The Role And Mechanism Of DNA Damage Repair Mediated By Long Non-coding RNA SCARNA2 In Colorectal Cancer Radiosensitivity

BackgroundColorectal cancer(CRC)is the third common cancer in the world.Although technological advances in early diagnosis and intervention have effectively improved the overall survival rate of colorectal cancer,the prognosis of patients with advanced colorectal cancer is still poor due to the high recurrence rate,high metastasis,and tolerance of anticancer treatments.Preoperative radiotherapy is one of the conventional treatments for advanced colorectal cancer,but nearly one-third of colorectal cancer patients shows hyposensitivity or complete resistance to radiotherapy.Therefore,radiotherapy resistance is still the main challenge in the treatment of colorectal cancer.In the era of "Precision Treatment",exploring new targets or new molecules to overcome radioresistance is an important tool for individualized treatment of colorectal cancer patients,and is of great significance for improving the overall survival rate of colorectal cancer patients.DNA damage response(DDR)is a precise signal cascade system designed to detect DNA damage and determine the fate of damaged cells.It is one of the important areas of tumor biology research,plays a pivotal role in the regulation of tumor sensitivity to radiotherapy.The important reason for tumor cells to develop radioresistance is that cancer cells can recognize DNA damage induced by DNA damage inducers or ionizing radiation,and repair these damages by activating various DNA repair pathways.Cancer cells with higher DNA repair activity are highly resistant to radiotherapy.The occurrence of colorectal cancer is also related to mutations of oncogenes,tumor suppressor genes,and genes related to DNA repair,such as KRAS,TP53,and BRCA1/2.These mutations can cause genome instability and promote progression of cancer.Studies have shown that targeted DNA damage repair can effectively improve the sensitivity of colorectal cancer to radiotherapy,but the regulatory mechanism of DNA damage repair is still unclear.It has found that many new regulatory molecules play an important role in DDR.Exploring for new targets or new molecules that are abnormally activated by DNA damage repair in tumors is of great significance for overcoming radioresistance and improving the cure rate.Long noncoding RNA(lnc RNA)can regulate a variety of biological processes,include cell cycle,cell differentiation,X chromosome inactivation,m RNA alternative splicing,RNA transcription and translation,etc.A number of studies have shown that lnc RNA dysregulation is one of the main factors in the occurrence of colorectal cancer.It affects the signal pathway,the metastasis and the radiosensitivity in colon cancer by regulating its target genes.lnc RNA plays an important role in DNA damage repair,it can interact with protein,DNA or RNA through direct or indirect transcription.Therefore,lnc RNA regulates the sensitivity of colorectal cancer to radiotherapy through the DNA repair pathway has become an important area of clinical research.The exploration of candidate lnc RNA and their molecular mechanisms that can regulate the radioresistance of colorectal cancer through DDR is of great significance to improve the clinical efficacy of patients with colorectal cancer.However,the functions of most lnc RNAs in DDR have not been clarified yet.ATR is the central transduction factor of the checkpoint signaling pathway,it can be activated by DNA damage and replication stress,and has a wide range of functions and important physiological significance in DNA damage repair,cell cycle regulation and cell apoptosis.Based on our previous research,we found that ATR,a key molecule for DNA damage repair,had the ability to bind lnc RNA.Our project employed RIP-seq assay to screen out lnc RNAs which bind to ATR,then through RIP and RT-PCR verification,identifyed that SCARNA2 with the highest binding abundance to ATR.Thence,we comprehensively exploreed the specific regulation effects and molecular mechanisms of colorectal cancer radiotherapy sensitivity from the perspective of DNA damage repair.It provides a novel scientific basis for the development of potential markers and therapeutic targets for predicting the radiosensitivity of colorectal cancer.Contents and Method1.The role of lnc RNA SCARNA2 in DNA damage repair(1)Screen out and verify ATR-bound lnc RNA through RIP-seq assayFirst,ATR antibody is selected to enrich the RNA which bound to ATR through RIP assay,and the lnc RNA bound to ATR is screened out through high-throughput secondgeneration sequencing.Then select the top 30 lnc RNAs,and verify their interaction with ATR through RIP and RT-PCR methods.It was found that SCARNA2 had the highest binding abundance with ATR,so SCARNA2 was identified as target molecule.By exploring the basic expression of SCARNA2 in 15 kind of cell lines,the research object was determined to be HCT116 and HT29 colon cancer cell lines.