The Role And Molecular Mechanism Of PAX6 In The Metastasis And Cisplatin Resistance Of Non-small Cell Lung Cancer

Background: Lung cancer is the leading cause of cancer-related deaths in men and women worldwide.It is also the most common malignancy with the highest morbidity and mortality.Non-small cell lung cancer(NSCLC)accounts for 80% of lung cancer,about 50% of which is lung adenocarcinoma.Although some advance has been made in the treatment of lung cancer,distant metastasis remains a major contributor to disease-related deaths.At present,the first-line treatment of most metastatic NSCLC is still limited to platinum-based chemotherapy,which also leads to the rapid development of drug resistance in lung cancer.Existing studies have shown that epithelial-to-mesenchymal transition(EMT)is closely related to tumor metastasis,and cancer stem cells play an important role in the chemotherapy drug resistance.Paired-box 6(PAX6)is an important transcription factor for differentiation of human central nervous system,stem cells and neuroectodermal cells.Moreover,recent studies have shown that PAX6 can be expressed in tumors and has carcinogenic effects.However,its role in NSCLC is poorly understood.Therefore,to study the PAX6-mediated molecular mechanism related to NSCLC metastasis and overcome tumor chemotherapy resistance are important for establishing an effective treatment system of NSCLC.Objectives: In order to explore the clinical significance of PAX6 gene in NSCLC patients,this study first detected the expression of PAX6 gene in NSCLC and the relationship between PAX6 gene and clinical prognosis of lung cancer patients in clinical specimens.To further explore the influence of PAX6 on the invasion and metastasis of cancer cells during the malignant progression of NSCLC,we examine the biological function of NSCLC cells and tumor metastasis situation after silencing and overexpression of PAX6.Then we test the role of PAX6 in NSCLC cisplatin resistance in vivo and in vitro.Finally,the tumor metastasis RT~2 Profiler PCR chip was used to analysis and find the protein ZEB2(zinc finger E-box-binding protein 2)that may have specific binding sites with PAX6,and the corresponding verification and molecular mechanism were tested.This study provides new targets and corresponding molecular basis for the treatment of NSCLC metastasis and cisplatin resistance.,Methods: 1.The expression of PAX6 in lung cancer and its relationship with the prognosis of patients GEO data set(GSE30219)of GPL570 platform was extracted based on the GEO database,and survival analysis was performed according to the data.Then,it was verified in the lung adenocarcinoma tissue chip,and the survival curve was established by Kaplan-Meier method.Log-rank test was used for analysis and comparison.2.PAX6 promotes the EMT process,invasion and metastasis of NSCLC cells PAX6 interfering si RNA was purchased to screen effective sequence for constructing PAX6-silenced lentivirus,and PAX6 overexpression lentivirus p GMLV-CMV-PAX6 was purchased.Overexpressed PAX6 and silenced PAX6 virus were transfected in two NSCLC cell lines(A549,SPC-A-1),the expression levels of PAX6 were detected by q RT-PCR and western blot.Cell migration ability was detected by wound healing assay and transwell cell migration assay,cell invasion ability was detected by transwell cell invasion assay,and the expression levels of EMT-related marker proteins were detected by western blot and immunofluorescence assay.A model of lung metastasis in the tail vein of nude mice was established to detect the effect of PAX6 on the ability of metastasis in vivo.3.PAX6 promotes cisplatin resistance in NSCLC cells by regulating stem cell differentiation The expression levels of lung cancer stem cell key markers CD44,CD133 and ALCAM were detected by western blot and immunofluorescence assay in two NSCLC cell lines(A549,SPC-A-1)transfected silenced and overexpressed PAX6 virus.CCK-8 and colony formation assay were used to detect cell proliferation and sensitivity to cisplatin.Flow cytometry assay was used to detect cell apoptosis and cisplatin resistance.A nude mouse subcutaneous tumor model was established to investigate the effect of PAX6 on NSCLC cisplatin resistance in vivo.4.The mechanism of PAX6 in promoting NSCLC cell metastasis The tumor metastasis RT~2 Profiler PCR chip was used for sequencing analysis,and q RT-PCR and western blot were used for verification.ZEB2,a protein that may have specific binding with PAX6,was found,and the binding activity of both was analyzed by