The Role And Mechanism Of DNA Damage Repair Inhibited By Long Non-coding RNA ATRBL2 In Colorectal Cancer Radiosensitivity

BackgroundColorectal cancer is the third leading cause of cancer death worldwide,with approximately 700,000 deaths from colorectal cancer each year.Radiation therapy(RT)followed by radical surgery is the standard effective treatment strategy for all cancers,including rectal cancer.Patients with rectal cancer respond differently to radiotherapy,and the radiosensitivity of cancer cells is the main reason for the outcome of radiotherapy.The radiosensitivity of tumor cells is closely related to factors such as DNA damage repair.the stronger the DNA damage repair ability,the more resistant the tumor cells will be to radiation.the main forms of DNA damage by ionizing radiation include DNA double strand breaks(DSB)and single strand breaks(SSB),of which DSB is the most serious one.The body can usually repair DNA damage effectively,but once repaired incorrectly it may lead to cancer.Therefore,it is important to explore the molecular mechanism of DNA damage repair to overcome the radiation resistance of tumors.DNA double-strand breaks(DSBs)are the most severe type of DNA damage caused by ionizing radiation.In eukaryotic cells,DSB repair is regulated by three main protein kinases:ATM,ATR and DNA-PKcs.There are two main repair pathways: non-homologous end joining(NHEJ)and homologous recombination(HR).NHEJ is a fallible repair modality,mainly mediated by DNA-PKcs;HR is a precisely regulated biological process,mainly mediated by ATM and ATR,and is considered as a repair modality to protect genome integrity.ATM and ATR recognize and bind at sites of DNA damage,recruit substrates,and activate downstream pathways.The sequence of ATR is similar to that of ATM and Rad3,and is therefore named ataxia capillaris rad3-related ATR belongs to the phosphatidylinositol3 kinase-related kinase family and plays a key role in DNA damage repair and the DNA replication stress response(RSR).ATR is essential for cell survival by inhibiting the excitation of replication initiation sites and inducing cell cycle arrest and DSB repair.Thus,targeting ATR to modulate DNA damage repair is an important strategy to effectively overcome resistance to radiotherapy.Long-stranded non-coding RNAs(lncRNAs)are defined as non-coding transcription products that exceed 200 nt and lack the potential to encode proteins.lncRNAs are aberrantly expressed in a wide range of cancers,regulate a variety of biological processes,and are implicated in the regulation of numerous physiological and pathological processes within cells,and are particularly closely associated with cancer development,progression and prognosis.Interestingly,some lncRNAs are specifically induced and involved in the DNA damage response.Studies have shown that lncRNAs regulate signaling events involved in cell cycle and DNA damage repair and play an important role in DDR.Despite these important findings,the role of lncRNAs in DDR,especially in rectal cancer radioresistance,is still poorly understood.In the previous study,the group found that ATR has the ability to bind lncRNA and reported for the first time an ATR-binding lncRNA(ATR Binding lncRNA),ANRIL,which can target ATR and regulate its protein stability and phosphorylation process.Based on the previous foundation,we screened lncRNA molecules with differential binding to ATR before and after ionizing radiation by RIP-seq,and validated by RIP and RT-qPCR to screen a lncRNA LOC107985304 with post-irradiation timedependence.since this lncRNA is unnamed and the second lncRNA we found that binds directly to ATR direct binding lncRNA,we named it ATRBL2.In this study,we firstly examined the expression and subcellular localization of ATRBL2 in various tumor cell lines and normal epithelial cell lines using RT-qPCR and fluorescence in situ hybridization(FISH)techniques;secondly,we verified the relationship between ATRBL2 expression levels and cellular radiosensitivity by constructing ATRBL2 overexpression and knockdown stable screen cell lines;In situ hybridization experiments were then performed using microarrays of collected rectal cancer radiotherapy-sensitive and radiotherapy-resistant tissues to corroborate the correlation between ATRBL2 