Inhibitory Effect Of FoxO3a On The Cell Stemness Of Non-small Cell Lung Cancer In Vitro

Lung cancer is the leading cause of cancer-related death worldwide,and non-small cell lung cancer（NSCLC）represents the major histological subtype of the disease.Small molecule tyrosine kinase inhibitors and immunotherapy which are commonly used in the treatment of NSCLC have bring great survival benefits to specific patients,but the overall cure rates and survival rates of NSCLC remains poor due to the high heterogeneity and drug resistance.Evidence suggested that the growth ability of tumor depends on the stem cell-like properties of tumor cells,and the stemness are maintained through changing the self-renewal pathway（Wnt/?-catenin,Notch and Hedgehog）or regulating the major transcription factors（such as Oct4,Sox2 and Nanog）that related to self-renewal ability of embryonic stem cell.Besides,the stemness allows tumor cells to have self-renewal capacity and multi-directional differentiation potential,which are responsible for the tumor’s metastasis and resistance.Lots of studies have shown that the stemness of tumor cells play key roles in the resistance,metastasis and recurrence of NSCLC.Therefore,targeting developmental pathways that affect the stemness of tumor cells may be the key to preventing tumor resistance and treating cancer.In addition to participating in cell processes such as proliferation and apoptosis,as a transcription factor,FoxO3a also regulate life development processes related to stem cell maintenance,cell differentiation and organ development.FoxO3a may play a crucial role in tumor resistance by maintain the stemness of tumor cells.Here,we explored the role of FoxO3a in the resistance of NSCLC cell lines.Based on this,we investigated the effects of FoxO3a on the stem cell-like phenotypes of tumor cells such as migration and invasion,colony formation,self-renewal and the expression of stemness marker of NSCLC cells.We expect to explore the possible mechanisms involved and provide promising therapeutic strategies for addressing NSCLC resistance though targeting tumor cell stemness.ObjectivesThe study aims to detect the expression of FoxO3a in non-small cell lung cancer cells（NSCLC）lines and analyze its role in NSCLC resistance.The effects of FoxO3a on the resistance of NSCLC cell lines and the stem characteristics of tumor cells will be investigated.In addition,the regulatory mechanisms involved will be further studied to determine the effect of FoxO3a on tumor cell stemness in NSCLC.Methods1 The expression of FoxO3a in NSCLC cell linesqPCR and Western blot were used to detect the mRNA and protein expression levels of FoxO3a in five NSCLC cell lines including PC9,A549,H358,H1299 and H1975.2 Comparison of sensitivity to gefitinib of different NSCLC cell linesMTT assay was used to detect the sensitivity to gefitinib of five NSCLC cell lines including PC9,A549,H358,H1299 and H1975.Growth inhibition curves were drawn and IC₅₀0 values were calculated to compare the sensitivity of different NSCLC cell lines to gefitinib.3 Effects of FoxO3a on drug resistance of NSCLC cell linesTo explore the role of FoxO3a in NSCLC resistance,we transfected FoxO3a recombinant plasmid and FoxO3a-specific siRNA into PC9,A549 and H1299 cells by lipofection,respectively.Fluorescence observation,qPCR and Western blot were used to detect the transfection efficiency.After cells meet the needs of subsequent experiments after transfection,MTT assay was used to detect the changes of sensitivity of NSCLC cells to gefitinib after FoxO3a was changed,and the growth inhibition curve was drawn.4 Effects of FoxO3a on migration and invasion of NSCLC cell linesTo explore the regulation of FoxO3a on the homeostasis of tumor cell stemness in NSCLC,we examined the stem characteristics after changing the expression of FoxO3a.In this study,transwell assays