RNA干扰UHRF1基因表达对脑胶质瘤U251细胞增殖和凋亡的影响

Effects of RNA interference on UHRF1 gene expression and proliferation and apoptosis of glioma U251 cells

目的探讨RNA干扰UHRF1基因表达对脑胶质瘤U251细胞增殖和凋亡的影响。方法采用脂质Attractene reagent将靶向沉默UHRF1的质粒LV-UHRF1-shRNA和阴性对照质粒LV-scramble-shRNA分别转染至U251细胞(LVUHRF1-shRNA组和LV-scramble-shRNA组),以仅转染脂质体的细胞为对照组;采用实时荧光定量PCR(QPCR)检测各组转染48 h后的UHRF1 mRNA水平,采用MTT法检测各组转染24、48、72 h的增殖情况,Brd U染色法检测转染12、24、48、72 h后的Brd U阳性率,Annexin V-FITC/PI双染流式细胞术检测转染48、72 h后的凋亡率,Western blotting检测转染72 h后的凋亡相关蛋白(Bax、caspase-3和Bcl-2)水平。结果 QPCR检测结果显示对照组、LV-scramble-shRNA组和LV-UHRF1-shRNA组的UHRF1水平分别为1.098±0.136、1.127±0.193和0.309±0.073,LV-UHRF1-shRNA组的UHRF1水平低于对照组和LVscramble-shRNA组(P<0.05)。与对照组和LV-scramble-shRNA组相比,LV-UHRF1-shRNA组在以上时间点细胞增殖率和Brd U阳性率均降低,差异有统计学意义(P<0.05);LV-UHRF1-shRNA组的凋亡率为(25.71±1.87)%,均高于对照组的(7.73±0.66)%和(7.86±0.59)%,差异有统计学意义(P<0.05);Western blotting检测结果显示与对照组和LV-scramble-shRNA组相比,LV-scramble-shRNA组的Bax、caspase-3水平升高,而Bcl-2水平降低,差异有统计学意义(P<0.05);对照组和LVscramble-shRNA组以上指标的差异均无统计学意义(P>0.05)。结论在U251细胞中的下调UHRF1可抑制增殖并诱导凋亡,UHRF1发挥类似癌基因的作用,可作为脑胶质瘤的治疗的候选基因。

Objective To investigate the effect of RNA interference on UHRF1 gene expression and proliferation and apoptosis of glioma U251 cells.MethodsThe plasmid LV-UHRF1-shRNA and negative control plasmid LV-scramble-shRNA were transfected into U251 cells(LV-UHRF1-shRNA group and LV-scramble-shRNA group)using lipid Attractene reagent,respectively.Cells only transfected liposomes were used as the control group.The UHRF1 mRNA level was detected by real-time fluorescent quantitative PCR(QPCR)at 48 h after transfection.MTT method was used to detect the proliferation of 24,48 and 72 h in each group.BrdU staining was used to detect the positive rate of BrdU after transfection of 12,24,48,72 h,and Annexin V-FITC/PI double staining dye flow cytometry was used to detect the apoptotic rate at 48 and 72 h after transfection.Western blotting was used to detect the levels of apoptosis related proteins(Bax,caspase-3 and Bcl-2)at 72 h after transfection.Results The results of QPCR test showed that the level of UHRF1 in the control group,the LV-scramble-shRNA group and the LV-UHRF1-shRNA group were 1.098±0.136,1.127±0.193 and 0.309±0.073,respectively.The UHRF1 levels in the LV-UHRF1-shRNA group was lower than those of the control group and the LV-scramble-shRNA group(P<0.05).Compared with the control group and the LV-scramble-shRNA group,the cell proliferation rate and the BrdU positive rate were decreased but the apoptosis rate increased in the LV-UHRF1-shRNA group(P<0.05).The apoptotic rate was(25.71±1.87)%in LV-UHRF1-shRNA group,higher than(7.73±0.66)%of the control group and(7.86±0.59)%of the LV-scramble-shRNA group,and the difference was statistically significant(P<0.05).The results of Western blotting showed that compared with the control group and the LV-scramble-shRNA group,the level of Bax and caspase-3 increased but the Bcl-2 level decreased in the LV-scramble-shRNA group,and the difference was statistically significant(P<0.05).There was no significant difference in the above indices between the control group and the LV-scramble-shRNA group(P>0.05).Conclusion The down-regulation of UHRF1 in U251 cells can inhibit proliferation and induce apoptosis.UHRF1 plays a role similar to oncogene,which can be used as a candidate gene for the treatment of glioma.