The Role Of CENPU In The Proliferation Of Lung Cancer Cells And The Immunosuppressive Function Of CD45~+ Erythroid Progenitor Cells

Part 1:The role of CENPU in the proliferation of lung cancer cells.Background:At present,lung cancer remains the leading cause of cancer-related death in the world.Non-small cell lung cancer(NSCLC)accounts for approximately 80-85%of lung cancers,and more than 50%of patients with NSCLC are initially diagnosed in advanced stages of the disease.Over the last decade,major progress has been made in the application of advanced medical technologies such as targeted therapies and immunotherapy to improve survival in patients with advanced NSCLC.Although the overall 5-year survival rate for lung cancer has increased,it remains less than 18%.Moreover,even for lung cancer patients diagnosed at an early stage,a higher recurrence rate cannot be ignored.Therefore,further elucidation of the molecular mechanism of lung cancer progression is important for exploring new effective therapeutic targets.Centromere protein U(CENPU),also known as myelodysplasia/myeloid leukemia factor 1 interacting protein(MLF1IP),Kaposi’s sarcoma-associated herpesvirus-latent nuclear antigen interacting protein 1(KL1P1),Cenp-50 or polo-box-interacting protein1(PBIP1),is a constitutive component of the centromere.It plays an important role in the process of cell cycle.It has been reported that CENPU could repress herpes simplex virus thymidine kinase promoter activity,while in ovarian cancer cells,CENPU could up-regulate the expression of HMGB2 and promote tumor cell proliferation,implying that CENPU could be a transcriptional repressor or promoter.Studies have shown that CENPU is highly expressed in glioblastoma cells and promotes glioma growth by inhibiting apoptosis of glioma cells;overexpression of CENPU predicting poor survival of breast cancer,and in prostate cancer,the expression of CENPU showed a large individual difference.However,the role and mechanism of CENPU in the development of NSCLC has not yet been explored.Further research of the expression and mechanism of CENPU in NSCLC is conducive to the discovery of new lung cancer therapeutic targets,and provides new ideas for improving the clinical prognosis of patients with lung cancer.Methods:1.Expression of CENPU in tumor tissues and normal lung tissues of NSCLC patients.(1)The online database UALCAN was used to analyze the the expression of CENPU in NSCLC samples.(2)Collect the NSCLC tumor tissues and normal lung tissues.(3)The m RNA and protein expression levels of CENPU in tumor tissues and normal lung tissues of NSCLC patients were detected by qRT-PCR and Western blot,respectively.2.The relationship between the expression level of CENPU and the prognosis of NSCLC patients.The online database PROGgene V2 and Kaplan Meier-plotter were used to analyze the relationship between the expression level of CENPU and the survival in NSCLC patients.3.Knockdown of CENPU expresiion could inhibit the proliferation of lung cancer cells.(1)Knock-down the expression of CENPU in lung cancer cells by siRNA transfection.(2)CCK-8 and Ed U staining were used to detect the effect of knockdown of CENPU on the proliferation of lung cancer cells.(3)The effect of knockdown of CENPU expression on the cell cycle process of lung cancer cells was detected by PI staining.4.Exogenous overexpression of CENPU promotes the proliferation of lung cancer cells.(1)Exogenous overexpression of CENPU in lung cancer cells by CENPU plasmid transfection.(2)The impact of up-regulation of CENPU on the proliferation of lung cancer cells was detected by Ed U staining.5.CENPU regulates the expression of the transcriptional molecule FOXM1 in lung cancer cells.(1)Download the expression data of CENPU and FOXM1 from the online database UCSC Xena,and performed correlation analysis by Graphpad Prism 6.0.(2)Knock-down the expression of CENPU in lung cancer cells by siRNA transfection.