World Health Organization(WHO)guidelines.RTPCR and immunohistochemistry were used to detect the expression of RPA3 in glioma tissues.4.The expression of RPA3 in glioma cell lines(U87,U251,A172)and human astrocytes(HA)was detected by Western Blot and RT-PCR.Results:1.In the database of Hiplot platform,the expression of RPA3 m RNA in low-grade gliomas(Low-grade gliomas,LGG)and high-grade gliomas(glioblastomas,GBM)was significantly higher than that in normal brain tissues.2.In the nine databases on the Gliovis website,in six databases,the survival time of patients with high expression of RPA3 was significantly lower than that of patients with low expression.There was no statistical significance between the expression of RPA3 and the survival time of patients in three databases.Meta-analysis of risk ratio(Hazard ratio,HR)of comprehensive data set showed that glioma patients with high expression of RPA3 had shorter survival time(Overall Survival,OS)than those with low expression.3.The expression of RPA3 m RNA in glioma tissue was significantly higher than that in control brain tissue,and the average expression level of RPA3 m RNA in GBM was higher than that in LGG group.4.The expression and protein levels of RPA3 m RNA in glioma cell lines(U87,A172,U251)were significantly higher than those in normal astrocytes.Conclusions:1.The expression level of RPA3 m RNA in glioma was significantly higher than that in normal brain tissue,and the expression level of RPA3 m RNA in GBM was higher than that in LGG.2.The expression level of RPA3 m RNA in glioma cells was significantly higher than that in normal astrocytes.3.The expression level of RPA3 is closely related to the survival time of glioma patients: the survival time of RPA3 high expression group is lower than that of low expression group,so RPA3 may be a potential new target for glioma therapy.Part Two Effects of RPA3 on proliferation,migration and invasion of glioma cellsObjective: To investigate the effect of RPA3 on the proliferation,migration and invasion of glioma cells.Methods:1.Small interference RNA(Small interfering RNA,si RNA)and RPA3 overexpression plasmids were used to knock down and up-regulate the expression of RPA3 in U251 glioma cell lines.They were divided into two groups: knock-down control group(si NC group)and knock-down RPA3group(si RPA3 group),empty plasmid group(pc NC group)and overexpression RPA3 group(pc RPA3 group).The expression of RPA3 protein in cells was detected by Western Blot and RT-PCR techniques.2.The effect of RPA3 on the proliferation of glioma cells was analyzed by cell proliferation test(CCK-8 test).3.The effect of RPA3 on the migration and invasion of glioma cells was analyzed by Transwell test and cell scratch test.Results:1.In U251 glioma cell line,the levels of RPA3 m RNA and protein in si RPA3 group were significantly lower than those in si NC group,while the levels of RPA3 m RNA and protein in pc RPA3 group were significantly higher than those in pc NC group(P < 0.01).2.RPA3 could significantly promote the proliferation,migration and invasion of glioma cells,and the inhibition of RPA3 expression could significantly inhibit the proliferation,migration and invasion of glioma cells.Conclusions: RPA3 can promote the proliferation,migration and invasion of glioma cells,and play a certain role in the carcinogenesis of gliomas.Part Three Effects of RPA3 on proliferation,migration and invasion of glioma cells through PI3K-AKT-mTOR pathwayObjective: To analyze the possible regulatory mechanism of the effect of RPA3 on the proliferation,migration and invasion of glioma cells.Methods:1.Through the bioinformatics analysis website,gene enrichment analysis(Gene set enrichment analysis,GSEA)and gene set variation analysis(Gene Set Variation Analysis,GSVA)were used to predict the signal pathways related to RPA3 promoting the occurrence and development of glioma.2.In U251 glioma cell lines with down-regulated and up-regulated RPA3 expression after transfection,Western Blot was used to detect the level of AKT/p-AKT,mTOR/p-mTOR,a protein related to PI3K-AKT-mTOR signaling pathway,in glioma cells after changing RPA3 expression.Results:1.GSEA and GSVA analysis showed that PI3K-AKT-mTOR signal pathway was significantly enriched in glioma related pathways,and the activation of this pathway was positively correlated with the expression of RPA3.2.The results of Western Blot showed that the level of AKT/p-AKT,mTOR/p-mTOR in glioma cells increased significantly after overexpression of RPA3,but decreased significantly after silencing RPA3,and the difference was statistically significant(P < 0.01).Conclusions: RPA3 can promote the proliferation,migration and invasion of glioma cells by activating PI3K-AKT-mTOR pathway.Therefore,we think that RPA3 may become a new target for glioma therapy in the future.