The Roles And Mechanisms Of NUSAP1 Regulating DNA Damage Repair Pathway In Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia(CLL)is a leukemia with the most frequent incidence in western countries.According to the latest SEER statistics,the incidence of the disease is 4.7/100,000,with more males than females(about 1.9:1),and the 5-year survival rate is about 87.9%.The disease characterized by varied clinical course and easy recurrence commonly occurs in elderly patients with a median age of 70 years.Del(17p)/TP53 mutation,NOTCH 1 mutation,CD49d,Immunoglobin Heavy Chain Variable Region(IGHV)mutation status,complex karotypes and staging systems(Rai stage and Binet stage)offer great value in clinical prognosis and treatment guiding.Novel drugs,including inhibitors of the B-cell receptor signaling pathway(idelalisib,acalabrutinib,ibrutinib and duvelisib)and inhibitors of the anti-apoptotic protein Bcl-2(Venetoclax),exhibit advantages over traditional chemotherapeutic immunotherapy regimens.Chimeric antigen receptor T cells(CAR-T)and allogenetic stem cell transplantation(alloSCT)can be used for cell immunotherapy in high-risk patients.In recent years,many genetic abnormalities have been identified to be associated with the course of disease invasion and resistance to genotoxic chemotherapy.Nucleolar and spindle associated protein 1(NUSAP1)is a microtubule-binding protein with a molecular weight of 55kD,which takes critical part in chromosome separation and spindle assembly.NUSAP1 is a substrate of SCFcyclin F and participates in ubiquitin-dependent proteolysis.In addition,NUSAP1 plays an important regulatory role in cancer biology.In previous studies,NUSAP1 was highly expressed in many human tumors,including colorectal cancer,prostate cancer and astrocytoma.NUSAP1 is considered a candidate biomarker for oral squamous cell carcinoma,esophageal squamous cell carcinoma,cervical cancer,breast cancer,hepatocellular carcinoma and prostate cancer.Up-regulation of NUSAP1 inhibits cell apoptosis,promotes tumor cell proliferation,invasion and metastasis by regulating Wnt/β-catenin,Hedgehog,PI3K/Akt and other pathways.DNA damage is a frequent phenomenon in the development of cancer.Defects in DNA damage response are the main factors that lead normal cells to acquire cancer-causing mutations.However,after tumorigenesis,malignant cells adapt to various mechanisms,such as repairing DNA damage caused by unexamined DNA replication,to manage their survival.The process of DNA repair refers to the repair of cells after activating DNA damage,which is usually manifested by cell cycle arrest,injury of activation to repair related pathways,cell adaptation to injury,or cell apoptosis.In addition,the ability of cancer cells to repair DNA damage induced by radiation or chemotherapeutic drugs is a potential mechanism for treatment resistance.DNA repair in CLL cells,which is thought to be a potential mechanism of drug resistance,limits the cytotoxic effects of chemotherapy.Based on these premises,inhibition of the DNA repair process has recently been considered as a promising approach to cancer treatment.On basis of NUSAP1 in CLL cell lines and abnormal expression of clinical specimens,this study aimed to explore its biological function of NUSAP1 in the development in CLL through the lenti-virus transfection to change NUSAP1 expression level.Furthermore,the molecular mechanism of NUSAPI regulate DNA damage repair pathways in CLL chemoresistance was also discussed to provide experimental foundation and theoretical basis for novel targets in CLL treatment improvement.Part I The biological role and clinical significance of NUSAP1 in the occurrence and development of chronic lymphocytic leukemiaObjective:Recent studies have shown that NUSAP1 is highly expressed in a variety of solid tumors and associated with poor cancer prognosis.However,the role and clinical significance of NUSAP1 in CLL remains unclear.The purpose of this study was to detect the expression of NUSAP1 in CLL cell lines and primary cells of patients,analyze the relationship between the its expression and the clinical prognosis of patients,for tentative exploration of its clinical significance.In addition,the cellular biological processes and signal transduction pathways of NUSAP1 enrichment in CLL cells were analyzed by RNA sequencing technology to predict its biological role.The expression level of NUSAP1 was bidirectional regulated by virus transfection technology in vitro experiments for further verification of effects of NUSAP1 on cell proliferation,apoptosis and cell cycle in CLL.Material and Methods:1.Bioinformatics analysis of GEO database;2.Case selection and specimen collection;3.Extraction of human peripheral blood mononuclear cells;4.Immunomagnetic bead sorting;5.Cell culture;6.RNA extraction,reverse transcription and real-time fluorescence quantitative polymerase chain reaction;7.Protein extraction and Western Blottingc;8.Lentiviral vector mediated silencing and overexpression of NUSAP1 gene;9.RNA-Seq sequencing technology;10.Annexin V-PE/7AAD assay was used to detect cell apoptosis.11.Cell Counting Kit-8(CCK8)assay for Cell proliferation;12.PI/RNase assay was used to detect cell cycle;13.Establishment of CLL xenograft mouse model;14.Statistical analysis.Results:1.Real-time