MAGE-D4 Expression, Serum Antibody Analysis In Non-small Cell Lung Cancer And Construction Of Gene Silenced Cell Line

OBJECTIVEThe purpose of this study was to detect the expression of MAGE-D4 protein in non-small cell lung cancer(NSCLC)and MAGE-D4 antibody in serum.The relationships between them and clinicopathological parameters of patients were further analyzed.Eukaryotic expression plasmid of MAGE-D4 was constructed and a cell line of lung cancer stably silencing MAGE-D4 gene was obtained to provide conditions for further study of the biological function of MAGE-D4.METHODS1.The expression of MAGE-D4 mRNA was analyzed by two independent lung cancer data sets in Oncomine oncogene chip database.The Hou Lung data set includes 91 lung cancer samples and 65 normal lung tissues.The Okayama Lung data set includes 226 lung cancer samples and 20 normal lung tissues.2.MAGE-D4 protein expression and cell localization was assayed by immunohistochemistry(IHC)in 81 NSCLC surgical specimens and 38 adjacent non-tumor tissues.The correlations between MAGE-D4 protein expression and clinicopathological parameters were evaluated.3.Sera from 74 NSCLC patients were tested for MAGE-D4 antibody by ELISA,and the correlations between the positive rate of MAGE-D4 antibody and clinicopathological parameters were evaluated.4.RT-PCR was used to detect the expression of MAGE-D4 in three lung cancer cell A549,H460 and PC9.Lentivirus with MAGE-D4/shRNA was used to infect lung cancer cell with high expression of MAGE-D4.A cell line stably silencing MAGE-D4 gene was obtained after puromycin screening.The interference efficiency of MAGE-D4 was detected by RT-qPCR and Western Blotting.5.DNA polymerase with high fidelity was used to amplify the full length sequence of MAGE-D4 coding region.T4 ligase linked target gene and pEGFP-C1 plasmid digested by Bgl II and Kpn I.Recombinant DNA was transformed into Escherichia coli DH5?.The plasmid of positive colonies was extracted and sequenced after kanamycin screening and identification by restriction enzyme digestion.Then the MAGE-D4/pEGFP-C1 plasmid was transfected into A549 cell with negative MAGE-D4 gene expression.RESULTS1.The results of oncogene chip database analysis showed that the expression of MAGE-D4 in lung cancer tissues was higher than that in normal lung tissues in two data sets(P < 0.001).2.MAGE-D4 protein was more frequently expressed in NSCLC(67.90%,55/81)than in adjacent non-tumor tissues(7.89%,3/38).The positive expression rate of MAGE-D4 protein was significantly correlated with Ki-67 > 10%(P < 0.05).There was no significant difference between MAGE-D4 expression and other clinicopathological parameters.MAGE-D4 protein was mostly localized in cytoplasm and in a few cases co-expressed in cytoplasm and nucleus.3.ELISA results showed that 18 of 74 patients with NSCLC were positive for MAGE-D4 antibody and the positive rate was 24.32%.The average titer of MAGE-D4 antibody in lung cancer patients' serum(0.8395±0.3588)was significantly higher than that in normal serum(0.6049±0.1848,P<0.05).There was no significant correlation between the positive rate of MAGE-D4 antibody and clinicopathological parameters.4.H460 cell with high expression of MAGE-D4 gene was selected as target cell for gene silencing experiment.H460 cell was successfully infected by lentivirus with MAGE-D4/shRNA and emitted green fluorescence.The interference efficiency of MAGE-D4 was 80% for mRNA and 61.3% for protein detected by RT-qPCR and WB,respectively.5.The inserted sequence in MAGE-D4/pEGFP-C1 was completely consistent with the full length sequence of MAGE-D4 coding region.The recombinant plasmid could express green fluorescent protein in A549 cell,which showed that the eukaryotic expression plasmid of MAGE-D4/pEGFP-C1 was successfully constructed.CONCLUSION: MAGE-D4 protein is highly expressed in NSCLC,and its expression may be related to the proliferative activity of the tumor.MAGE-D4 antibody can be detected in NSCLC serum,which suggests that MAGE-D4 may be a potential molecular marker for assistant diagnosis of lung cancer.Lung cancer cell with MAGE-D4 stable silencing and eukaryotic expression vector were successfully constructed,which laid a foundation for further study on the biological function and molecular mechanism of MAGE-D4.