小干扰RNA抑制垂体特异性转录因子-1对大鼠生长激素垂体腺瘤细胞生物学行为的影响

Effect of interfering RNA targeting pituitary specific transcription factor-1 on biological behaviors in rat growth hormone pituitary adenoma cells

目的 观察小干扰RNA（siRNA）抑制大鼠垂体生长激素腺瘤（GH3）细胞中垂体特异性转录因子-1（Pit-1）基因的表达对其生物学行为的影响.方法 设计合成针对Pit-1基因的siRNA,经脂质体包裹转染GH3细胞.采用Western blot、逆转录-聚合酶链反应（RT-PCR）法检测siRNA作用后,Pit-1蛋白和Pit-1 mRNA表达的变化；应用细胞计数试剂盒（CCK-8）进行细胞增殖-毒性检测法检测细胞生长增殖的变化；应用流式细胞仪检测细胞凋亡的变化；制备Transwell小室测定GH3细胞体外侵袭能力的变化.结果 siRNA显著抑制Pit-1蛋白和mRNA的表达,与阴性对照组比较siRNA组GH3细胞增殖能力降低,36、48 h吸光度（A）值分别减少了0.46、0.41；24、36、48 h凋亡率分别增加了3.17％、6.36％、8.46％；侵袭力减弱,24、36、48 h穿膜细胞数分别减少了38.43、46.54、38.13个,差异有统计学意义（P＜0.05）.结论 Pit-1 siRNA能够有效抑制GH3细胞的生长,促进其凋亡,降低侵袭力.

Objective To observe the effect of small interfering RNA （siRNA） silencing pituitary specific transcription factor 1 （Pit-1） gene expression on the biological behaviors in the rat pituitary growth hormone adenoma cells （GH3）.Methods Pit-l-targeted siRNA was designed and synthesized,and transfected into GH3 cells with mixed with liposome.Western blotting and reverse transcription polymerase chain reaction （RT-PCR） were applied to detect the expression levels of Pit-1 protein and mRNA after siRNA-Pit-1 transfection.Cell proliferation was examined by using cell counting kit-8 （CCK-8） cell proliferation-toxicity test.Cell apoptosis was detected by using flow cytometry.In vitro invasion ability changes in GH3 cells were detected by Transwell cell array.Results siRNA significantly inhibited the expression of Pit-1 protein and mRNA.As compared with negative control group,cell proliferation ability was decreased with the absorbance （A） values being respectively decreased by 0.46 and 0.41 at 36 and 48 h,apoptosis rate was increased by 3.17％,6.36％ and 8.46％,and the number of cells penetrating the membrane was decreased by 38.43,46.54 and 38.13 at 24,36 and 48 h in siRNA group,respectively （P ＜ 0.05 for all）.Conclusion Pit-1 siRNA can effectively inhibit proliferation,promote apoptosis and reduce invasiveness of GH3 cells.