Molecular Mechanism Of Downregulation Of EFEMP1 Expression And Hypermethylat Ion Of DNA In Nonsmall Cell Lung Cancer

Objective:To investigate the down regulation of EFEMP1 expression in CD4+T cells and tumor cells in non-small cell lung cancer(NSCLC)patients and the molecular mechanism of DNA hypermethylation.Methods: 1.mRNA levels of EMEFP1 and cytokines in CD4+Tcells are detected by reverse transcription-quantitative PCR(QRT-PCR).EMEFP1 expression is detected by flow cytometry.2.Using the Bisulfite sequencing method(Bisulfite sequencing PCR,BSP)to detect the presence of DNA methylation in the lung tissue of the lung and the normal lung tissue of NSCLC in patients with NSCLC.In Western blot,the expression level of the lung tissue,normal lung tissue,EFEMP1 protein was detected in NSCLC patients.3.A549 cell line is treated with 5-Azacytidine(5-Aza)for 72 hours.Flow cytometry is used to detect the EFEMP1 expression between 5-Aza group and Control group.mRNA levels of EMEFP1 and cytokines in A549 cells are detected by QRT-PCR.Enzyme linked immunosorbent assay(ELISA)areused to detect EFEMP1 expression in cell serum and protein,respectively.Results: 1.QRT-PCR results show much lower mRNA expression levels in CD4+T cells of NSCLC patients,compared to normal controls(P<0.05).This result is also consistent to the results of flow cytometry,which shows lower EFEMP1 expression in NSCLC patients.While mRNA expressions of TNF-??IL-6?IL-8(P<0.05)are obviously higher in NSCLC patients.2.The BSP results showed a significant increase in DNA methylation(P < 0.05)in NSCLC patient lung cancer tissue cell EFEMP1 gene,compared to the normal lung tissue control group in NSCLC patients.The Western blot results showed that the EFEMP1 protein was significantly lower in the NSCLC patient group than the normal control group.3.QRT-PCR results show that 5-Aza group has higher m RNA expression level of EFEMP1,compared to control group(P<0.05).However,lower mRNA expression levels of TNF-??IL-6and IL-8are found in 5-Aza group(P<0.05).Higher EFEMP1 expression are found in cell serum and A547 cell protein by ELISA and Western Blot,respectively.Conclusions:DNA hypermethylation level of EFEMP1 may contribute to the reduction of EFEMP1 in NSCLC lung cells.