Long Non-coding RNA ANRIL Promotes Homologous Recombination Repair Of Radiation-damaged Lung Cancer Cells By Maintaining ATR Protein Stability

Background Lung cancer is one of the most common malignant tumors in the world,and its morbidity and mortality ranks first among male malignant tumors,and the overall mortality rate of men and women also ranks first,and the overall incidence of men and women is second only to breast cancer.Radiation therapy is an important strategy for the treatment of lung cancer.Radiotherapy has a good effect on lung cancer in the early stage,but with the progress of treatment,some patients are clinically resistant to radiotherapy,resulting in poor local control or recurrence of lung cancer,which leads to treatment failure.Therefore,finding effective new targets for radiation sensitization of lung cancer has become an important scientific problem to be solved.The radiotherapy sensitivity of tumor cells is related to many factors such as cell oxygenation,cell cycle,proliferation activity,DNA damage repair,etc.Among them,the enhancement of tumor cell DNA damage repair ability is an important reason for radiotherapy resistance.The main target of ionizing radiation is DNA.By inducing DNA damage and blocking the DNA repair process,it has a cytotoxic effect on tumor cells,thereby blocking the cell cycle and promoting cell apoptosis.Studies have shown that long non-coding RNA(lncRNA)play an essential role in the initiation of DNA damage repair,which provides new ideas for explaining the molecular mechanisms of DNA damage repair and even tumor radiotherapy resistance from the perspective of lncRNA.A large number of studies have shown that lncRNA can participate in tumor radiotherapy resistance by regulating DNA damage repair,but most of the roles of lncRNA in DNA damage repair and radiotherapy resistance have not been fully studied and functionally explained,and their regulatory mechanisms are still unclear.We screened out the ionizing radiation-induced lncRNA ANRIL in our previous study,and found that ANRIL plays an important role in the sensitivity of lung cancer cells to ionizing radiation and is closely related to its regulation of DNA damage repair.Based on the previous work,this study intends to comprehensively reveal the effect of ANRIL on the DNA damage repair ability of lung cancer cells after ionizing radiation by using the I-Sce1-NHEJ/HR reporter system,RNA-binding protein immunoprecipitation,RNA-pull down and other methods.A mouse lung cancer tumor-bearing model was constructed to further verify the effect of ANRIL on ionizing radiation sensitivity and DNA damage repair of lung cancer cells in vivo.Finally,the ANRIL-binding protein and the exact functional domain that binds to it were identified,and the exact molecular mechanism of ANRIL’s regulation of HR repair was further explored.This study will provide new ideas for the study of ionizing radiation sensitivity of lung cancer cells from the perspective of lncRNA.Contents and Methods1.Effects of ANRIL on repair of homologous recombination in lung cancer cells after ionizing radiation(1)Expression level of ANRIL in lung cancer cells and its effect on ionizing radiation-induced DNA damage(1)The expression level of ANRIL in lung cancer cellsⅠ.Search the ANRIL expression level of lung cancer in TCGA database.Ⅱ.Select clinical lung cancer specimens,and detect the expression level of ANRIL in tumor and adjacent normal tissues by Real-Time PCR.Ⅲ.Lung cancer cells were treated with actinomycin D to block the synthesis of nascent RNA,and the expression level of ANRIL in lung cancer cells was detected by Real-Time PCR at different time points after treatment to judge the half-life of ANRIL in lung cancer cells.(2)Effects of ANRIL on DNA damage induced by ionizing radiation in lung cancer cellsⅠ.Select different lung cancer cell lines,construct ANRIL knockdown(KD)and ANRIL overexpression(OE)stable cell lines by lentiviral packaging plasmid system,and verify the efficiency of ANRIL knockdown and overexpression by Real-Time PCR.Ⅱ.The effects of ANRIL knockdown or overexpression on the formation ofγ-H2 AX foci at different time points in lung cancer cells after ionizing radiation were observed by immunofluorescence staining under confocal microscopy.(2)Effects of ANRIL on the repair of non-homologous end joining(NHEJ)and homologous recombination(HR)and their downstream signaling pathways in lung cancer cells after ionizing radiation(1)The effects of ANRIL knockdown on the repair efficiency of non-homologous end-joining(NHEJ)and homologous recombination(HR)in lung cancer cells after ionizing radiation were detected by I-Sce1-NHEJ and HR reporter systems.(2)The effects of ANRIL knockdown or overexpression on NHEJ and HR repair downstream signaling pathways in lung cancer cells at different time points after ionizing radiation were detected by Western Blot.(3)The effects of ANRIL knockdown or overexpression on the formation of HR repair key molecules ATR and RPA2 foci in lung cancer cells after ionizing radiation were observed under confocal microscope by immunofluorescence staining.