Expression Of Madd In Non-Small-Cell Lung Cancer Tissues And Its Effects On Proliferation And Apoptosis Of A549Cells

Lung cancer is the most common respiratory tract malignancy with the top incidence and mortality in China. Approximately30%of NSLC patients could be treated with surgery, but the five-year survival rate of them is no more than10%. In addition, the quality of postoperation life of NSLC patients is low. Moreover, patients at late stage can only treated with routine chemotherapy or radiotherapy, both of which show high toxicity and low curative effect. Therefore, to find novel therapy methods really has crucial and realistic meanings on NSLC treatment.TRAIL is one member of tumor necrosis factor super family which shows significant anti-tumor effects on several cancers without any side effects on normal tissue cells. At present, researches about TRAIL signaling pathway were focused on its effects on cancer treatment and many kinds of drugs, such as recombinant TRAIL protein and antibodies targeting death receptors, were already progessed to Phase II clinical trial. Combination therapy of TRAIL with other anticancer drugs shows excellent curative effects on enhancing cancer cell sensitivity and inhibiting tumor metastasis. But several tumor cell lines were inherently TRAIL resistant, which hinders the application of TRAIL in clinical trails. MADD is one of the six isoforms encoded by IG20gene. Cumulative evidences have proved that MADD was overexpressed in various cancer cells, including cells of lung cancer, and could inhibit tumor cell apoptosis via TRAIL-mediate signal pathway. Besides, knockdown of all IG20splice variants or the MADD variant not only induces spontaneous apoptosis but also shown to synergize with TRAIL. As a result, MADD can be regarded as a promising target on TRAIL resistance associated researches in lung cancer. However, researchs about the differential expression of MADD in lung normal and tumor tissues and its molecular mechanism on ADS proliferation and apoptosis are rare studied. In this study, both normal and tumor lung tissues belonging to different pathological types and stages were used to detect the expression of MADD. Plasmids carrying MADD gene or siRNA vectors targeting MADD were transfected into A549cells and the influence of MADD on proliferation and apoptosis of A549cell were analyzed.Methods:1. Immunohistochemistry was performed to detect MADD expression in lung normal and tumor tissues;2. RT-PCR were used to detect the expression of IG20in A549cells;3. Plasmids amplification;4. Lentivirus package, titer assay and amplification;5. Plasmids or vectors were effectively transfected into A549cells;6. Western blot was performed to detect the expression of MADD in transfected A549cells;7. MTT assay was used to detect the proliferation ability of transfected A549cells;8. Annexin V/FITC assay was determined to analyze cell apoptosis.Results:1. MADD was expressed in lung normal, SCC, and ADC tissues in which the levels ranked from low to high in sequence.2. Four splice variants of IG20, including MADD, were expressed in A549cells. 3. High purity plasmids were obtained.4. Plasmids or vectors were effectively transfected into A549cells.5. Western blot showed that both introduction of exogenous MADD gene and RNAi targeting MADD could effectively affect MADD expression in A549cells.6. MTT assay showed that MADD can promote the proliferation of A549cells.7. Annexin V/FITC assay demonstrated that over-expression of MADD could effectively inhibit A549cell apoptosis, and down-modulation of it could obviously enhance apoptosis.Conclusion:In this study, we found that MADD was over-expressed in Non-Small-Cell Lung cancer tissues compared with lung normal tissues. And MADD levels in tissues of different pathological types were various. Besides, transfection was performed to change the expression of MADD in A549cells and over-expression of it could promote the survival of lung adenocarcinoma cells while abrogation of it led to cell death. MADD improved cell survival rate by inhibiting cell apoptosis. MADD might be a potential therapeutic target spot for lung adenocarcinoma therapy.