expression levels and clinical tumor radiotherapy sensitivity.On this basis,we used comet electrophoresis,immunofluorescence,I-Sce1-NHEJ/HR reporter gene system and secondgeneration sequencing to comprehensively reveal the effect of ATRBL2 on cellular DNA damage repair ability,especially the regulatory role on the key link of HR repair;Finally,through immunoprecipitation and RNA-pull down mass spectrometry,the binding sites and functional domains of ATRBL2 were identified,and the molecular mechanism of ATRBL2 regulation of ATR was deeply explored to provide new ideas and new targets for medical protection against ionizing radiation and tumor radiotherapy sensitization research.Contents and Methods1.Screening for radiation-responsive/responsive lncRNA molecules that bind differentially to ATR before and after irradiationRNA immunoprecipitation(RIP)experiments were performed by ATR antibody to enrich the lncRNAs bound to ATR before and after irradiation,respectively,and the lncRNAs differentially bound to ATR were screened by high-throughput second-generation sequencing,and the top 10 lncRNAs with significant differences were selected,and then their interactions with ATR were verified by RIP and RT-qPCR methods.Meanwhile,RTqPCR was used to detect the expression of these lncRNAs at different time points after illumination,so as to select molecules with radiation-responsive expression levels as target molecules for the next study.2.Expression profile and subcellular localization of ATRBL2 in a variety of tumor cells and normal cellsBasal expression of ATRBL2 was examined in a variety of tumor cells(colorectal,lung,cervical and osteosarcoma)and normal epithelial cells(intestinal epithelial cells and lung epithelial cells)to determine the tissue expression specificity of ATRBL2.The subcellular localization of ATRBL2 in cells was examined using nucleoplasmic RNA isolation assays and fluorescence in situ hybridization(FISH)assays.3.Study on the regulatory effect of ATRBL2 on radiosensitivity of colorectal cancer cellsHCT116 and HT29 cells were selected to construct ATRBL2 overexpression and knockdown cell lines,and the proliferation viability,clone formation rate and apoptosis of the irradiated cells were examined by CCK8,clone formation,flow cytometry and Western blot to further clarify the effect of ATRBL2 on the radiosensitivity of tumor cells.4.Effect of ATRBL2 on DNA damage repair and signaling pathwayThe changes in the extent of DNA damage after ATRBL2 overexpression were detected by comet electrophoresis assay at different time points after irradiation,and the recruitment of γ-H2 AX foci,a key indicator of cellular DNA damage repair,was detected using immunofluorescence assay to determine the effect of ATRBL2 control on the extent of DNA damage in tumor cells.Finally,protein gel electrophoresis(Western blot,WB)method and immunofluorescence method were used to detect the expression and phosphorylation levels of key protein molecules in NHEJ and HR pathways,while the repair efficiency of NHEJ and HR pathways were detected using DDR reporter gene system to clarify the effects of ATRBL2 on DNA damage repair pathways and the specific molecular mechanisms.5.The regulatory role and mechanism of ATRBL2 on ATR and homologous recombination repair pathwayFluorescence in situ hybridization(FISH)combined with immunofluorescence laboratory was used to detect the co-localization between ATRBL2 and ATR.The effect of overexpression of ATRBL2 on the dynamic processes of p-ATR and key downstream molecules of HR,RAD51 and RPA2 foci in the nucleus was investigated by immunofluorescence assay.The effect of ATRBL2 on cell cycle progression in colorectal cancer cells after γ-irradiation was analyzed by flow cytometry.Finally,the effect of ATRBL2 on the interaction between ATR and HR pathway-related proteins was explored by immunoprecipitation experiments.6.Effect of ATRBL2 on the radiosensitivity of colorectal cancer subcutaneous graft tumors and analysis of clinical samplesA subcutaneous colorectal cancer radiotherapy model in nude mice was constructed to further clarify the radiosensitizing effect of ATRBL2 overexpression in colorectal cancer in vivo by tumor growth curve observation,tumor weight,pathological sections and immunohistochemistry,and