The Effects And Mechanism Of TIGAR On The Differentiation Of Bmil~+ Stem Cells And The Repair Of Radiation-induced Intestine Damage

PurposeTo investigate the proliferation and radiosensitivity effects after knocking down of NEK2 gene by siRNA in human lung cancer A549 cells and the corresponding mechanism.Methods and MaterialsThe differential expression level of NEK2 mRNA in non small-cell lung cancer(NSCLC)was explored in Oncomine,which was further confirmed by Western blot in NSCLC cells(A549,H460 and H1299)and normal human lung cells(HFLIII and HBE).Next we carried out a series of in vitro experiments to find out whether knock down of NEK2 can affect the radiosensitivity of lung cancer cells:(1)NEK2 siRNA was transfected into A549 cells with liposome and NEK2 expression level was measured by Western blot;(2)The proliferation and radiosensitivity was detected by clone formation assay;(3)Cell cycle and apoptosis were analyzed by flow cytometry;(4)?-galactose glucoside enzyme dyeing experiment was used to detect the cellular scenescence;(5)Immunofluorescence experiment was used to detect the repair of DNA double strand break and to survey the mitotic and nuclear morphology;(6)Finally Western blot was performed to investigate the expression changes of apoptosis,cell cycle and DNA damage related proteins.ResultsThe expression level of NEK2 increased significantly in NSCLC.In vitro studies showed that:(1)The result of Western blot showed that transfection with two NEK2 siRNAs for 24 or 48 h could both successfully suppress the NEK2 expression in A549 cells;(2)NEK2 down-regulation inhibit the proliferation ability of A549 cells(p<0.05);(3)NEK2 down-regulation can improve the radiosensitivity of A549 cells,the DO values declined from 2.34 Gy to 2.03 Gy(siRNAl)and 1.65Gy(siRNA2),and the sensitizing enhancement ratio(SER)was 1.16(siRNA1)and 1.42(siRNA2);(4)As compared to negative control group,the apoptosis rate was enhanced after irradiation(from 10.42%to 16.68%),Western blot showed the decrease of the total PARP,Caspase3 and Bcl2 proteins,while the cleaved level of PARP and Caspase3 increased.The results were consistent with that in the flow cytometry;(5)The proportion of cells in G2/M phase rose in A549 cells transfected with NEK2 siRNA prior to irradiation as compared with the cells exposed to X ray alone.Western blot showed that down-regulation of NEK2 could decrease the expression levels of phospho-CDC25C and CDK1;(6)?-galactosidase staining showed that NEK2 knock down could promote the cellular senescence,the percentage increased from 11.70%to 26.65%;(7)As Compared with IR alone,down-regulation of NEK2 before IR could increase the radiation induced y-H2AX foci formation,while hinder the DNA damage repair(p<0.05).The results of Western blot showed that Rad51 protein was suppressed by siRNA,which were consistent with the y-H2AX experiment;(8)Immunofluorescence was carried out using the spindle and centrosome markers,the anti-a-tublin and anti-y-tublin antibodies respectively in A549 cells,the results indicated that the proportion of chromosomal segregation errors and the proportion of abnormal nuclei induced by radiation increased dramatically(17.82%to 44.64%).ConclusionsThe expression level of NEK2increased in NSCLC,and NEK2 siRNA could successfully suppress the NEK2 expression in A549 cells.NEK2 siRNA reduced the proliferation of A549 cells,it could also increased the radiosensitivity in A549 cell line.The radiosensitizing effects of NEK2 knock down were caused by enhancing the rate of apoptosis,cellular senescence and possibly mitotic catastrophe,which may be related to the prolonged G2/M arrest and the suppression of DSB repair.