(4)The effects of ANRIL knockdown on the gene expression of DNA damage response-related signaling pathways in lung cancer cells after ionizing radiation were analyzed by RNA sequencing.(3)Effects of ANRIL on ionizing radiation sensitivity and HR repair of a tumor-bearing model of lung cancer(1)Effects of ANRIL knockdown on ionizing radiation sensitivity and HR repair in a tumor-bearing model of lung cancer.Ⅰ.To construct a subcutaneous tumor-bearing model of ANRIL knockdown lung cancer cells in nude mice.Ⅱ.The effect of ANRIL knockdown on tumor proliferation after ionizing radiation was evaluated by measuring tumor growth curve,tumor weight and tumor Ki67 immunohistochemical staining.Ⅲ.To evaluate the effect of ANRIL knockdown on tumor in situ apoptosis after ionizing radiation by TUNEL staining of lung cancer tumor-bearing tissues.Ⅳ.The effect of ANRIL knockdown on DNA damage after ionizing radiation was evaluated by immunohistochemical staining of γ-H2 AX in lung cancer tumor-bearing tissues.Ⅴ.By immunohistochemical staining of ATR,RPA2 and Rad51 in lung cancer tumor-bearing tissues,the effect of ANRIL knockdown on HR repair after tumor ionizing radiation was evaluated.(2)Effects of ANRIL overexpression on ionizing radiation sensitivity in a tumor-bearing model of lung cancer.Ⅰ.To construct a subcutaneous tumor-bearing model of ANRIL-overexpressing lung cancer cells in nude mice.Ⅱ.To evaluate the effect of ANRIL overexpression on tumor proliferation after ionizing radiation by measuring tumor growth curve,tumor weight and tumor Ki67 immunohistochemical staining.2.Molecular mechanism of ANRIL binding to ATR and regulating its protein stability(1)Study on the interaction between ANRIL and ATR protein(1)The binding of ANRIL to ATR protein was detected by RNA-pull down.(2)The binding of ANRIL to ATR protein was verified by RIP,and the effect of ATR phosphorylation level on the binding of the two was investigated.(3)Construct different truncations of ATR,and detect the exact functional domain of ANRIL binding to ATR in lung cancer cells by RIP.(4)The secondary structure of ANRIL was predicted by RNA fold web server online software,and the exact ANRIL fragment bound to ATR in lung cancer cells was verified by RNA-pull down.(2)The molecular mechanism of ANRIL regulating the stability of ATR protein(1)The effect of ANRIL knockdown on the stability of ATR protein in lung cancer cells after ionizing radiation,and the effect of ATR inhibitor(VE821)pretreatment on the expression level of ANRIL in lung cancer cells after ionizing radiation.(2)The effect of ANRIL knockdown on the expression level of ATR m RNA in lung cancer cells after ionizing radiation.(3)Effects of ANRIL knockdown on ATR protein stability after ionizing radiation in lung cancer cells after pretreatment with a proteasome inhibitor(MG132)to block protein ubiquitination.(4)The effect of ANRIL knockdown on the ubiquitination level of ATR in lung cancer cells after ionizing radiation was detected by ATR-specific antibody immunoprecipitation.(3)Effects of ATR overexpression in ANRIL knockdown lung cancer cells on the sensitivity of lung cancer cells to ionizing radiation(1)To construct a transfected cell line overexpressing ATR in ANRIL knockdown cells,and to verify the expression level of ATR by Western Blot.(2)The effect of ATR overexpression in ANRIL knockdown cells on the proliferation of lung cancer cells after ionizing radiation was assessed by the clone formation assay.(3)The effect of ATR overexpression in ANRIL knockdown cells on the formation of γ-H2 AX foci in lung cancer cells after ionizing radiation was observed under confocal microscope by immunofluorescence staining.(4)The effect of ATR knockdown in ANRIL-overexpressing lung cancer cells on the sensitivity of lung cancer cells to ionizing radiation(1)Construct an ATR-knockdown cell line in ANRIL-overexpressing cells,and use Western Blot to verify the expression level of ATR.(2)To evaluate the effect of ATR knockdown in ANRIL-overexpressing cells on the proliferation of lung cancer cells after ionizing radiation by the clone formation assay.(3)The effect of knockdown of ATR in ANRIL-overexpressing cells on the formation of γ-H2 AX foci in lung cancer cells after ionizing radiation was observed under confocal microscope by immunofluorescence staining.Results1.ANRIL promoted homologous recombination repair in radiation-damaged lung cancer cells ANRIL was highly expressed in lung cancer cells,and its half-life was about 6-8hours.ANRIL knockdown aggravated the DNA damage of lung cancer cells after ionizing radiation,and reduces HR repair efficiency by about 60%,but has no significant effect on NHEJ repair efficiency.ANRIL overexpression reduces the degree of DNA damage in lung cancer cells after ionizing radiation.HR repair is mainly mediated by ATR and ATM molecules.Further studies show that ANRIL knockdown inhibits the