miR-7沉默表皮生长因子受体对胶质瘤细胞周期的影响

目的 研究miR-7阻遏脑胶质瘤细胞增殖周期由G1期向S期转化的内在机制.方法 脂质体转染miR-7表达质粒进入人脑胶质瘤细胞株U251,原位杂交和RT-PCR方法检测外源miR-7基因的整合效果;流式细胞术检测细胞周期变化,Western blot方法检测EGFR、CyclinD1、CyclinE、CDK2、CDK4、CDK6、P16、P21和P27的蛋白表达;SPSS13.0软件进行统计学分析.结果 RT-PCR结果显示miR-7基因被成功整合入U251胶质瘤细胞,原位杂交结果示miR-7主要定位于细胞核和细胞质;转染后瘤细胞增殖速度减慢,且大部分阻滞于G1期,处于分裂期S期的细胞比例明显减少(P＜0.05);miR-7转染组EGFR、CyclinD1、CyclinE、CDK2、CDK4和CDK6蛋白表达水平均有不同程度下降(P＜0.05),其中EGFR、CyclinD1、CDK4和CDK6的降低程度更为显著(P＜0.01),P16水平显著升高(P＜0.01),P21和P27的表达没有明显变化(P＞0.05).结论 miR-7可能通过有效沉默癌基因EGFR的表达途径,抑制细胞周期G1期调节蛋白复合体CyclinD1/CDK4/6的活性,并接受P16的协同作用,共同阻遏胶质瘤由G1期向S期转化,进而抑制肿瘤增殖;miR-7有望成为一种新的胶质瘤治疗候选药物.

Effect of epidermal growth factor receptor gene silence by miR-7 on inhibiting glioma cell cycle

Objective To study the internal mechanism of inhibition of glioma cell cycle from G1 phase into S phase by miR-7.Methods After transfecting plasmid containing miR-7 into U251 glioma cell line,hybridization in situ and real-time PCR were used to detect the gene integration,and the cell cycle was detected by flow cytometry.The protein expression level of EGFR,CyclinD1,CyclinE,CDK2,CDK4,CDK6,P16,P21 and P27 were detected by Western blot.The corrclation among them was analyzed by software SPSS 13.0.Results The real-time PCR showed that miR-7 gene was successfully integrated into U251 glioma cells,and it was mainly localized in the nucleus and cytoplasm from in situ hybridization results.After miR-7 gene transfection,the cell proliferation rate slowed down,and most of them were inhibited in G1 phase in mitotic cells.As meanwhile,the percentage of S phase cells decreased significantly ( P ＜0.05).The expression level of miR-7 was negatively correlated with EGFR,CyclinD1,CDK4,and CDK6 ( P ＜ 0.01 ),and was negatively correlated with CyclinE and CDK2 (0.01 ＜ P ＜0.05),but was positively correlated with P16 ( P ＜0.01 ).There was no correlation with P21 and P27 ( P＞ 0.05 ).Conclusion Through effective silencing the expression of oncogene EGFR,miR-7 may inhibit the activity of protein complex CyclinD1/CDK4/6,which is an important cell cycle G1 phase regulatory.With the P16 synergies,they inhibit glioma cell cycle from G1 phase into S phase.So miR-7 is expected to become a new drug candidate for glioma treatment.