FHL2通过MGMT影响胶质母细胞瘤U87细胞对替莫唑胺的耐药性

FHL2 affects the resistance of glioblastoma U87 cells against temozolomide via MGMT

目的:探讨干扰四个半LIM结构域蛋白2(FHL2)的表达对胶质母细胞瘤U87细胞中O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)表达的影响,以及对U87细胞替莫唑胺(TMZ)耐药性的影响。方法:利用慢病毒感染技术分别将携带不同FHL2干扰序列的慢病毒(shFHL2-1#、shFHL2-4#)及其阴性对照(shN)感染U87细胞,分别命名为shFHL2-1#、shFHL2-4#和shN组;采用siRNA转染技术将siMGMT-1#、siMGMT-4#和siN转染至U87细胞,为siMGMT-1#、siMGMT-4#和siN组,qPCR和WB法验证FHL2或MGMT的敲低效果。用TMZ处理上述各组细胞(以DMSO处理组为对照),随后以CCK-8法和细胞克隆形成实验检测TMZ处理前后FHL2或MGMT敲低组细胞的增殖情况,FCM检测TMZ处理前后FHL2敲低组细胞的凋亡情况,WB法和免疫荧光法检测敲低FHL2对U87细胞中MGMT表达的影响,WB法检测TMZ处理对各组细胞中FHL2和MGMT表达水平的影响。结果:成功构建敲低FHL2或MGMT表达的U87细胞。与shN组相比,shFHL2-1#、shFHL2-4#组U87细胞的增殖能力减弱、凋亡水平升高(均P<0.01),MGMT表达水平明显降低(均P<0.01)。经TMZ处理后,与相应的DMSO处理组相比,shN组细胞中FHL2和MGMT的表达水平显著升高(均P<0.05),而细胞的增殖和凋亡均无显著变化(均P>0.05);shFHL2-1#、shFHL2-4#组细胞中FHL2和MGMT的表达水平无显著改变(均P>0.05),但细胞增殖能力进一步显著降低、凋亡水平进一步显著升高(均P<0.01)。敲低MGMT使U87细胞增殖减慢(P<0.01),而siMGMT-1#、siMGMT-4#组细胞经TMZ处理后增殖能力进一步降低(均P<0.01)。结论:干扰FHL2表达使得U87细胞增殖减慢而凋亡加剧、MGMT表达下调,提示FHL2可能通过影响MGMT的表达调控U87细胞对TMZ的耐药性。

Objective:To investigate the effect of interfering the expression of four and a half LIM-only protein 2(FHL2)on the expression of O~6-methylguanine DNA methyltransferase(MGMT)and temozolomide(TMZ)resistance of glioblastoma U87 cells.Methods:Lentiviruses carrying different sequences of FHL2 interference sequences(shFHL2-1#, shFHL2-4#)and negative control(shN)were infected into U87 cells, namely shFHL2-1# group, shFHL2-4# group, and shN group, respectively. siMGMT-1#, siMGMT-4#,and siN were transfected into U87 cells by siRNA transfection technology, namely siMGMT-1# group, siMGMT-4# group, and siN group, respectively. The FHL2 or MGMT knockdown efficiency was verified by qPCR and WB. The above groups of cells were treated with TMZ(the DMSO treatment group was used as the control), and then the proliferation of cells with FHL2 or MGMT knockdown before and after TMZ treatment was detected by CCK-8 method and cell clone formation assay, the apoptosis of cells in FHL2 knockdown group before and after TMZ treatment was detected by flow cytometry. WB method and immunofluorescence method was used to detect the effect of FHL2 knockdown on the expression of MGMT in U87 cells. WB method was used to detect the effect of TMZ treatment on the expression levels of FHL2 and MGMT in each group of cells. Results:Glioblastoma U87 cells with FHL2 or MGMT knockdown were successfully constructed. Compared with the shN group, the proliferation ability in cells of shFHL2-1# or shFHL2-4# group was significantly reduced while the apoptosis rate was significantly elevated(all P<0.01), and the expression of MGMT were significantly reduced(all P<0.01). After TMZ treatment, the expression levels of FHL2 and MGMT in the shN group were significantly increased(both P<0.05), while the proliferation and apoptosis of the cells were not significantly changed(all P>0.05)compared with the corresponding DMSO treatment group. The expression levels of FHL2 and MGMT in the cells of shFHL2-1# and shFHL2-4# groups did not change significantly(all P>0.05), but the cell proliferation capacity was significantly reduced, and the apoptosis level was significantly increased(all P<0.01). Knockdown of MGMT slowed down the proliferation of U87 cells in shN group(P<0.01), while the proliferation capacity of cells of siMGMT-1# and siMGMT-4# groups was further reduced after TMZ treatment(all P<0.01). Conclusion:Interfering with FHL2 expression weakened the proliferation ability of U87 cells and increased the apoptosis rate, and downregulated the expression of MGMT, suggesting that FHL2 may regulate the resistance of U87 cells to TMZ by affecting the expression of MGMT.