CHIP experiment,then the correlation between the two was detected by lung cancer tissue chip.STRING online analysis tool was used to predict ZEB2 preliminarily and the regulatory effect of PAX6 on ZEB2 and E-cadherin was investigated by q RT-PCR and western blot.The regulatory effect of PAX6 on the PI3K/AKT signaling pathway was further detected.Results: 1.The expression of PAX6 in lung cancer and its relationship with the prognosis of patients PAX6 expression was negatively correlated with the overall survival of NSCLC patients and the expression level of E-cadherin.In addition,PAX6 was highly expressed in NSCLC tissues and was associated with poor prognosis.Notably,PAX6 expression was correlated with advanced clinical stage and regional lymph node spread based on Chi-square test.PAX6 was an independent risk factor for the prognosis of NSCLC patients based on multivariate Cox regression analysis.2.PAX6 promotes the EMT process,invasion and metastasis of NSCLC cells We first established A549 and SPC-A-1 cell lines that were constitutively and stably silenced or overexpressed PAX6.PAX6 overexpression markedly reduced the levels of the epithelial marker E-cadherin and increased mesenchymal markers N-cadherin,Vimentin,and FSP-1,whereas PAX6 knockdown had the opposite effects,suggesting that PAX6 can promote EMT in NSCLC cells.Similarly,PAX6 overexpression significantly enhanced the ability of cell migration,invasion and intrapulmonary metastasis,whereas PAX6 knockdown suppressed these abilities.3.PAX6 promotes cisplatin resistance in NSCLC cells by regulating stem cell differentiation PAX6 overexpression significantly increased the expressions of CD44,CD133 and ALCAM,key markers of lung cancer stem cells,while PAX6 knockdown inhibited the expressions of the above genes,indicating that PAX6 has the role of regulating the stem cell transformation.Further studies showed that PAX6 could promote NSCLC cell proliferation,inhibit cell apoptosis,and reduce its sensitivity to cisplatin.The results of subcutaneous tumor model in nude mice also showed that PAX6 could promote the formation of subcutaneous tumor nodules and cisplatin resistance.4.The mechanism of PAX6 in promoting NSCLC cell metastasis ZEB2,the downstream regulatory target of PAX6,was screened by sequencing of human tumor metastasis RT~2 Profiler PCR chip,and the result was verified by q RT-PCR and Western blot.PAX6 can directly bind to the ZEB2 promoter region.ZEB2 was negatively correlated with the overall survival of NSCLC patients and the expression of E-cadherin,which was consistent with the previous trend of PAX6 results.Moreover,PAX6 knockdown could downregulate the expression of ZEB2 and upregulate the expression of E-cadherin,whereas PAX6 overexpression showed the opposite results.PAX6-mediated ZEB2 upregulation at the protein and m RNA levels were obviously reversed by PAX6 knockdown,whereas E-cadherin downregulation was reversed by PAX6 knockdown.Furthermore,the levels of phosphorylated PI3K(p-PI3K)and phosphorylated AKT(p-AKT)were dramatically increased by PAX6 overexpression,whereas opposite effects were observed in PAX6 knockdown.The increased levels of p-PI3 K and p-AKT observed after PAX6 overexpression were significantly reduced by PAX6 knockdown,and was significantly inhibited after the addition of PI3K-AKT inhibitor LY294002,while the levels of total PI3 K and AKT remained unchanged.Conclusions: We determined that PAX6 was highly expressed in NSCLC patients and negatively correlated with the overall survival of patients,and PAX6 could be used as an independent risk factor for the prognosis of NSCLC patients.PAX6 could promote the EMT process,cell migration,invasion and intrapulmonary metastasis of NSCLC cells at the cellular and animal levels.In addition,PAX6 enhanced the resistance of NSCLC cells to cisplatin by regulating the stem cell transformation of tumor cells,and promoted the proliferation of NSCLC cells and the formation of subcutaneous tumor nodules in nude mice.Moreover,it was found to regulate the transcriptional activity of ZEB2 and to directly bind to the promoter region of ZEB2.The PAX6-ZEB2 axis promotes metastasis by mediating E-cadherin downregulation through the PI3K/AKT signaling pathway,thereby mediating cell migration,stem cell transformation,and cisplatin resistance,ultimately affecting survival in NSCLC patients.Therefore,PAX6 can be used as a predictor for clinical prognosis of NSCLC,and PAX6 is expected to be a new therapeutic target to overcome NSCLC metastasis and cisplatin resistance.