were used to detect the migration and invasion ability of PC9,A549 and H1299 cells,and the number of migrated and invasive cells were counted using Image J software.5 Effects of FoxO3a on colony formation of NSCLC cell linesAfter overexpressing and silencing FoxO3a in PC9,A549 and H1299 cells,plate colony-forming assay and soft agar colony-forming assay were used to analyze the size and number of colony formation of NSCLC cell lines.The impact of FoxO3a on the independent growth ability and anchor-independence growth ability of NSCLC cell lines were compared.6 Effects of FoxO3a on self-renewal ability of NSCLC cell linesAfter overexpressing and silencing FoxO3a in PC9,A549 and H1299 cells,tumor sphere formation assay was used to detect the changes of self-renewal ability of NSCLC cell lines.7 Effects of FoxO3a on the expression of surface stemness markers in NSCLC cell linesAfter overexpressing and silencing FoxO3a in PC9,A549 and H1299 cells,CD133 and CD44 were selected as surface stemness markers of NSCLC cancer stem cells.After staining,flow cytometry was used to detect the proportion of CD133⁺CD44⁺in NSCLC cell lines.8 Regulate effects of FoxO3a on stem cell-related genes in PC9 cellsSeven stem cell-related genes PRR11,NONO,RYBP,MYCN,HMG-2,IFITM1 and LGR6 were screened out though pubmed and functional query.To investigate the regulation of FoxO3a on each gene,qPCR was used to detect the mRNA expression of them after overexpressing and silencing FoxO3a in PC9 cells.9 Prediction and verification of the binding of FoxO3a to stem cell-related genes in PC9 cellsThe potential binding sites to FoxO3a of three stem cell-related genes obtained by qPCR were predicted by the JASPAR database.The highest-scoring binding sites were selected for ChIP primer design.ChIP-qPCR and 1%agarose gel electrophoresis were used to confirm the enrichment efficiency of FoxO3a for each stem cell-related genes and the binding of FoxO3a to each gene’s promoter was analyzed.Results1 The expressions of FoxO3a in gefitinib sensitive cells and gefitinib resistant cells were different in NSCLCPC9 is sensitive to gefitinib while A549,H358,H1299 and H1975 are resistant to gefitinib.The results of qPCR and Western blot showed that the mRNA and protein expression levels of FoxO3a in H358,H1299,H1975 and A549 cells were lower than those in PC9 cells（P<0.01）.2 The sensitivities of different NSCLC cell lines to gefitinib were differentThe results of MTT showed that the growth inhibition rate of gefitinib against sensitive strains was significantly higher than that of resistant strains,that is,PC9was significantly more sensitive to gefitinib than other NSCLC cell lines.3 FoxO3a enhances sensitivity of NSCLC cell lines to gefitinibIn MTT experiments,FoxO3a overexpression significantly enhanced the inhibition of gefitinib on PC9 cells in a concentration-dependent manner with IC₅₀value decreased.The IC₅₀0 values of A549 and H1299 cells were also decreased.While silencing of FoxO3a significantly reduced the inhibition of gefitinib on PC9cells in a concentration-dependent manner with IC₅₀0 value increased.The IC₅₀0 values of A549 and H1299 cells were also increased.4 FoxO3a inhibits the ability of migration and invasion of PC9,A549 and H1299 cellsThe results of transwell migration assay showed that after overexpression FoxO3a,the migrated cell numbers of PC9,A549 and H1299 cells decreased from249.0?29.1,473.3?38.4,248.7?17.8 to 55.7?9.0（P<0.001）,53.0?1.0（P<0.01）,51.0?15.0（P<0.001）,respectively.While after silencing FoxO3a,the migrated cell numbers of PC9,A549 and H1299 cells increased from 363.0?30.1,452.0?40.6,274.7?34.0 to 477.0?44.2（P<0.05）,859.0?135.5（P<0.001）,684.7?40.2（P<0.001）,respectively.The results of transwell invasion assay showed that after overexpression FoxO3a,the invading cell numbers of PC9,A549 and H1299 cells decreased from324.3?34.3,478.3?62.5,269.67?27.1 