(2)The effect of DNA damage on the expression level of SCARNA2HCT116 and HT29 cells were treated with ionizing radiation(IR),DNA damage inducers such as camptothecin(CPT)or etoposide(ETO).RNA was extracted at different time points.The changes of SCARNA2 transcription level were detected by RT-PCR.HCT116 and HT29 cells were incubated with DNA-PKcs inhibitor Nu7441,ATM inhibitor Ku-55933,or ATR inhibitor VE-821,respectively.Then,the cells were treated with IR,CPT or ETO,respectively.RNA was extracted at which SCARNA2 showed kenspeckle response to DNA damage,and RT-PCR was used to detect the effect of DDR pathway inhibitors on the transcription level of SCARNA2.(3)The influence of SCARNA2 interference on DNA damage repairSCARNA2 knockdown(sh-SCARNA2),CRISPR Cas9 knockout(sg-SCARNA2)and overexpression(SCARNA2-OE)stable transfection cell lines were constructed through the lentiviral packaging plasmid system,and the down-regulation and overexpression efficiency were measured by RT-PCR.Comet electrophoresis assay was used to detect the severity of DNA damage after irradiation.Immunofluorescence was applied to detect the dynamic changes of ?H2AX and 53BP1 foci formed in SCARNA2-knockout cells at 0,0.5,4,8,12 and 24 h after irradiation.Finally,Western Blot was applied to detect the changes in the expression levels of DDR pathway-related proteins after were treated with IR,CPT or ETO.2.The regulatory effect and mechanism of lnc RNA SCARNA2 on ATR and homologous recombination repair pathways(1)The effect of SCARNA2 down-regulation on gene transcriptome after irradiationApplied RNA-seq assay to compare the gene transcriptome changes in control and SCARNA2 down-regulated cells after non-irradiation and irradiation treatment.(2)The influence of SCARNA2 on NHEJ and HR repairThe I-Sce I-HR/NHEJ reporter gene system was constructed to determine the repair efficiency of NHEJ or HR in SCARNA2 down-regulatied cells.The effect of SCARNA2down-regulation on RAD51 and RPA2 foci formation in the nucleus was detected by immunofluorescence.(3)Screening and identification of SCARNA2 binding proteinRNA pull down experiment is used to screen the protein complexes which bind to SCARNA2 in HCT116 cells,and the proteins were identified by mass spectrometry.(4)The influence of SCARNA2 interference on the interaction between ATR and HR pathway related proteinsCo-immunoprecipitation was used to explore the effects of SCARNA2 interference on the interaction and modification of related proteins in the ATR and HR pathways.3.Lnc RNA SCARNA2 promotes DNA damage repair and regulates colorectal cancer radiosensitivity(1)The effect of SCARNA2 on the radiosensitivity of colon cancer cellsSCARNA2 down-regulated and over-expressing cell lines were treated with IR,CPT or ETO.Then,the survival rate,proliferation ability,cell viability,cell apoptosis rate and the expression level of apoptosis pathway related proteins were detected by clone formation rate,CCK-8,Annexin-FITC/PI double staining flow cytometry,and Western Blot.Flow cytometry and Western Blot were used to detect changes in cell cycle progression and changes in protein levels related to cycle pathways after cells were processed by IR,CPT or ETO.(2)The effect of SCARNA2 down-regulation on the radiosensitivity of Cell derived xenograft(CDX)modelConstruct nude mouse subcutaneous tumor-bearing model(Cell derived xenograft,CDX)of a human tumor cell line,and explore the effect of SCARNA2 on cell proliferation by measuring the volume of the nude mouse tumor-bearing tumor after irradiation.By performing TUNEL staining on the tumor tissue to detect the apoptosis of SCARNA2 tumor cells.Through immunohistochemical staining of tumor tissues to explore the effect of SCARNA2 on the expression level of HR pathway related molecular proteins.Through the above in vivo experiments,we explored the regulatory effect of SCARNA2 on the radiotherapy sensitivity of subcutaneous tumors in nude mice.