(3)qRT-PCR and Western blot were used to detect the expression of FOXM1 after knockdown of CENPU expression in lung cancer cells.(4)Knock-down the expression of FOXM1 in lung cancer cells by siRNA transfection.(5)qRT-PCR and Western blot were used to detect the expression of CENPU after knockdown of FOXM1 expression in lung cancer cells.(6)Ed U staining were used to detect the change of proliferation in lung cancer cells after co-transfection of si CENPU and FOXM1 expression plasmids.6.Knockdown of FOXM1 expression could inhibit the proliferation of lung cancer cells.(1)Download the expression data of FOXM1,PCNA and Ki-67 from the online database UCSC Xena,and performed correlation analysis by Graphpad Prism 6.0.(2)Knock-down the expression of FOXM1 in lung cancer cells by siRNA transfection.(3)CCK-8 and Ed U staining were used to detect the effect of knockdown of FOXM1on the proliferation of lung cancer cells.(4)The effect of knockdown of CENPU expression on the cell cycle process of lung cancer cells was detected by PI staining.7.Overexpression of FOXM1 predicts poor survival in patients with NSCLC patients.The online database PROGgene V2 and Kaplan Meier-plotter were used to analyze the relationship between FOXM1 expression and the survival of NSCLC patients.Results:1.CENPU was overexpressed in tumor tissues of NSCLC patients.(1)CENPU was up-regulated in tumor tissues,as compared to expression in normal lung tissues of NSCLC patients based on online database analysis.(2)The m RNA and protein expression levels of CENPU in tumor tissues of NSCLC patients were higher than in normal lung tissues,as detected by qRT-PCR and Western blot.2.Overexpression of CENPU predicted poor survival of NSCLC patients.High expression of CENPU was significantly associated with decreased OS in NSCLC patients based on the analysis from online database PROGgene V2 and Kaplan Meier-plotter.3.Knockdown of CENPU expression inhibited the proliferation of lung cancer cells.(1)qRT-PCR and Western blot confirmed that transfection of si CENPU could effectively knock down the expression of CENPU in lung cancer cells.(2)Knockdown of CENPU expression inhibited the proliferation of lung cancer cells,as measured by CCK-8 and Ed U assay.(3)Knockdown of CENPU expression induced the G1-M arrest of lung cancer cells.4.Exogenous overexpression of CENPU promoted the proliferation of lung cancer cells.(1)Transfection of CENPU plasmid could successfully up-regulate the expression of CENPU in lung cancer cells.(2)Overexpression of CENPU promoted the proliferation of lung cancer cells.5.CENPU regulated the expression of the transcriptional molecule FOXM1 in lung cancer cells.(1)Online analysis showed that the expression of CENPU was positively correlated with FOXM1 expression levels in tumor tissues of NSCLC patients.(2)RNA interference experiments confirmed that CENPU knockdown can down-regulate the expression of FOXM1.(3)Knockdown of FOXM1 did not affect the expression level of CENPU.(4)Over-expression of FOXM1 significantly reversed the inhibition of proliferation caused by CENPU knockdown.6.Knockdown of FOXM1 expression inhibited the proliferation of lung cancer cells.(1)Online analysis showed that the expression of FOXM1 was positively correlated with the expression level of PCNA or Ki-67 in tumor tissues of NSCLC patients.(2)qRT-PCR and Western blot confirmed that transfection of si FOXM1 can effectively knock down the expression of FOXM1 in lung cancer cells.(3)Knockdown of FOXM1 expression inhibited the proliferation of lung cancer cells,as measured by Ed U assay.