| Purpose:PDIA6 was the differentially expressed protein screened by two-dimensional gel electrophoresis(2D-DIGE)combined with mass spectrometry(MS)technique between the non-small cell lung cancer(NSCLC)tisssues and the adjacent normal tissues in our previous studies.The aimed of this study was to further elucidate the clinical relevance,biological functions,and molecular mechanisms of PDIA6 in NSCLC,and to provide new insights for the diagnosis and treatment of NSCLC.Method:(1)Expression and clinical significance of PDIA6 in NSCLC:a.To detect the differential expression of PDIA6 in 18 pairs of NSCLC and the adjacent lung tissues,lung normal cell and NSCLC cells by using Western blotting method.b.To detect the expression of PDIA6 in NSCLC tissue microarrays by using immunohistochemistry(IHC),and to analysis the expression of PDIA6 and survival prognosis of NSCLC patients by using statistical method.(2)Cell proliferation assay:a.To establish stable cell lines for PDIA6 overexpression or knockdown.b.To investigate the effects of PDIA6 on NSCLC cell proliferation in vitro by using CCK8 and clone formation array.c.To further validate the NSCLC cell proliferation of PDIA6 in vivo by using nude mice tumor xenograft model.(3)Cell apoptosis assay:a.Cisplatin was used to establish the apoptosis cell model.To detect the effect of PDIA6 on cell apoptosis in NSCLC by using flow cytometry analysis(FCM),transmission electron microscopy(TEM),DAPI staining,Western blotting method.c.To observe the effect of PDIA6 on cell apoptosis in NSCLC after pretreatment with or without 20 ?M Z-VAD-FMK by using AO/EB staining and Western blotting method.(4)Cell autophagy assay:a.To detect the effect of PDIA6 on cell autophagy in NSCLC by using TEM,MDC staining,GFP-LC3 plasmid,Western blotting method.(5)The molecular mechanism of PDIA6 involvement in cell apoptosis and autophagy in NSCLC cells:a.To detect the effect of PDIA6 on JNK/c-Jun cell signaling pathway in NSCLC by using human phospho-kinase array,Western blotting,luciferase reporter assay and immunofluorescence assay.b.To detect the effect of PDIA6 on JNK/c-jun cell signaling pathway in NSCLC after pretreatment with or without JNK-specific inhibitor SP600125 by using FCM analysis and mRFP-GFP-LC3 dual fluorescent autophagy indicator system.c.To detect the expression of apoptosis and autophagy-related proteins in NSCLC after transfected with siRNA JNK by using Western blotting method.d.To detecte the interaction and co-localization between PDIA6 and MAP4K1 by using Co-immunoprecipitation(Co-IP)and Confocal microscopy analysis.e.To detect the expression of MAP4K1/JNK/c-Jun cell signaling pathway related proteins after transfected with siRNA MAP4K1 by using Western blotting method.Results:1.Expression of PDIA6 in NSCLC and clinical significanceIn this study,PDIA6 protein levels were also higher in NSCLC tissues and NSCLC cell lines than paired adjacent normal tissues and two normal lung cell lines(p<0.05),respectively,as measured by western blotting.Combined with the clinical pathological features of tissue microarrays,the expression of PDIA6 was not correlated with the NSCLC patients' the gender,age,tumor size,and pathological grade,but was correlated with the TNM stages(p=0.005),histological type(p=0.034),and lymph node metastasis(p=0.019).NSCLC patients with high expression of PDIA6 had much shorter overall survival than those with low PDIA6 expression by Kaplan-Meier analysis(p=0.0043).There was also a significant relationship between the high expression of PDIA6 and the shorter overall survival of patients with stage I(p=0.0386).Furthermore,PDIA6 expression was also as an independent prognostic predictor for overall survival in NSCLC patients as showed by multivariate analysis(p=0.009).2.Effect of PDIA6 on cell proliferation,apoptosis and autophagy of NSCLC cells(1)Construction of stable cell linesA549 cell line was used as model for overexpression of PDIA6,NCI-H520 and Anip973 cell lines were used as model for knockdown of PDIA6.(2)Cell proliferation assay:a.CCK8 and Clone formation array showed that PDIA6 promotes NSCLC cell proliferation in vitro.b.Nude mice tumor xenograft model further showed that PDIA6 promotes NSCLC cell proliferation in vivo.