The Effect And Mechanism Of MSI2 On Radiation Sensitivity In Non-small Cell Lung Cancer

BackgroundCancer has overpowered other diseases to rank first among the cause of death worldwide.With both highest incidence rate(11.4%)and mortality rate(18%),lung cancer becomes the most lethal malignant tumors.Non-small cell lung cancer is a heterogeneous collection of lung cancers,including squamous cell carcinoma,large cell lung cancer,and the most common adenocarcinoma.In addition to traditional surgical resection,radiation therapy is becoming one of the common treatments for clinicians due to the advancement of technology and treatment protocol.However,NSCLC patients are still plagued by radiotherapy complications and radiotherapy resistance.Complications such as radiation-induced pneumonia strictly limit dose and frequency of radiotherapy,while resistance of lung carcinoma to radiotherapy can greatly reduce therapeutic effect.Considering there is no effective method of either protection or treatment for radiotherapy complications,increasing radiation sensitivity of carcinoma is the best solution to not just alleviate the suffering of patients from complications but also enhance the effectiveness of radiotherapy.The radiation sensitivity of tumor cells can be attributed to several factors,such as tumor microenvironment,cell hypoxia,apoptosis and DNA damage repair,among which the variation of DNA damage repair is a crucial factor in tumor resistance to radiotherapy.When cells are exposed to ionizing radiation,their DNA molecules may suffer severe damage,which can induce cytotoxic effects,cell cycle arrest and,in some cases,cell apoptosis.Various of studies have confirmed that tumor radiotherapy resistance is highly correlated with the enhancement of DNA damage repair,and specific inhibition of DNA damage repair in radiotherapy can greatly enhance the killing effect on tumor cells.RNA binding protein(RBP)refers to a class of proteins that can recognize and specifically bind RNA,and it’s an important regulator involved in the cascade regulation of RNA metabolism and gene expression.Studies have shown that RNAbinding proteins can affect the sensitivity of tumor cells to radiation by regulating DNA damage repair.In addition to participating in the regulation of DNA damage-induced post-transcriptional modification,some RNA-binding proteins can also be directly recruited to the damage site to participate in the repair process.Although the regulatory effect of certain RBPs on DNA damage repair and tumor radiotherapy and chemotherapy resistance have been clarified,the mechanism of most RNA-binding proteins in DNA damage repair has not been elucidated.Our group has found one particular RNA binding protein,MSI2,which has high expression in lung carcinoma tissues,not only promotes radiation resistance in lung cancer cells,but also participates in affecting DNA damage repair.Based on previous work,we plan to further clarify the specific effect of MSI2 on radiation sensitivity,and find out the underlying mechanism of MSI2 regulating DNA damage repair.This project aims to provide new radiotherapy resistance biomarker and DNA damage repair inhibitors targeting MSI2 for radiosensitization of NSCLC cells.Contents and Method1.The effect of MSI2 on radiation sensitivity of NSCLC cells(1).Construction of MSI2-knockdown H1299 cell lineUtilizing Crispr Cas9 technology,gRNA sequences targeting MSI2 fragment were designed,and the plasmids carrying gRNA were transfected with lipo3000 in H1299 cells overexpressing Cas9 protein.Following 48 to 72 hours of transfection,the cells were laid with mono-clones,and cell protein extracted from the grown mono-clone cell line was verified by Western Blot.Subsequently,the mono-clone with the most obvious knockout effect was selected for further experiments.(2).The effect of knocking out MSI2 on the sensitivity to ionizing radiationBoth MSI2-knockout and control cell lines were treated with γ-rays radiation,then the cell proliferation ability,cell viability,cell cycle arrest and cell apoptosis rate were observed separately by colony formation,CCK-8 assay,PI single staining flow cytometry and PI/FITC staining flow cytometry.We also constructed nude mice subcutaneous tumor model to verify radiation resistance effect of MSI2.2.The mechanism of MSI2 affecting radiation sensitivity of NSCLC cells(1).The influence of knocking out MSI2 interference on severity of DNA damage and DNA damage repair processComet assay,immunofluorescence and Western Blot were used to verified severity of DNA damage and variation of DDR-related proteins between MSI2-knockout and control cell lines after irradiation.