Relationship Between Radiotherapy Resistance And Cytoplasmic MtDNA Phase Separation

Objective: Radiotherapy is one of the main methods to treat cancer.However,in clinical treatment,many cancers are resistant to radiotherapy,which has become a concern of many researchers.In recent years,many studies have shown that radiotherapy resistance is closely related to the weakening of immune response mediated by c GAS-STING pathway.Mitochondria play an important role in the process of radiotherapy,and mtDNA released after radiation can also activate c GAS-STING pathway.Due to the large number of mitochondria in the cell,the mitochondrial DNA released after radiation may occupy a considerable part of the cytoplasmic DNA.At the same time,DNA can also be used as the main component of phase separation during the formation of phase separation in cells.Based on the above background,we speculate that mtDNA released after radiation may be involved in the formation of phase separation,thus promoting the binding and activation of c GAS.The low level of phase separation in radiotherapy resistant cells may lead to a weak activation level of c GAS,thus affecting the effect of radiotherapy.So we designed a series of experiments.Methods: 1.In the process of nucleoid self-assembly,ecDNA binding chromatin DNA and various factors to form transcription centers,and automatic aggregation of ecDNA to form ecDNA centers,circular DNA is used as the skeleton.Therefore,we speculated that,due to the special topology of circular DNA,it may be easier than simple linear DNA to combine with other circular DNA,linear DNA and various factors,and then aggregate into networks and form phase separation.Therefore,in order to verify whether circular DNA can participate in the formation of phase separation in the cytoplasm,we constructed circular DNA with the Cy5 label in vitro.It was then transfected into cells and its distribution in the cytoplasm was observed by fluorescence microscopy.2.In addition,considering the natural ring structure of the plasmid,we labeled pc DNA3.1,p CDH and p ET-28a(+)with 500 n M EDC,and took the bacteria for clicking chemical verification of the labeling effect.Then,we collected labeled plasmids and transfected them into cells,and observed the distribution of plasmids within cells by clicking chemical reactions.3.If the mitochondrial DNA fragments released after radiation are likely to cyclize,it is more likely that mtDNA will form phase separation in the cytoplasm.In order to verify whether linear DNA can cyclization in cells,we designed corresponding primers and attached biotin labels to upstream primers.Then,Hela genome was used as the template for PCR,and the obtained fragments were transfected into cells,and cell lysate was taken and streptavidin magnetic beads were used for IP reaction.Then,linear DNA and IP products were used as templates for fluorescence quantitative PCR to verify the cyclization.4.In order to better observe the difference between radiotherapy resistant cells and control cells,low dose X-ray was used to repeatedly irradiate U2 OS cells at intermittent intervals to establish radiation resistance.After that,the cells obtained after repeated irradiation and the control cells were irradiated by X-ray at the same time,and the radiation resistance effect was detected by MTT assay.5.In the literature,methods for labeling mtDNA in cells include immunofluorescence staining for mitochondrial transcription factor A(TFAM),DNA polymerase γ(DNA POLγ),and Brd U,as well as click chemical reactions for EDU and PDG.In order to observe whether the mtDNA released after radiation is involved in the formation of phase separation,we used 2μM EDC to label the cells for seven days in combination with existing laboratory conditions.The distribution of mtDNA in the cytoplasm after radiation was examined by clicking chemistry and the differences between the two types of cells were compared.6.In order to further study whether mtDNA can promote the activation of Cgas-STING pathway by participating in the formation of phase separation,we irradiated EDC-labeled cells with X-rays,then performed click chemistry and incubated c GAS antibodies to observe whether they were co-located.Meanwhile,RNA was extracted from control cells and radiation-resistant cells after radiation,and qPCR was performed to observe the expression of IFN-β,the downstream product of c GASSTING pathway.Experimental results:1.qPCR results showed that circular DNA was successfully constructed.Many red droplets were observed under a fluorescence microscope 24 hours after transfection of the constructed circular DNA into the cells,while the number of red droplets decreased significantly 2 minutes after the cells were treated with 1.5% 1,6-hexadiol.Proof that circular DNA may form phase separation.2.The results of the click-chemical reaction after transfection showed that the plasmids formed phase separation droplets in the cytoplasm.And after 1,6-hexadiol treatment,the number of droplets decreased significantly.This further proves that circular DNA may have the ability to form phase separation in cells.3.The results of qPCR using linear DNA and IP products as templates showed that CT values were not detected in linear DNA,while CT values of IP products were 13.17,12.73 and 14.29,respectively,indicating the existence of circular DNA in IP products,which proved that linear DNA could be cycled in cells.Therefore,mitochondrial DNA fragments released after radiation may be cyclified,which increases the possibility of its involvement in the formation of phase separation.4.The results of the MTT experiment showed that,under the same irradiation conditions,the cell vitality of the cells obtained by repeated X-ray irradiation was significantly higher than that of the control cells,indicating that we successfully constructed the U2 OS cell line with a certain radiation resistance.5.The click-chemical results after EDC labeling showed that DNA could form phase separation in cytoplasm after radiation,and the amount of phase separation was less in radiation-resistant cells.It should be noted that in addition to mtDNA,EDC-labeled DNA may also contain other cytoplasmic DNA.However,due to the large number of mitochondria,mitochondrial DNA released after radiation may occupy a considerable part of cytoplasmic DNA,so the majority of DNA revealed through EDC may be mitochondrial DNA.Therefore,we speculate that mtDNA may be involved in phase separation in the cytoplasm after radiation,and the amount of phase separation in radiation-resistant cells may be less than in control cells.6.The immunofluorescence detection showed that c GAS and DNA were co-located after radiation separation.At the same time,qPCR results showed that radiation resistant cells had lower IFN-β expression than control cells.Conclusions: Through transfection in vitro to construct circular plasmid DNA and EDC tags,we proved that the circular DNA may have the ability to form the phase separation in the cytoplasm.At the same time,after the linear DNA transfection qPCR results show that the radiation into the extranuclear mtDNA fragments possible cyclization.In addition,the EDC tag mtDNA experiments show that the radiation after separating the DNA can be formed in the cytoplasm,and radiation resistance less the number of phase separation in the cell.Although EDC-labeled DNA may contain other cytoplasmic DNA except mtDNA,mtDNA may account for a large proportion in cytoplasmic DNA,so most of the DNA manifested through EDC may be mtDNA.So we speculated that after radiation mtDNA may participate in the formation of phase separation in the cytoplasm,and the number of phase separation in radiation resistant cells less.Finally,immunofluorescence results show that after radiation c GAS,positioning with DNA separation.And qPCR experimental results show that the radiation and radiation resistance amount of IFN-beta expression in the cell is lower than in control cells.In conclusion,we speculate that mtDNA released after radiation may be involved in the formation of phase separation,thus promoting the binding and activation of c GAS.Radiation resistant cells due to the low level of formation of phase separation,may result in c GAS-STING pathway mediated immune response weaker than in control cells,making the poor efficacy of radiotherapy.