quantitative PCR and Western Blotting results presented that the mRNA and protein expression levels of NUSAP1 were significantly increased in the samples of 51 CLL patients and CLL cell lines(MEC-1,EHEB)compared with control group(p<0.001)2.The high expression of NUSAP1 was statistically correlated with the high level of WBC count,high Binet stage,high Rai stage,high level of β2-microglobulin,IGHV unmutation,11 q/17p deficiency and p53 mutation.A significant difference between its expression level and overall survival was confirmed in the GSE22762 database(HR=3.15,p<0.05).3.In RNA sequencing results,KEGG pathway enrichment analysis results indicated that NUSAP1 was enriched in tumor pathway,such as PI3K-Akt signaling pathway,p53 signaling pathway,DNA repair pathway and cell cycle pathway.As indicated by the GO functional annotation,NUSAP1 is closely related to biological processes such as cell proliferation,cell cycle and stimulus response.GSEA gene enrichment analysis showed that NUSAP1 was related to DNA replication,mismatch repair,nucleotide excision and repair,base excision and repair,homologous recombination,and cell cycle.4.CLL cell lines were transfected with lentivirus to bidirectional regulation of NUSAP1 expression.Compared with the control group,cell proliferation activity of ShNUSAP1 group was significantly reduced(p<0.01),the ratio of apoptotic cells increased significantly(p<0.01),the cell cycle was arrested in G0/G1 phase(p<0.01).5.It was showed in vivo experiments that the tumor size of cells implanted with ShNUSAP1 was significantly lower than that in the Shcontrol group(p<0.01).Conclusions:NUSAP1 expression was abnormally elevated in CLL samples and cell lines,and was associated with poor prognosis of the patients.RNA sequencing analysis predicted that NUSAP1 might affect the occurrence and development of CLL by participating in cell proliferation,apoptosis,cell cycle and other biological functions.After NUSAP1 knockdown,the proliferation activity of MEC-1 cells was significantly reduced,the cell cycle of G0/G1 phase was arrested,apoptosis was increased,and subcutaneous tumorigenesis of mice was inhibited.The results of this study will provide a new theoretical basis for the study of the pathogenesis of CLL,accurate prognosis assessment and specific targeted therapy.Part Ⅱ NUSAP1 regulates drug resistance via DNA damage repair pathway in chronic lymphocytic leukemiaObjective:DNA repair in CLL cells,which is thought to be a potential resistance mechanism,limits the clinical efficacy of refractory/relapsed CLL patients.Inhibiting the DNA repair process has recently been considered a promising approach to cancer treatment.The purpose of this study was to regulate the expression of NUSAP1 in CLL cells by lentiviral transfection technology,and further explore the influence mechanism of NUSAP1 on downstream cell signaling pathway and cell drug resistance,so as to provide a theoretical basis for seeking new therapeutic targets to overcome CLL relapse and chemoresistance problems.Material and Methods:1.Cell culture;2.Lentiviral vector mediated silencing and overexpression of NUSAP1 gene;3.Domain plasmid transfection:4.Protein extraction and Western Blotting detection;5.Immunofluorescence staining;6.Cell Counting Kit-8(CCK8)assay for Cell proliferation;7.Co-immunoprecipitation(Co-IP);8.Statistical analysis.Results:1.Western blotting validation showed that protein levels of P-CHK2 and y H2AX were increased in NUSAP1 knockdown group compared with control group,and the protein levels of P-ATM and RAD51 were surpressed,which meaned DNA damage response activation.2.RAD51 protein expression in CLL cells of the overexpressed NUSAP1 group was enhanced compared with the control group through COIP,which proved that NUSAP1 could combine with RAD51 to perform effectively.3.Compared with the control group,the fluorescence of RAD51 staining was decreased in the down-regulated cells with NUSAP1 expression,while the staining of RAD51 cells was increased after the overexpression of NUSAP1.NUSAP1 was mainly expressed in the nucleus,while RAD51 was mainly expressed in the cytoplasm.4.It was proved by COIP that NUSAP1 bound to RAD51 both endogenous and exogenous,and directly bound to RAD51 through C-terminal domain.With transfection missing SAP domain plasmid to CLL cells,the expression level of RAD 51 decreased in NUS AP1-△ SAP transfection group compared with NUSAP1 upregulation group.5.After NUSAP1 knockdown,the viability of cells treated with fludalabine and ibrutinib was lower than that of the control group(p<0.01).After treated with drugs on the basis of transfecting RAD51 plasmid,the cell viability of MEC-1 and EHEB cells recovered after 48 h.Conclusions:In conclusion,NUS API regulate the DNA signaling pathway to affect the DNA damage repairing in CLL cells.Interrupting the expression of NUSAP1 gene increased the level of DNA damage in CLL cells,while overexpression of NUS API gene enhanced the DNA damage repairing in CLL cells.It is suggested that NUSAP1 could promote the growth of CLL cells through DNA damage repairing to strengthen the chemoresistance in CLL.The results of this study will provide new theoretical basises for the pathogenesis of CLL,accurate prognosis assessment and specific targeted therapy.