Overexpression of LRIG1 gene enhances chemosensitvity of human bladder cancer T24 cell line to cisplatin

Abstract

Objective To investigate the effects and mechanisms of LRIG1 overexpression on chemosensitivity of human bladder cancer T24 cell line to cisplatin. Methods Human bladder cancer T24 cell line was used in the experiment since December 2012. Firstly, human bladder cancer T24 cells which were capable of highly expressing exogenous LRIG1 gene were established and identified. The T24 cells were divided into 3 groups: experimental group (transfected with Ad–Surp–LRIGl), control group (transfected with Adeno–SP–EGFP), blank control group (untransfected). Secondly, experiments of CDDP–induced apoptosis were divided into 4 groups: control group (untreated), CDDP group (treated with CDDP), CDDP/Adeno–SP–EGFP group (treated with CDDP/Adeno–SP–EGFP and CDDP), CDDP/Ad–Surp–LRIG1 group (treated with CDDP/Ad–Surp–LRIG1 and CDDP). The expression of mRNA and protein of LRIG1 and EGFR were detected by RT–PCR and Western blot. The effect of LRIG1 and/or CDDP on the expression of EGFR and pEGFR of T24 cells were measured by Western blot. The cellular sensitivity to CDDP was evaluated by cell counting Kit–8 (CCK–8). The cells apoptosis rates were examined by flow cytometry. Results The mRNA expression of LRIG1 and EGFR in experimental group, control group and blank control group were (2.79±0.15) and (0.65±0.05), (0.76±0.09) and (1.98±0.07), (0.66±0.05) and (2.05±0.04); the protein expression of LRIG1 and EGFR in experimental group, control group and blank control group were(1.98±0.12) and (0.92±0.05), (0.88±0.07) and (2.51±0.07), (1.00±0.08) and (2.42±0.09). The differences were significant between experimental group and the other groups(P 0.05). The IC50 values in CDDP group, CDDP/Adeno–SP–EGFP group and CDDP/Ad–Surp–LRIGl group were (30.96±0.57) μg/ml, (31.55±0.48) μg/ml and (14.09±0.31) μg/ml. The difference were significant between CDDP/Ad–Surp–LRIGl group and the other groups(P 0.05). The apoptosis rate in CDDP/Ad–Surp–LRIGl group [(69.77±2.14)%] was significantly higher than that in CDDP/Adeno–SP–EGFP group [(48.15±1.53)%], CDDP group [(46.82±1.23)%] and control group [(3.17±0.21)%] (P 0.05). The expression of total EGFR protein and nuclear pEGFR protein in control group、CDDP group、CDDP/Adeno–SP–EGFP group, CDDP/Ad–Surp–LRIGl group were (2.52±0.11) and (1.76±0.08), (1.22±0.05) and (2.75±0.15), (1.25±0.05) and (2.82±0.13), (0.32±0.02) and (1.08±0.04). The differences between CDDP group and control group, CDDP/Adeno–SP–EGFP group and CDDP/Ad–Surp–LRIGl group were significant(P 0.05). Conclusions LRIG1 promotes the cisplatin–induced apoptosis of T24 cells and enhances the sensitivity to cisplatin by down–regulating the expression of EGFR and pEGFR. Key words: Leucine–rich repeats and immunoglobulin–like domains–1 gene; Cis–diaminodichloroplatinum; Bladder neoplasm; Apoptosis