利用慢病毒介导的shRNA在GT1-7细胞中敲低Srsf1及其对GnRH、Kiss1的影响

目的:探讨慢病毒介导短发夹RNA(shRNA)敲低丝氨酸/精氨酸富集剪接因子1(Srsf1)基因对小鼠GT1-7细胞中性发育相关基因促性腺激素释放激素(GnRH)、Kiss1的影响。方法:实验分为3组,即空白对照组、阴性对照组及shRNA干扰组。用Srsf1 shRNA慢病毒稳转GT1-7细胞,Real-time PCR和Western blot检测GT1-7细胞中Srsf1在mRNA和蛋白水平的变化,并检测稳转株中GnRH、Kiss1基因的表达情况。结果:慢病毒介导的shRNA成功感染了GT1-7细胞,与空白对照组及阴性对照组相比,shRNA干扰组中Srsf1在mRNA水平降低了45%(P0.001),在蛋白水平敲低了42%(P0.05)。在稳定低表达Srsf1的GT1-7细胞中,GnRH、Kiss1基因在mRNA水平也显著降低(P0.05)。结论:成功的构建了Srsf1基因敲低的细胞株。在GT1-7细胞中敲低Srsf1基因会抑制GnRH、Kiss1基因的表达。

Effect on GnRH, Kiss1 Gene with Lentivirus-shRNA Mediated Knock-down Srsf1 in GT1-7

ABSTRACT Objective: Investigating the effect of lentivirus-mediated shRNA knock-down serine/arginine-rich splicing factor 1(Srsf1) gene on gonadotropin-releasing hormone (GnRH), Kiss1 in the GT1-7 cell lines. Methods: In this experiment, control, negative control, and shRNA interference groups were designed. Stable cell clones were established by transfecting the lentivirus into the GT1-7 cells. Real-time PCR and Western blot were performed to detect the changes of the mRNA and protein levels of Srsf1. Meanwhile, the mRNA expression levels of GnRH and Kiss1 were also determined. Results: Lentiviral with Srsf1-shRNA was successfully infected into the GT1-7 cells. Compared tocontrol and negative control groups, the mRNA expression level of Srsf1 was reduced by 45% (P<0.001) in shRNA interference group andthe protein expression level was also decreased by 42% (P<0.05). In stable cell clones, GnRH and Kiss1 genes were also significantly reduced in mRNA levels (P<0.01). Conclusion: The stable cell clones were established successfully. It is confirmed that the mRNA expression levels of GnRH and Kiss1 decreased in GT1-7 cell lines with the Srsf1 knockdown.