(2).The mechanism of MSI2 RNA binding domain affecting DNA damage repair in NSCLC cellsConstruct plasmid containing MSI2 RNA binding domain(RBD),and rescue MSI2 RBD in MSI2-knockout mono-clone through lentivirus infection.After the expression level of MSI2 RBD was confirmed by western blot,we performed colony formation and immunofluorescence to observe sensitivity to ionizing radiation and severity of DNA damage.The transcriptional changes of related genes and signal pathways before and after irradiation in both MSI2-knockout group and control group were detected by RNA-seq assay,and verified by RT-PCR.Results1.The influence of MSI2 on radiation sensitivity of NSCLCAfter knocking out MSI2,we observed inhibition of independent viability and cell proliferation according to clone formation and CCK8 assay after irradiation.Also knocking out MSI2 significantly enhanced the apoptosis rate of NSCLC cells 24 hours after irradiation,indicating that MSI2 can reduce radiation sensitivity.However,knocking out MSI2 was proved to have no genuine influence on cell cycle process.After receiving local irradiation,the tumor volume of MSI2-knockout group decreased more significantly than control group,proving that MSI2-knockout significantly decrease radiotherapy resistance of NSCLC cells.2.The mechanism of MSI2 affecting radiation sensitivity of NSCLCAfter irradiation,we performed comet assay and found out knockout of MSI2 can cause more severe DNA damage compared to control cell line.Moreover,immunofluorescence showed that knocking out MSI2 promoted formation of γ-H2AX foci after y-ray irradiation.Western blot assay revealed that knockout of MSI2 highly inhibited the activation of DNA damage response(DDR),especially ATR and its downstream HR pathway.3.MSI2 reduces radiation sensitivity and promote DNA damage repair in NSCLC cells after irradiation through RNA recognition motifWe confirmed successful construction of rescued-MSI2-RBD cell lines by western blot assay.Clone formation assay showed that rescuing MSI2 RBD can restore cell survivability and proliferation after irradiation.Immunofluorescence proved that rescuing MSI2 RBD significantly reduced DNA damage compared to MSI2-knockout cell line.RNA-seq revealed that knocking out MSI2 could affect transcriptional level of genes related to cell cycle,cell apoptosis,FoxO and p53 pathway.Also RT-PCR confirmed differential expression of related genes after irradiation.Discussion and analysisResistance to radiotherapy in lung cancer is an important factor in inhibition of therapeutic effect and tumor recurrence,and it also remains a research hotspot and difficulty.Our study shows that MSI2 plays an important part in radiation sensitivity and radiation-induced DNA damage repair of NSCLC cells.Knockout of MSI2 can increase the sensitivity of NSCLC cells to ionizing radiation by increasing cell apoptosis,inhibiting cell proliferation and growth capacity of nude mice tumor.We also revealed that knocking out MSI2 can aggravate DNA damage and inhibit Homologous recombination repair.In addition,Rescuing MSI2-RBD can alleviate the suppression of cell proliferation and DNA damage caused by MSI2-knockout.Knocking out of MSI2 can affect the expression of genes related to apoptosis,FoxO,p53 and other signaling pathway.MSI2 may exert radiation resistance by affecting the transcription of CDC25C,TP53INP1,KIF20A,and PIK3R3.ConclusionAbove all,this study clarifies that MSI2 can promote radiation resistance and activate DNA damage repair pathway in NSCLC,also we elucidate mechanism underlying MSI2 affecting tumor radiation sensitivity,and find out target genes and signaling pathways regulated by MSI2 transcription.This study suggests that MSI2 is likely to become a new target for radiotherapy sensitization and a prognostic biomarker of NSCLC radiotherapy.