The Regulatory Mechanism Of PP2A As The Key Rate-limiting Enzyme Of The APE1/DNA-PKcs/hnRNPA1 Regulatory Axis In Telomere Protection And Its Clinical Value

Telomeres are unique structures at the ends of chromosomes,which can protect chromosomes and maintain genome stability.The telomere length in tumor cells remains stable during mitosis,which is one reason why tumor cells escape senescence and program death and continue to increase.Research evidence in recent years has shown that heterogeneous nuclear ribonucleoprotein A1(hnRNPA1)has a critical telomere function at the end of tumor cell chromosomes.In addition to helping telomerase maintain the effective length of telomeres,it can also help the replication protein A(RPA)dissociate from the telomere single-stranded DNA,thereby promoting the protection of telomere protein 1(POT1)to wrap the telomere single-stranded DNA.It also effectively avoids the exposure of the sticky ends of telomere DNA and the abnormal activation of DNA damage response(DDR)and ultimately avoids the decrease of genome stability caused by chromosome fusion.The research group's previous research found that apurinic/apyrimidine endonuclease/redox factor(APE1)can phosphorylate hnRNPA1 in the M phase by activating DNA-dependent protein kinase(DNA-PKcs).The phosphorylation status of hnRNPA1 determines it's the main characteristics of affinity with telomere single-stranded DNA and competitive inhibition of RPA.However,at the same time,we also found that POT1 cannot be directly recruited to telomere single-stranded DNA and replace hnRNPA1 to complete telomere capping.Therefore,we speculate that a certain phosphatase may dephosphorylate hnRNPA1.This process helps it dissociate from telomere single-stranded DNA and promotes the binding of POT1 to telomere ss DNA,which is an essential limitation of the telomere protection regulatory pathway Speed enzyme.Inhibition of this phosphatase can cause genome instability by destroying telomere structure and can increase the sensitivity of cells to radiotherapy.Also,oxidative stress caused by radiotherapy will exert an effect on immune cells in the tumor microenvironment through APE1.Based on the evidence of appeal,it is suggested that APE1 and phosphatase may have synergistic radiosensitization effects.The main research content and research results are as follows:1.Phosphatase PP2 A is the key phosphatase that acts on hnRNPA1 and acts on the Ser-95 and Ser-192 sites of hnRNPA1.Proteomics mass spectrometry analysis was used to identify the phosphatase that interacts with hnRNPA1 mimic phosphorylation mutant and the mimic dephosphorylated mutant egg.It was found that the structural subunit PPP2R1 A of phosphatase PP2 A interacts with hnRNPA1 and changes with the level of phosphorylation.Furthermore,it further confirmed that PPP2R1 A is the basis for the biological effects of phosphatase PP2 A.Subsequently,by studying the activation of the upstream pathway of hnRNPA11 after knocking down PPP2R1 A,it was confirmed that hnRNPA1 is the direct substrate of phosphatase PP2 A.And hnRNPA1's Ser-95 and Ser-192 sites are the active sites of PP2 A.2.The phosphorylation of hnRNPA1 in the M phase is sequentially regulated by DNA-PKcs and PP2 A.This dynamic process facilitates the dissociation of hnRNPA1 from telomere DNA.By comparing the changes in the affinity of PPP2R1 A and hnRNPA1 in different cell cycles by PLA,it was found that the binding of PPP2R1 A and hnRNPA1 in the late stage of M was the highest,and then it was confirmed by CO-IP that the combination of the two in the M phase increased with time.The phosphorylation level of hnRNPA1 is dynamically regulated by kinase and phosphatase.3.PP2A-dependent dephosphorylation of hnRNPA1 can promote POT1 binding and protect telomere DNAThrough in vitro telomere ss DNA affinity experiments,it was found that after knocking down PPP2R1 A,the ability of POT1 on telomere ss DNA to replace RPA was reduced,and it was confirmed by immunofluorescence that knocking down PPP2R1 A could also reduce the ability of telomere ss DNA to recruit POT1.The results prove that PP2 A is the key rate-limiting enzyme that phosphorylates the APE1/DNA-PKcs/hnRNPA1 regulatory axis.4.PP2 A contributes to the specific re-accumulation of TERRA expression level in the M phaseThe expression of TERRA in cells changes with the cell cycle,and this change can help the recruitment of hnRNPA1 in the M phase.We found through q RT-PCR that the phosphatase PP2 A contributes to the specific re-accumulation of TERRA expression levels in the M phase.Highly expressed TERRA It can chelate free hnRNPA1 to help POT1 bind to telomere ss DNA.This result proves that PP2 A can also restrict the APE1/DNA-PKcs/hnRNPA1 regulatory axis through TERRA.5.PP2 A subunit PPP2R1 A has telomere protection during mitosisThrough western blotting,it was found that knockdown of PPP2R1 A would lead to the activation of ATR-related DNA damage pathways and the presence of DNA damage signal ?H2AX at the telomeres.Finally,it was found through telomere fluorescence in situ hybridization that knockdown of PPP2R1 A would lead to increased telomere malformations.The results proved that dephosphorylation of hnRNPA1 associated with PPP2R1 A could maintain genome stability.6.PPP2R1 A has clinical value as a key rate-limiting enzyme in the APE1/DNA-PKcs/hnRNPA1 regulatory axisThrough bioinformatics analysis,it is found that PPP2R1 A is highly expressed in tumor cells,and in clinical pathological specimens,it is further found that the expression of PPP2R1 A increases with the increase of tumor clinical TNM stage,which has the potential to predict prognosis.Simultaneously,further clone formation experiments found that knocking down PPP2R1 A can enhance the sensitivity of radiotherapy.7.Oxidative stress in the tumor microenvironment after radiotherapy can induce APE1-dependent M2 macrophage pyrolysisRadiotherapy can directly kill tumor cells while causing oxidative stress on the tumor microenvironment.APE1 is likely to participate in this mechanism in the tumor microenvironment as an oxidative stress response protein.We found that under oxidative stress,APE1 has a significant effect on M2.The phages play a role in promoting cell pyrolysis.Moreover,through experimental methods such as Elisa and EMSA,it is found that APE1 promotes the assembly of NLRP3 inflammasomes through the transcription factor NF-?B and finally leads to the pyrolysis of M2 macrophages.In summary,this study found that phosphatase PP2 A is the rate-limiting enzyme of the APE1/DNA-PKcs/hnRNPA1 regulatory axis during tumor cell mitosis,and it plays a role in telomere protection.PP2 A can directly act on the s95 and s192 sites of hnRNPA1.It can help hnRNPA1 dissociate from telomere single-stranded DNA by dephosphorylating hnRNPA1 and recruit POT1 and telomere units in the telomere guard protein complex.The combination of strands of DNA ultimately plays a role in maintaining the stability of the genome.We further found that PP2 A has the potential to predict prognosis and increase the sensitivity of radiotherapy.While studying the sensitivity of radiotherapy,it is also found that in the oxidative stress microenvironment after radiotherapy,the excessive activation of APE1 can promote the activation of inflammasomes and ultimately promote the pyrolysis of TAMs and weaken the tumor immune suppression effect.This study is the first to fully elucidate the dynamic changes of hnRNPA1 phosphorylation in tumor cells to precisely regulate its protective effect on chromosome ends and confirm that PP2 A is the key rate-limiting enzyme of the APE1/DNA-PKcs/hnRNPA1 regulatory axis,which enriches APE1 in The regulatory mechanism in the tumor immune microenvironment.It will provide the experimental and theoretical basis for the future development of radiosensitization treatment schemes targeting phosphatase PP2 A or combined with APE1.