to 122.7?10.1（P<0.001）,91.0?5.6（P<0.001）,50.0?43.1（P<0.001）,respectively.While after silencing FoxO3a,the invading cell numbers of PC9,A549 and H1299 cells increased from 434.3?15.1,549.3?49.2,254.7?40.3 to 686.7?26.2（P<0.001）,856.3?53.6（P<0.001）,487.3?45.8（P<0.001）,respectively.5 FoxO3a inhibits colony forming ability of PC9,A549 and H1299 cellsThe results of plate colony-forming assays showed that compared with the control group,the colonies formed by the cells after overexpression of FoxO3a were smaller,and the colony numbers of PC9,A549 and H1299 cells decreased from112.0?11.3,440.0?36.6,75.7?6.0 to 57.0?3.0（P<0.01）,187.7?4.9（P<0.01）,5.0?1.0（P<0.001）,respectively.While the colonies formed by cells in each group were larger after silencing FoxO3a,and the colony numbers of PC9,A549 and H1299 cells increased from 101.3?3.5,509.0?35,66.7?3.05 to 210.3?12.5（P<0.001）,1392.3?4.5（P<0.001）,116.0?6.0（P<0.001）,respectively.The results of soft agar colony-formng assays showed that compared with the control group,the colonies formed by the cells after overexpression of FoxO3a were smaller,and the colony numbers of PC9,A549 and H1299 cells decreased from 42.0?4.0,69.3?6.1,49.3?6.5 to 18.3?3.1（P<0.01）,34.7?0.6（P<0.001）,24.3?4.7（P<0.01）,respectively.While the silence of FoxO3a,the colonies formed by each group were larger,and the colony numbers of PC9,A549 and H1299 cells increased from 51.3?5.1,82.0?2.7,66.3?6.7 to 88.0?4.6（P<0.001）,139.3?2.5（P<0.001）,91.0?4.6（P<0.01）,respectively.6 FoxO3a inhibits self-renewal ability of PC9,A549 and H1299 cellsIn tumor sphere experiments,FoxO3a overexpression significantly reduced the sphere size of cells,and the sphere numbers of PC9,A549 and H1299 cells decreased from 5.0?1.0,6.7?2.1,8.0?1.0 to 0.3?0.6,1.0?0.0,0.7?0.6（P<0.05）,respectively.while FoxO3a silenced significantly increased the sphere size of cells,and the sphere numbers of PC9,A549 and H1299 cells increased from 7.7?2.5,3.3?1.2,3.7?0.6 to 15.3?4.2（P<0.05）,12.0?4.0（P<0.05）,20.3?2.5（P<0.05）,respectively.7 FoxO3a affects the expression of stemness markers in PC9,A549 and H1299 cellsIn flow cytometry experiments,FoxO3a overexpression decreased the proportion of CD133⁺CD44⁺in PC9,A549 and H1299 cells from 25.4%,74.7%,66.6%to 15.4%,42.6%,29.5%,respectively.While FoxO3a silenced increased the proportion of CD133⁺CD44⁺in PC9,A549 and H1299 cells from 0.1%,0.3%,35.8%to 1.1%,4.9%,62.4%,respectively.8 FoxO3a regulates the expression of stem cell-related genes in PC9 cellsThe results of qPCR showed that FoxO3a promotes the mRNA expression of PRR11 and NONO while inhibits the mRNA expression of IFITM1;FoxO3a affects the expression of MYCN,HMG-2 and LGR6 in mRNA levels,but the regulatory trends are inconsistent.FoxO3a has no regulatory effect on the expression of RYBP in mRNA levels.PRR11,NONO and IFITM1 can be selected as the focus in further study.9 FoxO3a directly binds to the promoter region of the stem cell-related geneThe predicted results of the JASPAR database showed that FoxO3a has potential binding sites in the promoter regions of PRR11,NONO and IFITM1 genes.The results of ChIP-qPCR and 1%agarose gel electrophoresis showed that FoxO3a has high enrichment efficiency at the binding sites selected by each gene and the fold change of enrichment（FoxO3a/IgG）of each gene is more than 5,which suggested ChIP positive.Conclusion1 FoxO3a is highly expressed in gefitinib-sensitive NSCLC cell lines and low in gefitinib-resistant NSCLC cell lines.2 FoxO3a enhances the sensitivity of NSCLC cell lines to gefitinib.3 FoxO3a inhibits the migration,colony formation,self-renewal ability and the expression of surface stemness markers of NSCLC cells.4 FoxO3a promotes the expression of PRR11 and NONO in mRNA levels while inhibits the expression of IFITM1 mRNA.5 FoxO3a directly binds to the promoter regions of the PRR11,NONO and IFITM1 genes.