Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standa...Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standard chemotherapeutic for GBM,but TMZ treatment benefits are compromised by chemoresistance.This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance.Methods:CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM.Western blot,realtime PCR,flow cytometry,and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1(RAD51AP1).Results:Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells.Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment.Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells.Knockdown of E2F1 increased sensitivity to TMZ.Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1,mediates TMZ resistance,and has a potential E2F1 binding site on the promoter.Knockdown of RAD51AP1 enhanced the sensitivity of TMZ;however,overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells.Furthermore,RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase(MGMT)expression.The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated,but not MGMT-unmethylated TMZ-treated GBM patients.Conclusions:Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment.RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair.Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.展开更多
Background:The DNA damage repair mechanism plays a crucial role in the occurrence and development of hepatocellular carcinoma(HCC),and RAD51-associated protein 1(RAD51AP1)has received increasing attention as an import...Background:The DNA damage repair mechanism plays a crucial role in the occurrence and development of hepatocellular carcinoma(HCC),and RAD51-associated protein 1(RAD51AP1)has received increasing attention as an important protein in the homologous recombination repair pathway.However,the role of RAD51AP1 and its molecular regulatory mechanism in HCC still need further investigation.Methods:We first analysed RAD51AP1 expression,functional enrichment and prognostic value in HCC.Then,the miRWalk,miRDB,and Encyclopedia of RNA Interactomes databases were used to predict the corresponding microRNAs and long noncoding RNAs of RAD51AP1,and their expression levels and prognostic value were analysed.Results:RAD51AP1 was upregulated in the majority of cancers include HCC.The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that RAD51AP1 was mainly involved in pathways related to the cell cycle and repair in HCC.Moreover,the expression level of RAD51AP1 was significantly correlated with T stage,pathologic stage,histologic grade and the level of alpha-fetoprotein.In addition,RAD51AP1 was an independent risk factor significantly and had a high predictive value in HCC.Based on ceRNA network,RAD51AP1 may be regulated by upstream MSC-AS1 and hsa-miR-23c to affect the HCC occurrence and development.Conclusions:High expression of RAD51AP1 plays an important biological role in the cell cycle and repair pathways,and has important diagnostic and prognostic value in HCC.Based on the regulatory mechanism of ceRNA network,we speculate that lncRNA MSC-AS1 acts on hsa-miR-23c and regulates DNA damage repair of HCC through RAD51AP1.It provides a new perspective for further study of DNA damage repair mechanism and potential related treatment of HCC.展开更多
基金supported by the Science and Technology Project of Tianjin Municipal Health Commission(Grant Nos.TJWJ2022MS003 and TJWJ2021ZD008)the Tianjin Science and Technology Plan Project(Grant Nos.21JCYBJC01520 and 20JCYBJC01070)。
文摘Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standard chemotherapeutic for GBM,but TMZ treatment benefits are compromised by chemoresistance.This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance.Methods:CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM.Western blot,realtime PCR,flow cytometry,and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1(RAD51AP1).Results:Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells.Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment.Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells.Knockdown of E2F1 increased sensitivity to TMZ.Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1,mediates TMZ resistance,and has a potential E2F1 binding site on the promoter.Knockdown of RAD51AP1 enhanced the sensitivity of TMZ;however,overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells.Furthermore,RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase(MGMT)expression.The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated,but not MGMT-unmethylated TMZ-treated GBM patients.Conclusions:Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment.RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair.Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.
基金the financial support from Shandong Traditional Chinese Medicine Science and Technology Project(No.2020M139)the Scientific Research Project of Shandong College of Traditional Chinese Medicine(No.2021FY02)the Development Plan of Laizhou Science and Technology Project(No.2022L01).
文摘Background:The DNA damage repair mechanism plays a crucial role in the occurrence and development of hepatocellular carcinoma(HCC),and RAD51-associated protein 1(RAD51AP1)has received increasing attention as an important protein in the homologous recombination repair pathway.However,the role of RAD51AP1 and its molecular regulatory mechanism in HCC still need further investigation.Methods:We first analysed RAD51AP1 expression,functional enrichment and prognostic value in HCC.Then,the miRWalk,miRDB,and Encyclopedia of RNA Interactomes databases were used to predict the corresponding microRNAs and long noncoding RNAs of RAD51AP1,and their expression levels and prognostic value were analysed.Results:RAD51AP1 was upregulated in the majority of cancers include HCC.The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that RAD51AP1 was mainly involved in pathways related to the cell cycle and repair in HCC.Moreover,the expression level of RAD51AP1 was significantly correlated with T stage,pathologic stage,histologic grade and the level of alpha-fetoprotein.In addition,RAD51AP1 was an independent risk factor significantly and had a high predictive value in HCC.Based on ceRNA network,RAD51AP1 may be regulated by upstream MSC-AS1 and hsa-miR-23c to affect the HCC occurrence and development.Conclusions:High expression of RAD51AP1 plays an important biological role in the cell cycle and repair pathways,and has important diagnostic and prognostic value in HCC.Based on the regulatory mechanism of ceRNA network,we speculate that lncRNA MSC-AS1 acts on hsa-miR-23c and regulates DNA damage repair of HCC through RAD51AP1.It provides a new perspective for further study of DNA damage repair mechanism and potential related treatment of HCC.
