Study On The Radiation Sensitization Effect Of GDF11 In Human Lung Cancer Cells

Objective: Growth differentiation factor 11, also known as bone morphogenetic protein 11, is a member of the TGFβ superfamily and bone morphogenetic protein subfamily proteins. It is widely expressed in the spleen, pancreas, small intestine, kidney,muscle,developing nervous and other tissues,it is circulating in the blood, and decreased expression with increasing age. In the early embryo development, GDF11 is involved in the formation and differentiation of bone, kidney, pancreas, retina, olfactory nerves and other tissues and organs through the negative regulation of MAPK / AKT /m TOR signaling pathway, which is believed to be the key signal in the normal development of the embryo. In recent years, the study found that GDF11 in improving cognitive, reversing myocardial hypertrophy, regulating metabolism of skeletal muscle has very important function, it shows that GDF11 has a very important biological functions and potential applications in the mediation of cell aging.At present, the biological function of GDF11 is not clear, especially in tumor cells, rarely reported. In this study, we observed the expression of GDF11 in Lung cancer cells and normal cells; investigate the relationship between the GDF11 expression and ionizing radiation; explore the effect of up-regulated the expression of GDF11 on the radiation sensitivity of lung cancer cells. The objective of the study is to reveal the molecular mechanism and function of GDF11 in enhancing the radiosensitivity of lung cancer.Methods:1. HBE, BEAS-2B, H1299 and A549 cell lines as the research object, and detecte the content of GDF11 protein by western blot.2. Cells were treated with X- ray irradiation, and detecte the content of GDF11 protein by western blot.3. GDF11 primer sequences was designed and synthesized, and synthesized the target gene fragment by RT-PCR. Digested pc DNA3.1 plasmid was connected with GDF11 fragment. The plasmid was verified by agarose gel electrophoresis and sequenced. The constructed plasmid transfected into A549 cells,and the change of GDF11 protein was detected by western blot.4. A549 cells were transfected with GDF11 plasmid, X-ray irradiation and GDF11 combined with X-ray irradiation respectively, observed radiosensitivity by colony formation; the effect of GDF11 transfection on the growth and proliferation of A549 cells was analyzed by MTT assay; Annexin V / PI apoptosis kit, Flow cytometry analysis, western blot to detect the expression of Bcl-2, Bax, Puma to detection of apoptosis.Results:1. GDF11 expression in common lung cells,but the content of GDF11 was different in different cells, the expression of GDF11 in normal cells was higher than that in tumor cells2.The expression of GDF11 was induced by ionizing radiation.After ionizing radiation, the expression of GDF11 changes with time in 48h; In terms of dose, the expression of GDF11 increased with the increase of the dose in the 0~6Gy.3. Sequencing results confirmed that the GDF11 plasmid sequence correct, and successfully transfected into A549 cells.The expression results were verified by blot western, and the over expression rate was 200%.4. Clone formation experiment, transfected A549 cells, clone forming ability decreased significantly compared with the experimental control group, the colony formation rate decreased, an increased radiosensitivity about 35%; determined by MTT show that, GDF11 significantly inhibited the growth of A549 cells, the inhibition rate was about 22.3%;Flow cytometry display,In 24 h, the apoptosis rate of GDF11 transfection group was 27.17% higher than that of the control group 18%, In 48 h, the apoptosis rate of GDF11 combined irradiation group was 39.63% higher than that in the irradiation group 34.99%;Western blot detected the Bcl-2 expression was significantly reduced, the expression of Bax and Puma are up-regulated.Conclusion:1. GDF11 expression in common lung cells.2. Ionizing radiation increases the expression of GDF11, which was influenced by time and dose.3. Sequencing results confirmed that the GDF11 plasmid sequence correct, and successfully transfected into A549 cells.4. Overexpression of GDF11 significantly inhibite the growth of A549 cells,down regulation of Bcl-2, up regulation of Bax and Puma to promote apoptosis and radiosensitivity of A549 cells.