Experimental Study Of EGFL7 Mediated Hypoxia-induced Multidrug Resistance In Non-small-cell Lung Cancer

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The correlation of multidrug resistance and EGFL7 expression in non-small-cell lung cancerObjective To explore the serum level and specimen expression of EGFL7 and MDR1/P-gp in non-small-cell lung cancer(NSCLC)patients,and further investigate the relationship between EGFL7 and P-gp,which according to the clinicopathologic features.Methods 93 NSCLC patients were collected in this experiment,which were further divided into surgery group and chemotherapy group.30 healthy people were collected as normal group.The serum levels of EGFL7 in each groups was examined by ELISA assay.The expression of EGFL7 and P-gp proteins in surgery group was also detected by IHC.The serum levels of EGFL7 m RNA and MDR1 m RNA in chemotherapy group were examined by RT-PCR and q RT-PCR.The correlations between EGFL7 and P-gp and their relationships with the clinicopathologic characteristics of NSCLC were also analyzed.Results Higher serum level of EGFL7 secretion in NSCLC group was observed than the normal group;the serum level of EGFL7 was conspicuous lower in surgery group than chemotherapy group.Surprisingly,the serum level of EGFL7 was further decreased in surgery group after operation;however,the serum level of EGFL7 was significantly increased in chemotherapy group which underwent a two-period Cisplatin-contained regimen treatment.The expression of EGFL7 and P-gp in NSCLC tissue were higher than in para-carcinoma tissue,which displayed a significant correlation.Furthermore,the expression levels of EGFL7 and P-gp in NSCLC were closely related with TNM staging,but had no significant relation with sex and age.Levels of EGFL7 m RNA and MDR1 m RNA were significantly increased in peripheral blood after chemotherapy inchemotherapy group.Moreover,EGFL7 m RNA was positive correlated with MDR1 m RNA.Conclusions EGFL7 was highly expressed in NSCLC patients,it was further increased after chemotherapy,which was positive correlated with MDR.EGFL7 maybe related to the multidrug resistance in NSCLC chemotherapy.Part? The correlation of EGFL7 and hypoxia-induced multidrug resistance in non-small-cell lung cancer cellObjective To further explore the effects of EGFL7 in NSCLC MDR in vitroMethods A549 NSCLC cell was cultured in cisplatin-containing medium from 0.05ug/ml to 2ug/ml to induce MDR cell(A549/CDDP).The crossing resistant and chemo-sensitivity characteristics of MDR cell and other NSCLC cells(PC9?SPC-A1?A549)after hypoxia culturing were evaluated by CCK-8 and Annexin V-PE/7-AAD assay.Meanwhile the expressions of EGFL7 and P-gp in those cells were detected by Western blot.Moreover,MDR cell and NSCLC cells were infected with LV-EGFL7-si RNA or LV-EGFL7 to further study MDR.Results The 2 ug/ml cisplatin-resistant cell line A549/CDDP was successfully established,it showed crossing resistance to Carboplatin,Taxol and Gemcitabine.Both CCK-8 and Annexin V-PE/7-AAD demonstrated that hypoxia could conspicuously decrease the chemo-sensitivity in MDR cell and NSCLC cells;Western blot showed that hypoxia could significantly upregulate the expressions of EGFL7 and P-gp(especially displaying a peak expression with a 4h hypoxia culturing).The level of P-gp and chemo-resistance in A549 was increased after infected with LV-EGFL7,which was opposite in A549/CDDP after infected with LV-EGFL7-si RNA.Conclusion Hypoxia could induce NSCLC cell chemotherapy resistance,which was related with the up-regulation of EGFL7.EGFL7 maybe participate in hypoxia-induced multidrug resistance in non-small-cell lung cancer cell.Part ? The mechanisms of EGFL7 mediated multidrug resistance in non-small-cell lung cancer cellObjective To study the underline mechanism of EGFL7-related multidrug resistance in NSCLCMethods A549 and A549/CDDP were infected with LV-EGFL7 or LV-EGFL7-si RNA.The changes of membrane proteins efflux function were detected by Rhodamine123,apoptosis was measured Caspase8,9 Kits.PI3K/Akt and Ras/Raf/MEK/ERK/ELK-1 signal pathways were detected in those cells by Western blot.Results The efflux of Rhodamine123 was conspicuously enhanced and Caspase 9 activities were significantly downregulated(without influencing Caspase 8 activities)in A549 cell after infected with LV-EGFL7;the level of Bax was significantly decreased in A549 cell after infected with LV-EGFL7,meanwhile Bcl-2,Mcl-1 and Survivin were conspicuously increased in A549 cell after infected with LV-EGFL7.Moreover,Akt/pm TOR/p70S6K1,Akt/GSK-3?/P-gp,Ras/Raf/MEK/ERK/ELK-1 signal pathways were activated in A549 cell after infected with LV-EGFL7.These results were reversed in A549/CDDP cell after infected with LV-EGFL7-si RNA.Conclusions EGFL7 modulates NSCLC MDR via regulating membrane protein transport and apoptosis which influenced by PI3K/Akt and Ras/MAPK signal pathway.EGFL7 maybe an important molecular target for predicting NSCLC MDR and a systematic target for the NSCLC MDR treatment.Part ? Silencing the expression of EGFL7 reverses the multidrug resistance of non-small-cell lung cancer cell in vivoObjective To investigate the phenotype of multidrug resistance in NSCLC through silencing the EGFL7 expression in vivo.Methods A549 was inoculated subcutaneously in ethylic nude mice to establish a subcutaneous xenotransplanted tumor model.Tumor-bearing mice were further divided into: control group,CDDP group(treated with CDDP),CP group(treated with CDDP and PBS),CLG group(treated with CDDP and LV-GFP),CLE group(treated with CDDP and LV-EGFL7-si RNA).Tumor size was measured per 4 days,the growth curve of tumorvolume was depicted according to the change of time;tumor weights were calculated in the 36 th day after mice were sacrificed.The expressions of EGFL7 and P-gp were detected by IHC.Results We successfully established the NSCLC cells A549 ethylic nude mouse xenofrafts,which was confirmed by H&E staining.In compare with other groups,LV-EGFL7-si RNA plus CDDP treatment could significantly inhibit the tumor growth and decline the tumor weight.IHC results showed that the expressions of EGFL7 and P-gp were conspicuously upregulated in xenofrafts tumor tissues,while the expressions of the above two proteins were significantly downregulated by LV-EGFL7-si RNA treatment.Conclusions Silencing the expression of EGFL7 could obviously restore the chemosensitivity of xenofrafts.Hence,on the basis of prior in vitro experiments,we further confirmed that EGFL7 was an important systematic target for predicting and treating NSCLC MDR.