phosphorylation of ATR downstream signaling molecules RPA2 and CHK1 after ionizing radiation in lung cancer cells,but has no significant effect on the phosphorylation of ATM downstream signaling molecules KAP1 and CHK2.ANRIL overexpression enhances the phosphorylation of ATR,RPA2,CHK1 and P21 in lung cancer cells after ionizing radiation,but has no significant effect on the phosphorylation of KAP1.It is suggested that ANRIL promotes DNA damage repair in lung cancer cells after ionizing radiation through ATR-mediated HR repair.We further verify this result by immunofluorescence staining.ANRIL knockdown reduces the numbers of ATR and RPA2 foci in lung cancer cells after ionizing radiation,while ANRIL overexpression increases the numbers of ATR and RPA2 foci in lung cancer cells after ionizing radiation.In addition,we find by RNA sequencing that ANRIL knockdown affects the gene expression of DNA damage response-related signaling pathways(PPAR signaling pathway,PI3K-Akt signaling pathway,and p53 signaling pathway)in lung cancer cells after ionizing radiation.Next,we used a lung cancer tumor-bearing model to verify that ANRIL down-regulates the radiosensitivity of lung cancer cells and promotes HR repair in vivo.ANRIL knockdown aggravates the inhibition of growth and proliferation,increases in situ apoptosis,increases DNA damage,and down-regulates the expression of ATR,RPA2,and Rad51,key HR repair molecules,after ionizing radiation in lung cancer tumor-bearing tissues.ANRLI overexpression alleviates the growth and proliferation inhibition of lung cancer tumor-bearing tissues after ionizing radiation.2.ANRIL maintains its protein stability by directly binding to ATR and inhibiting the ubiquitination pathway The above findings suggest that ANRIL promotes HR repair by promoting the phosphorylation and activation of ATR and its downstream signaling molecule RPA2.In order to clarify the interaction between ANRIL and ATR and RPA2,we found that ANRIL could bind to ATR but not to RPA2 by RNA pull down assay.It was further confirmed by RNA-binding protein immunoprecipitation that ANRIL binds to ATR and was not affected by the phosphorylation level of ATR.Further studies showed that ANRIL could bind to the N-terminus of ATR but not to the C-terminus of ATR.In addition,we also found that the 5’-region(1-880 nt and 881-1640nt)of ANRIL can bind to ATR,while its 3’-region(1641-2480 nt and 2481-3857nt)can not.To further investigated the exact effect of ANRIL on ATR,we found that ANRIL knockdown decreased the total ATR protein in lung cancer cells after ionizing radiation by western blot analysis.Further studies found that ANRIL knockdown did not affect the expression of ATR m RNA in lung cancer cells after ionizing radiation,indicating that ANRIL may have post-translational regulation of ATR protein.We further pretreated ANRIL-knockout lung cancer cells with a proteasome inhibitor(MG132),and found that ANRIL-knockdown lung cancer cells pretreated with MG132 recovered the ATR protein decrease after ionizing radiation.We also detected ubiquitination levels in protein complexes immunoprecipitated by ATR-specific antibodies by co-immunoprecipitation and found that ANRIL knockdown increased ubiquitination levels in lung cancer cells after ionizing radiation.These results indicated that ANRIL knockdown caused the decrease of total ATR protein in lung cancer cells after ionizing radiation was regulated by ubiquitination degradation,and ANRIL binding to ATR could block its ubiquitination degradation process.Finally,we verified that the interaction between ANRIL and ATR can reduce the sensitivity of lung cancer cells to ionizing radiation.Overexpression of ATR in ANRIL-knockdown lung cancer cells can improve the proliferation ability of lung cancer cells after ionizing radiation and reduce the degree of DNA damage.Knockdown of ATR in ANRIL-overexpressing lung cancer cells can reduce the proliferation ability of lung cancer cells after ionizing radiation and aggravate the degree of DNA damage.Conclusion This study reveals that ANRIL can promote HR repair by promoting the phosphorylation and activation of ATR and its downstream signaling molecules after ionizing radiation in lung cancer cells at the cellular and animal levels,and reduce the degree of DNA damage,thereby reducing the radiation sensitivity of lung cancer cells.ANRIL can directly bind to ATR to play a regulatory role.The binding of ANRIL and ATR is not affected by the phosphorylation level of ATR,and ANRIL only binds to the N-terminus of ATR,but not to the C-terminus of ATR.Additionally,the 5’-regions of ANRIL(1-880 nt and 881-1640nt)are essential in ANRIL binding to ATR.The binding of ANRIL to ATR helps to maintain its protein stability,and its mechanism is to inhibit the post-translational ubiquitination of ATR protein.ANRIL reduces the radiosensitivity of lung cancer cells by interacting with ATR.Our findings suggest that ANRIL holds promise as a novel target for overcoming radiotherapy resistance in lung cancer.