(3)The expression difference of SCARNA2 in radio-sensitive and radio-resistant colon tumor tissuesFISH assay was applied to compare the expression level of SCARNA2?ATR and the co-localization of the two in radio-sensitive and radio-resistant colon tumor tissues.Results1.Lnc RNA SCARNA2 promotes DNA damage repair of colon cancer cells(1)The expression of SCARNA2 in colon cancer cells is relatively high,and it responds to the DNA damage response which was induced by IR,CPT or ETO in a time-dependent manner.ATM and ATR inhibitors significantly inhibited the response of SCARNA2 to DNA damage,indicating that the up-regulation of SCARNA2 induced by DNA damage is regulated by ATM/ATR kinase.(2)Down-regulation of SCARNA2 aggravates DNA damage caused by ionizing radiation,delay the repair process of DNA damage,and inhibits the activation of ATR-Chk1 pathway induced by IR,CPT or ETO.2.Lnc RNA SCARNA2 repairs DNA damage through HR pathway(1)SCARNA2 repairs DNA damage through HR pathway.Down-regulation of SCARNA2 can inhibit the recruitment of RAD51 and RPA2 to the damage site after IR exposure,thereby delaying the process of DNA damage repair.(2)SCARNA2 binding proteins were mainly enriched in pathways which related to cell cycle,DNA damage repair,DNA replication and synthesis,RNA transcription and translation.Down-regulation of SCARNA2 will not only affect the cell cycle,apoptosis and the response to DNA damage related gene spectrum,but also inhibit the interaction between ATR and HR pathway related proteins such as Top BP1,NBS1,Mre11,Chk1,etc.(3)Deletion of SCARNA2 inhibits the phosphorylation of Chk1,which is the downstream target of ATR.The mechanism may be related to the methylation or ubiquitination modification of Chk1.3.Lnc RNA SCARNA2 regulates the radiosensitivity of colorectal cancer by promoting DNA damage repair pathways(1)Down-regulation of SCARNA2 reduces the survival ability,proliferation ability and cell viability of colon cancer cells,and promotes cell apoptosis,inhibits G2/M cycle arrest after IR or DNA damaging inducers treatment.(2)Down-regulation of SCARNA2 significantly inhibits the tumorigenic ability of colon cancer cells in nude mice.Through multi-point injection of sg-SCARNA2 lentiviral vector into the transplanted tumors,the expression level of SCARNA2 in tumor cells was successfully down-regulated.It was found that down-regulation of SCARNA2 inhibits the growth rate of the tumor after irradiation,promotes the apoptosis of tumor cells,and inhibits the HR repair of tumor cells after irradiation.(3)The expression level of SCARNA2 in radiosensitive colorectal cancer tissues is about 80% higher than that in radiotresistant colorectal cancer tissues(p <0.00001).ConclusionIn this study,we screened out lnc RNA SCARNA2 that bind to ATR through RIP-seq.Through a series of in vivo and in vitro biological experiments,it is found that SCARNA2 is closely related to the DNA damage repair and radiosensitivity of colon cancer cells.The deletion of SCARNA2 can inhibit the DNA damage repair ability of colon cancer cells.When used in combination with DNA damage inducers or IR,it can promote tumor cell apoptosis and increase the sensitivity of colon cancer cells.Through the exploration of mechanism,it is found that SCARNA2 promotes DNA damage repair through HR pathway.Deletion of SCARNA2 inhibits the phosphorylation of Chk1 targets,and the mechanism may be related to the methylation or ubiquitination modification of Chk1.At the same time,SCARNA2 down-regulation inhibits the phosphatase activity of CDC25 C and the activation of Wee1,which inhibits the G2/M arrest by affecting the activity of the Cyclin B/CDC2 complex.It is clear that SCARNA2 regulates the cell cycle process by activating the ATRChk1-CDC25C/Wee1 pathway.In summary,SCARNA2 has the potential to become a new target for colorectal cancer radiosensitization.In addition,the expression of SCARNA2 in radiosensitive and radioresistant colorectal cancer tissues is significantly different,and it is also expected to be a potential prognostic biomarker for colorectal cancer radiotherapy.