(3)Cell apoptosis assay:a.DAPI staining assay that PDIA6 knockdown significantly increased apoptosis rate(p<0.05),while PDIA6 overexpressed cells significantly decreased apoptosis rate(p<0.05).b.Transmission electronic microscopic showed that PDIA6 knockdown led to obviously ultrastructural changes,including microvillus disappearance,chromatin condensation and nuclear fragmentation.c.Western blotting showed that knockdown of PDIA6 noticeably decreased Bcl-2 expression and induced the activation of caspase-3 and caspase-9(p<0.05),whereas overexpression PDIA6 had the opposite effects on expression of these proteins,when compared to corresponding control groups.Meantime,expression of caspase-8 and caspase-12,respectively,did not have any significant changes upon PDIA6 knockdown or overexpression.d.After pretreatment with 20?M Z-VAD-FMK,the apoptotic ability of PDIA6 knockdown cell was weakened.(4)Cell autophagy assay:a.The TEM data revealed that the number of autophagosomes were increased in PDIA6 knocked down cells versus control cells.b.MDC data showed the staining intensity was increased in PDIA6 knocked down cells,but decreased in PDIA6 overexpressed cells.c.Transfection of GFP-LC3 plasmid data showed the number of GFP-LC3 puncta in PDIA6 knockdown NSCLC cells was increased.d.Western blotting showed that knockdown of PDIA6 noticeably decreased SQSTM1/p62 expression and upregulated levels of LC3II,Beclinl,and Atg5,whereas overexpression PDIA6 had the opposite effects on expression of these proteins.3.The molecular mechanism of PDIA6 involvement in cell apoptosis and autophagy of NSCLC cellsa.Human Phospho-Kinase Array combined with Western blotting showed that the phosphorylation levels of JNK and c-Jun were higher in the PDIA6 interference group than in the control group,whereas overexpression PDIA6 had the opposite effects.b.Luciferase reporter assay revealed that the JNK/c-Jun signaling activity was obvious decreased in PDIA6 overexpression cells.c.Immunofluorescence staining showed that phospho-c-Jun expression was significantly increased in the nucleus.d.The FCM analysis showed that depletion of PDIA6 decreased apoptosis rate after pretreatment with JNK-specific inhibitor SP600125.e.The mRFP-GFP-LC3 dual fluorescent autophagy indicator system data showed that SP600125 treatment significantly reduced either the yellow dots(autophagosomes)or red-only dots(autolysosomes)induced by PDIA6 knockdown.f.Western blotting data further revealed that inhibition of JNK activity using SP600125 or transfection with siRNA JNK obviously downregulated the levels of c-Jun phosphorylation,Cleaved caspase-3,and LC3II,in PDIA6 knocked down cells.g.Co-IP combined with MS to identify MAP4K1 as a protein that interacts with PDIA6.h.Confocal microscopy analysis data showed that PDIA6 and MAP4K1 have a co-localization between them in the cell cytoplasm.i.Knockdown of PDIA6 noticeably upregulated levels of p-JNK,p-MKK7,p-TAK1 and p-MAP4K1,whereas overexpression PDIA6 had the opposite effects on expression of these proteins.j.After transfection with siRNA MAP4K1,the expression level of MAP4K1,p-MAP4K1 and p-JNK were decreased in PDIA6 knockdown cells after transfection with siRNA MAP4K1.Conclusion:1.Expression of PDIA6 in NSCLC and clinical significance(1)PDIA6 was upregulated in NSCLC(2)The expression of PDIA6 was positively correlated with lymph node metastasis and TNM staging of NSCLC.(3)The expression of PDIA6 was negatively correlated with the survival time of NSCLC patients.(4)PDIA6 may be an independent prognostic factor for overall survival of NSCLC patients.2.Effect of PDIA6 on proliferation,apoptosis and autophagy of NSCLC cells(1)PDIA6 promotes NSCLC cell proliferation.(2)PDIA6 inhibits NSCLC apoptosis via intrinsic apoptosis pathway.(3)PDIA6-mediated autophagy contributes to apoptosis in NSCLC cells3.The molecular mechanism of PDIA6 involvement in cell apoptosis and autophagy of NSCLC cells(1)PDIA6 inhibits NSCLC cell apoptosis and autophagy through JNK/c-Jun signaling pathway(2)PDIA6 interacts with MAP4K1 to inhibit JNK/c-Jun signaling pathway... |
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