siRNA干扰蛋白质精氨酸甲基转移酶5表达对人胃癌SGC7901细胞增殖和克隆形成能力的影响

Effect of siRNA targeting protein arginine methyl transferase 5 on the proliferation and colony-formation in human gastric cancer SGC7901 cells

目的探讨小干扰RNA(siRNA)靶向抑制蛋白质精氨酸甲基转移酶5(PRMT5)表达后对人胃癌SGC7901细胞增殖和克隆形成能力的影响。方法设计靶向抑制PRMT5表达的特异siRNA(siRNA-1、siRNA-2),瞬时转染胃癌SGC7901细胞(干扰组),同时设置转染无义序列的阴性对照组。在瞬时转染48 h后采用Western blotting实验评估siRNA对PRMT5的干扰效果;采用CCK-8法检测siRNA抑制PRMT5表达后胃癌SGC7901细胞的增殖情况;采用Ed U法检测siRNA抑制PRMT5表达后胃癌SGC7901细胞的DNA复制情况;采用平板克隆形成实验检测siRNA抑制PRMT5表达后对胃癌SGC7901细胞克隆形成能力的影响。结果在胃癌SGC7901细胞中,瞬时转染siRNA-1和siRNA-2后PRMT5蛋白的表达水平均显著低于阴性对照组(P<0.01),表明两个siRNA具有良好的干扰效果,可用于后续实验。与阴性对照组相比,PRMT5表达干扰后的胃癌SGC7901细胞增殖速率显著下降,差异有统计学意义(P<0.01)。转染siRNA序列后,PRMT5干扰组(siRNA-1和siRNA-2)的Ed U阳性细胞百分比为(16.0±2.0)%和(19.5±3.0)%,远低于阴性对照组的(38.0±4.0)%,差异有统计学意义(P<0.01)。PRMT5干扰组(siRNA-1和siRNA-2)的克隆形成数分别为(50±6)个和(68±7)个,远低于阴性对照组的(120±11)个,差异均有统计学意义(P<0.01)。结论通过siRNA抑制PRMT5表达后可显著抑制胃癌SGC7901细胞的增殖和克隆形成能力,为胃癌的临床治疗提供新的策略和理论依据。

Objective To explore the effect of small interfering RNA（siRNA） targeting protein arginine methyhransferase 5 （PRMT5） on the proliferation and colony-formation in human gastric cancer SGC7901 cells. Methods siRNA-1 and siRNA-2 de- signed for specific targeting PRMT5 were transiently transfected into SGC7901 cells. Meanwhile, antisense sequence was used as nega- tive control（NC）. 48 h after transfection, Western blotting method was used to confirm the interfering efficacy to ensure that siRNA can be used for following experiments. CCK-8 assay was used to detect the proliferation of SGC7901 ceils after gene-specific siRNA was transfected. EdU assay was used to examine the DNA replication of SGC7901 ceils and plate colony-formation assay was used to evalu- ate the colony-formation of SGC7901 ceils when PRMT5 was silenced by siRNA. Results The level of PRMT5 protein was significant- ly decreased in SGC7901 cells when siRNA-1 and siRNA-2 were transfected, suggesting that siRNA-1 and siRNA-2 could be used for further studies. The proliferation rate of SGC7901 cells was significantly reduced when PRMT5 was silenced（P〈0. 01 ） in comparison with NC. Compared with NC, the positive rates of EdU cells interfered by siRNA-1 and siRNA-2 in PRMT5 group were（ 16. 0±2.0）% and （ 19. 5 ± 3.0） %, much lower than （ 38. 0± 4.0） % in NC with significance （ P 〈 0.01 ）. Furthermore, the numbers of SGC7901 colonies in PRMT5 group interfered by siRNA-1 and siRNA-2 were 50±6 and 68±7, lower than 120± 11 of NC with significance （P〈 0. 01）. Conclusion Knockdown of PRMT5 by gene-specific siRNA can inhibit the proliferation and colony-formation abilities in hu- man gastric cancer SGC7901 cells, which provide new methods and theoretical basis for the treatment of gastric cancer.