The Role And Mechanism Of Radioresistant Gene GOLPH3 In Mediateing Radiation-induced Bystander Effect

Radiation-induced bystander effect(RIBE)may be a potentially harmful event in radiotherapy(RT).There are increasing evidences indicating RIBE is tightly associated with the occurrence of secondary cancer.Although the direct damage in normal tissues is effectively reduced by using precise radiotherapy technique,RIBE is not inhibited.Furthermore,with the significant prolongation of survival time of cancer patients treated with RT,the potential health risks induced by RIBE raise concerns after RT.Therefore,developing the strategies to modulate RIBE will be indispensible.It is well known that radioresistance is one of the main obstacles limiting the therapeutic efficacy and prognosis of patients.In recent years,many radiosensitizers were developed and enhances RT-induced cancer cell death,thereby prolonging the survival time of the patients.Given that induction of secondary cancers is more obvious for long-term survivors,both improving tumor radiosensitivity and suppressing RIBE should been taken into account in the design of radiotherapy treatment plans.Some genes involved in both radioresistance and RIBE are the ideal targets for radiotherapy.Targeting these genes maybe lead to "killing two birds with one stone":it improves the efficacy of radiotherapy and reduces the occurrence of secondary cancer induced by RIBE.Increasing evidences suggest that some cytokines secreted from irradiated cells are involved in the intercellular signal transmission of RIBE.Inhibition of signaling molecules in protein secretion pathways maybe block the secretion of cytokines mediating RIBE.Meanwhile,some signaling molecules in protein secretion pathways were also reported to promote the radioresistance.Therefore,there may be some signaling molecules mediating both RIBE and radioresistanc in protein secretion pathways.In our study,a candidate gene set for involvement in radioresistance was generated by combining several methods,including screening based on a radioresistant cell line,screening based on database and searching literatures.Subsequently,6 candidate genes possibly involved in both radioresistance and RIBE were obtained by intersecting aforementioned radioresistance candidate gene set with protein secretion gene set in the Gene Ontology(GO)database.Furthermore,the role and molecular mechanism of GOLPH3,one of the 6 candidate genes,in RIBE was explored.The research results mainly include the following four aspects:(1)The radioresistant cell line was established by simulating the clinical conventional fractionated radiotherapy.We found that the radioresistant cell line was more malignant than the parent cell line,displaying accelerated proliferation,enhanced migration and anti-apoptosis ability and decreased background ROS level.In addition,compared with the parental cell line,the radioresistant cell line exhibited significantly elevated mitochondrial respiration and anaerobic glycolysis.(2)Combination of screening by mRNA sequencing based on the radioresistant cell line and the high-throughput data of tissue samples from GEO database,the collected genes in radiosensitivity regulation factors database(dbCRSR)and the related literatures,a candidate gene set containing 657 genes associated with radioresistance was generated.Furthermore,six potential genes(ANXA1,ERP29,HIF-1A,P2RX7,VGF and GOLPH3)possibly involved in both radioresistance and RIBE were obtained by intersecting the candidate gene set with the protein secretion gene set in GO database.(3)The role of GOLPH3 in the occurrence of RIBE was discovered.In the vitro model of RIBE,GOLPH3 knockdown significantly inhibited RIBE,and re-expression of GOLPH3 also restored the RIBE,confirming that GOLPH3 plays a key role in RIBE.In the mouse model of RIBE,GOLPH3 knockdown significantly inhibited the occurrence of RIBE in testis tissue,which suggested that GOLPH3 is involved in the occurrence of RIBE in vivo.(4)Molecular mechanism of GOLPH3-mediated RIBE was explored.Firstly,we found the secreted TNF-α was the intercellular signaling molecule which was involved in RIBE regulated by GOLPH3.Then our results suggested that GOLPH3 promoted the transcription of TNF-α in response to irradiation by activating the ERK1/2-EGR1 signal pathway,thereby increasing the secretion of TNF-α.In summary,we discovered that GOLPH3 promoted the radiation-induced release of TNF-α by ERK1/2-EGR-1-TNF-α axis,consequently triggering RIBE.In conclusion,we established a radioresistant cell line and found that the degree of malignancy of radioresistant cells was higher than the parent cells.Based on this radioresistant cell line,we obtained the radioresistance-related genes by mRNA sequencing.Combination of the results of mRNA sequencing,analysis of high-throughput data from the GEO database and the collected genes in dbCRSR and the related literatures,we constructed a candidate gene set associated with radioresistance.Then six potential genes possibly involved in both radioresistance and RIBE were obtained by intersecting the candidate gene set with the protein secretion gene set in GO database.Furthermore,we confirmed that GOLPH3,one of the six potential genes,was involved in the occurrence of RIBE.Mechanismly,it was found that GOLPH3 promoted radiation-induced TNF-α release by ERK1/2-EGR-1-TNF-αaxis,thereby inducing RIBE.Our findings provided a theoretical and practical basis for the further development of drugs targeting GOLPH3,which have dual functions in radiosensitization and suppression of RIBE.