Depleting PTOV1 Sensitizes Non-small Cell Lung Cancer Cells To Chemotherapy Through Attenuating Cancer Stem Cell Traits

Objectives:Lung cancer is the most common diagnosed cancer worldwide.Chemotherapy is an important component of treatment for all stages of non-small cell lung cancer(NSCLC).However,chemo-resistance represents one of the most significant barriers to improve outcomes of the patients.Prostate tumor overexpressed 1(PTOV1)has been reported to be upregulated in types of tumors,and its upregulation associates with tumor aggressiveness and a poor prognosis.However,its role in NSCLC has not been investigated.The aim of this study was to investigate the expression of PTOV1 in NSCLC and its correlation with the prognosis of patients with NSCLC,and further explore the effects and potential mechanisms of PTOV1 on chemosensitivity of NSCLC cells.Contents:To investigate the expression of PTOV1 in NSCLC and to analyze the correlation between the expression of PTOV1 and the prognosis of patients with NSCLC.2 SgRNAs(named Sg1 and Sg2 respectively)were designed to deplete PTOV1 using CRISPR/Cas9 system in NSCLC cell lines H460 and Calu3。The plate cloning assay,CCK8 assay,Annexin V-FITC apoptosis assay,Western blotting and other experimental methods were used to detect the sensitivity of tumor cell silencing PTOV1 to chemotherapeutic drugs.It was also explored that inhibition of PTOV1 can sensitize NSCLC cells by attenuating the stem cell traits of tumor cells.Finally,the nude mice were used for tumor formation experiments and histochemical experiments to further verify that inhibition of PTOV1 can sensitize NSCLC cells.Methods:Analysis of PTOV1 gene using multiple microarrays in the GEO database(https://www.ncbi.nlm.nih.gov/gds/)and RNA arrays or RNA seq data from the TCGA database(https://cancergenome.nih.gov/)Expression in normal lung tissue and lung cancer tissue.Survival rates were assessed for all lung cancer patients or subgroups of patients receiving chemotherapy with different PTOV1 expression levels using the Kaplan-Meier website(http://kmplot.com/analysis/).A total of 150 NSCLC patients who underwent histopathology and clinical diagnosis at the Cancer Institute and Hospital of Tianjin Medical University from December 2012 to January 2014 were used to analyze the correlation between PTOV1 expression and patient survival.Plate cloning assay,CCK8 assay,Annexin V-FITC apoptosis assay,Western blotting and other experimental methods were used to detect the sensitivity of tumor cell silencing PTOV1 expression to chemotherapeutic drugs.Application of sphere formation assays,Quantitative Real-Time PCR,flow cytometry,GSEA analysis,immunofluorescence and other experimental techniques to explore whether silencing PTOV1 can sensitize NSCLC cells by attenuating the stem cell traits of tumor cells.Finally,the nude mice were used for tumor formation experiments and Immunohistochemistry experiments to further verify that inhibition of PTOV1 can sensitize non-small cell lung cancer cells.Results:1.The PTOV1 gene was significantly upregulated in NSCLC.2.PTOV1 expression was an independent and poor prognostic factor in patients with NSCLC.3.Inhibition of PTOV1 expression increased the chemosensitivity of NSCLC cells in vitro.4.Inhibition of PTOV1 inhibited NSCLC cell migration and invasion,and increased chemotherapy-induced apoptosis.5.Depleting PTOV1 attenuated the stem cell-like traits of tumor cells and the β-catenin protein signal of NSCLC cells.6.Inhibition of PTOV1 expression sensitized NSCLC cells to chemotherapy in vivo.Conclusion:This study shows that PTOV1 is a poor prognostic factor in patients with NSCLC.Inhibition of PTOV1 can sensitize non-small cell lung cancer cells by attenuating the stem cell traits of tumor cells.Therefore,targeting PTOV1 may be a strategy to increase the sensitivity of NSCLC chemotherapy.