to verify its inhibitory effect on DNA damage repair.Tissue microarrays were collected from rectal and paracancerous tissues of the hospital’s Department of Anal Surgery and classified into radiotherapy-sensitive and resistant tumors according to the tumor regression grade(TRG)score.The expression level of ATRBL2 was detected by digoxin fluorescence in situ hybridization to determine its expression in tumor tissues and the relationship between it and radiotherapy sensitivity or resistance.Results1.LncRNA ATRBL2 increases the radiosensitivity of colorectal cells(1)By RIP experiments and high-throughput sequencing,ATRBL2,an lncRNA molecule with differential binding to ATR before and after irradiation,was screened,and this molecule responded to the irradiation response in a time-dependent manner,with its expression level gradually decreasing over time,indicating that ATRBL2 is a radiationresponsive lncRNA molecule that binds ATR protein.(2)Fluorescent quantitative PCR results revealed that ATRBL2 is specifically low expressed in a variety of tumor cells and highly expressed in the corresponding normal epithelial cells.a TRBL2 is predominantly found in the nucleus,and this subcellular localization determines that it may act by binding large molecules such as DNA or proteins.(3)ATRBL2 overexpression significantly inhibited the cell viability and clonogenic ability of irradiated tumor cells and enhanced the apoptosis level of irradiated cells,and these results suggest that ATRBL2 has a promotional role in the radiosensitivity of colorectal cancer cells.2.LncRNA ATRBL2 regulates DNA damage repair through the HR pathway(1)ATRBL2 overexpression causes colorectal cancer cells to have more severe DNA damage after irradiation;(2)ATRBL2 inhibited the expression and phosphorylation of key molecules in HR repair,but had no effect on the NHEJ pathway;the DDR reporter system confirmed that ATRBL2 overexpression reduced the efficiency of HR repair by about 35%;furthermore,overexpression of ATRBL2 inhibited the recruitment of RPA2 and RAD51 foci,key molecules downstream of HR;(3)ATRBL2 overexpression significantly reduced the percentage of G2/M phase cells in post-illumination colorectal cancer cells,and ATRBL2 was found to inhibit phosphorylation of CHK1,which is associated with G2/M phase block;(4)ATRBL2 overexpression affects the transcriptome of genes related to cell cycle,apoptosis,and DNA damage repair.In addition,overexpression of ATRBL2 inhibits the interaction of ATR with MRE11,a key protein of the HR pathway.3.ATRBL2 overexpression exerts a significant radiosensitizing effect in colorectal cancer cells treated with radiotherapy in vivo(1)ATRBL2 overexpression in colorectal cancer cells in vivo enhanced the radiosensitization effect,significantly reduced the post-irradiation volume of tumors in vivo,inhibited the post-irradiation proliferation of tumors in vivo,increased the post-irradiation apoptosis rate of tumors,and inhibited the post-irradiation DNA damage repair of tumors in vivo;(2)ATRBL2 was specifically low expressed in the tumors of rectal cancer patients,while ATRBL2 was more low expressed in radiation therapy-resistant cancer tissues relative to radiation therapy-sensitive rectal cancer tissues.It indicates that ATRBL2 is lowly expressed in rectal cancer and the higher the expression level,the greater the sensitivity to radiotherapy.ConclusionWe first observed the radiosensitizing effect of ATRBL2 in tumor cells,and further studies showed that ATRBL2 affects DNA damage repair mainly by influencing the HR pathway.Mechanistically,we demonstrated that ATRBL2 can bind to ATR and block its recruitment to the MRN complex,thereby inhibiting radiation-induced MRN-ATR-RPA2 and MRN-ATR-CHK1 signaling,leading to impaired DNA damage sensing,processing and repair,and ultimately causing increased sensitivity of tumor cells to radiation treatment.In addition ATRBL2 was specifically low expressed in rectal cancer tissues,while its low expression level indicated a higher resistance to radiotherapy in patients.In summary,we identified ATRBL2 as a binding lncRNA for ATR and revealed the molecular mechanism of ATRBL2 in the DNA damage response,providing strong evidence for potential therapeutic strategies for rectal cancer.