Effect And Mechanism Of LRIG1Gene On Proliferation And Radiosensitivity Of Glioma Cells

Objective:To up-regulate LRIG1gene expression in human glioma cells with liposomal transfection. To investigate the effect and mechanism of LRIG1gene on the cell cycle, apoptosis, DNA repair, cell proliferation and radiosensitivity of human glioma cells.Methods:Got the LRIG1overexpression human glioma U251cells by the method of liposome-mediated transiently transfection. Selected and set up a stable LRIG1overexpression cell line by G418. Detected the changes of LRIG1, P27, Bax, Bcl-2and Rad51expression in the cells with Western blot or RT-PCR. The cell growth inhibition rate was detected by CCK-8method. The cell cycle distribution and apoptosis rate were detected by flow cytometry after staining. The cell radiosensitivity was detected by clonogenic assay. The repairment of DNA damage before and after radiation were detected by comet assay method. Results:The LRIG1line was set up successfully. There was expression of green fluorescent protein in the LRIG1over-expression U251cells, observed by the fluorescence microscope. The expression of LRIG1protein in the stable LRIG1overexpression cell line was higher than the blank vector control cell line (P<0.05). Transiently transfected with plasmid encoding LRIG1protein, the expression of LRIG1mRNA in human glioma cells was increased (P<0.05). The over-expression of LRIG1significantly inhibited the proliferation of human glioma cells. After transfected with the LRIG1gene72hours, the inhibition rate of cell proliferation was up to26.4%. The over-expression of LRIG1up-regulated the expression of cell cycle inhibitor P27(P<0.01) and increasd the cell ratio of G0/G1phase from (38.5±2.1)%to (61.2±3.1)%(P<0.01). Thus the cell proliferation index was reduced by the over-expression of LRIG1. The over-expression of LRIG1increased the expression of pro-apoptotic protein Bax (P<0.05), while significantly reducd the expression of anti-apoptotic protein Bcl-2(P<0.01). Compared with (8.8±0.3)%of the control group, the cells apoptosis rate of the LRIG1over-expression group was up-regulated to (16.6±0.8)%(P<0.01). The radiosensitivity of human glioma cells was significantly enhanced by the over-expression of LRIG1in the clonogenic assay. The SER was1.448. DNA repair protein Rad51was significantly inhibited by LR1G1before and after radiation(P<0.01, P<0.01). The DNA damage repairment of human glioma cells was inhibited by LRIG1in the comet assay(P<0.05).Conclusion:The human glioma U251cell line stably expressing LRIG1protein was successfully set up in the experiment. The cell cycle inhibitor P27was up-regulated by the LRIG1overexpression. The the ratio of human glioma cells in the G0/G1phase was increased, and then cell growth was inhibited. The overexpression of LRIG1promoted the apoptosis of human glioma cells by the up-regulation of pro-apoptotic protein Bax and the down-regulation of anti-apoptotic protein Bcl-2. The expression of DNA repair protein Rad51was inhibited by the overexpression of LR1G1in human glioma cells before and after radiation, and then the repairment of DNA damage before and after radiotherapy was down-regulated. In summary, LRIG1can suppress the proliferation and enhance the radiosensitivity of human glioma cells. The research provided new evidences for the clinical application of LRIG1treating human gliomas.