(4)Knockdown of FOXM1 expression induced the G1-M arrest of lung cancer cells.7.Overexpression of FOXM1 predicted poor survival in patients with NSCLC.High expression of FOXM1 was significantly associated with decreased OS in NSCLC patients based on the online analysis.Conclusions:1.CENPU was abnormally up-regulated in tumor tissues of NSCLC patients.2.Overexpression of CENPU predicted a poor survival in patients with NSCLC.3.Knockdown of CENPU expression inhibited the proliferation of lung cancer cells.4.CENPU promoted the proliferation of lung cancer cells via FOXM1.5.Knockdown of FOXM1 expression in lung cancer cells could inhibite the proliferation of lung cancer cells.6.Overexpression of FOXM1 predicted a poor survival in NSCLC patients.Part 2:The immunosuppressive function of CD45~+erythroid precursor cells.Background:Patients with advanced malignant tumors often exhibited immunocompromised status,and were prone to viral or bacterial infections.Most of them complicated with anemia.Wether malignant tumor combined with anemia was the cause of impaired immune function?Our previous research found that advanced tumor bearing mice with anemia could induce the spleen producing a group of CD45~+Ter119~+CD71~+erythroid progenitor cell(EPC).These group of CD45~+EPCs accounts for about 15-20%of spleen nucleated cells,but the specific function and mechanism of this cells were still unclear.We will illustrate the function and mechanism of these cells,in order to explore a new ways of anti-tumor immunotherapy.Methods:1.The effect of CD45~+EPCs from spleen of advanced tumor-bearing mice with anemia on the proliferation of CD8~+T cells.(1)Flow-sorted CD45~+Ter119~+CD71~+EPCs from the spleen of LLC bearing mice or MMTV-Py MT spontaneous breast cancer mice with anemia.(2)Magnetic beads were used to sort the CD8~+T cells from the spleen of na(?)ve C57mice and labeled with CFSE.(3)Co-culture of CD45~+EPCs and CD8~+T cells in vitro.(4)The proliferation of CD8~+T cells was detected by flow cytometry after 3 days of co-culture.2.The effect of CD45~+EPCs from spleen of advanced tumor-bearing mice on the killing ability of antigen-specific CD8~+T cells.(1)Flow-sorted spleen CD45~+EPCs from spleen of advanced tumor-bearing mice.(2)Spleen antigen-specific CD8~+T cells were sorted on day 8 after LCMV-CL13infection.(3)CD45~+EPCs combined with antigen-specific CD8~+T cells were co-cultured with peptide coated target cells.(4)The killing ability of antigen-specific CD8~+T cells was detected by flow cytometry.3.Performed the RNAseq analysis of spleen CD45~+EPCs from tumor-bearing mice,anemia mice and newborn mice.(1)Sorted the spleen CD45~+EPCs from advanced tumor-bearing mice,anemia mice and newborn mice respectively,and sorted the spleen MDSCs of tumor-bearing mice as a control.(2)RNAseq detection and data analysis.4.Detection the ability to synthesize ROS of CD45~+EPC.(1)RT-PCR was used to detect the expression of NOX-2 in CD45~+EPC and CD45~-EPC.(2)Flow cytometry was used to detect the production of ROS in CD45~+EPC.5.Effect of blocking ROS on the function of CD45~+EPCs.(1)Sorted the spleen CD45~+EPCs of advanced tumor-bearing mice and co-cultured with CFSE-labeled CD8~+T cells.Blocked the production of ROS by apocynin.(2)CD45~+EPCs combined with antigen-specific CD8~+T cells were co-cultured with peptide coated targe cells,and blocked the production of ROS by apocynin.(3)The effect of apocynin treatment on CD45~+EPCs were detected by flow cytometry.Results:1.Spleen CD45~+EPCs from advanced tumor-bearing mice and MMTV-Py MT spontaneous breast cancer mice could significantly inhibit the proliferation of CD8~+T cells.2.Spleen CD45~+EPCs from advanced tumor-bearing mice and MMTV-Py MT spontaneous breast cancer mice could significantly inhibit the killing ability of antigen-specific CD8~+T cells.3.The transcriptome of CD45~+EPCs from advanced tumor-bearing mice closely resembled that of MDSCs4.Blocking ROS pathway could inhibit the immunosuppressive function of CD45~+EPCs.Conclusions:1.CD45~+EPCs possed the function of inhibiting the proliferation and killing ability of CD8~+T cells.2.Excessive ROS production is the primary mechanism for CD45~+EPCs mediated T cell immunosuppression.