RNA干扰Hedgehog信号通路对胃癌细胞增殖和凋亡影响及其机制的探讨

Effect of Hedgehog signaling pathway interfered by siRNA in gastric cancer cell proliferation and apoptosis and its mechanism

目的:观察RNA干扰Hedgehog信号通路关键因子Gli1对胃癌细胞的生物学行为的影响并分析相关机制。方法:人工合成靶向Hedgehog信号通路活化关键因子Gli1的siRNA(Gli1 siRNA)和对照无关siRNA(Con siRNA),应用脂质体将其转入人胃癌细胞SGC-7901,48 h后收集细胞,分别应用MTT法和流式细胞仪检测对细胞增殖、周期和凋亡的影响,同时应用TaqMan探针实时定量PCR检测靶基因Gli1、Hedgehog活化标志基因PTCH1、细胞周期负调控蛋白基因CDKN1A(p21)和抗凋亡基因Bcl-2的表达变化。结果:以单纯转染剂对照组为参照,siRNA转染后48 h,Gli1 siR-NA和Con siRNA的抑制率分别为(53.33±6.06)%和(13.33±6.11)%,两组差异有统计学意义,t=5.701,P=0.005,n=3;G0/G1期分别为(80.67±4.51)%和(66.00±3.10)%,两组差异有统计学意义,t=5.500,P=0.005,n=3;凋亡率分别为(15.97±1.76)%和(7.77±1.11)%,两组差异有统计学意义,t=4.671,P=0.01,n=3;Gli1siRNA转染细胞内Gli1、PTCH1、Bcl-2和CD-KN1A(p21)基因的相对(参照单纯转染剂)表达分别为对照的0.24±0.11、0.43±0.09、0.52±0.13和2.10±0.30。分别与Con siRNA相比4条基因表达均差异有统计学意义,t=9.759,P=0.001,n=3;t=8.645,P=0.001,n=3;t=4.940,P=0.008,n=3;t=5.962,P=0.004,n=3。结论:应用RNA干扰技术可以阻断Hedgehog信号通路关键因子Gli1的表达,抑制Hedgehog信号通路活化,从而有效地抑制胃癌细胞的增长,诱导凋亡,可能成为治疗胃癌新的生物靶向治疗途径。

OBJECTIVE： To study the effect of the small interfering RNA targeting Glil （Glil siRNA）, and the key factor of Hedgehog signaling pathway, on the bio logical behavior of gastric cancer cells and its mechanism. METHODS： Synthetic Glil siRNA and the irrelevant control siRNA （Con siRNA） were transferred into gastric cancer SGC-7901 cells. After 48 h the cell proliferation and the cell cycle and apoptosis were detected by MTT and flow cytometry. Simultaneously, the mRNA expression of the relevant genes including Glil, PTCH1, CDKNIA（p21）and BcI-2 were measured by TaqMan probe Rea-Time PCR. RESULTS： Glil siRNA and Con siRNA produced that the cell growth inhibition rates were （53.33± 6.06） % and （13.33±6.11）% （significant difference： t 5. 701, P= 0.005, n=3）, the G0/G1 phase were （80.67±4.51） and （66.00%±3.10）%（significant difference： t=5.500, P= 0.005, n= 3）, the apoptosis rate were （15.97 ± 1.76）% and （7.77±1.11）% （significant difference： t= 4. 671, P=0.01, n：3）, respectively. The mRNA relative quantity expression （RQ value refer to the blank transfec tion） of Glil, PTCH1, Bcl 2 and CDKN1A （p21） genes were0.24±0.11, 0.43±0.09, 0.52±0.13 and 2.10±0.30 in the Glil siRNA transferred cells with significant difference from the ConsiRNA （t 9. 759, P=0.001,n= 3; t=8.645, P=0.001,n=3;t=4.940, P=0.008,n=3; t= 5. 962, P = 0. 004, n = 3, respectively）. CONCLUSIONS： Application of RNA interference targenting to the key factor （Glil） of Hedgehog signaling pathway can effectively inhibite the growth of cancer cells, induce apoptosis, which may become a new way of biological treatment for gastric cancer.