CAJ | 학술논문

背景与目的：原癌基因Bmi-1是多梳基因家族中的一员，能调节正常干细胞和肿瘤干细胞的自我更新能力。近年来发现其在多种恶性肿瘤中表达上调。本文旨在观察Bmi-1基因沉默对肺腺癌A549细胞体内外增殖的影响，并初步探讨其机制。方法：根据本实验室设计的4条针对Bmi-1的小干扰RNA(siRNA)序列，选择一条已经证实最有效的序列作为靶序列和一条随机序列作为阴性对照，构建重组逆转录病毒siRNA表达载体并将其转染入A549细胞中；应用RT-PCR和蛋白质印迹法(Western blot)检测对Bmi-1基因的沉默效果；应用MTT比色法、台盼蓝拒染法及平板克隆形成实验检测Bmi-1-siRNA对A549细胞体外增殖的影响；利用流式细胞仪分析各组细胞的细胞周期；通过裸鼠腋窝皮下接种各组细胞，观察Bmi-1-siRNA对A549细胞在裸鼠体内的致瘤能力的影响；Western blot检测PTEN、p-AKT、cyclin D1、P21、P27蛋白表达。结果：Bmi-1-siRNA有效地沉默了Bmi-1基因mRNA和蛋白的表达；沉默Bmi-1基因的表达能够抑制A549细胞的体内外增殖能力，使干扰组细胞的细胞周期阻滞于G1期；沉默Bmi-1基因的表达后，干扰组细胞中PTEN、P21、P27蛋白增加，p-AKT、cyclin D1蛋白表达降低。结论：Bmi-1-siRNA通过使细胞周期阻滞于G1期来抑制肺腺癌A549的体内外增殖能力，这种抑制作用涉及cyclin D1和p-AKT表达下降以及P21/P27和PTEN的表达上调。
배경여목적：원암기인Bmi-1시다소기인가족중적일원，능조절정상간세포화종류간세포적자아경신능력。근년래발현기재다충악성종류중표체상조。본문지재관찰Bmi-1기인침묵대폐선암A549세포체내외증식적영향，병초보탐토기궤제。방법：근거본실험실설계적4조침대Bmi-1적소간우RNA(siRNA)서렬，선택일조이경증실최유효적서렬작위파서렬화일조수궤서렬작위음성대조，구건중조역전록병독siRNA표체재체병장기전염입A549세포중；응용RT-PCR화단백질인적법(Western blot)검측대Bmi-1기인적침묵효과；응용MTT비색법、태반람거염법급평판극륭형성실험검측Bmi-1-siRNA대A549세포체외증식적영향；이용류식세포의분석각조세포적세포주기；통과라서액와피하접충각조세포，관찰Bmi-1-siRNA대A549세포재라서체내적치류능력적영향；Western blot검측PTEN、p-AKT、cyclin D1、P21、P27단백표체。결과：Bmi-1-siRNA유효지침묵료Bmi-1기인mRNA화단백적표체；침묵Bmi-1기인적표체능구억제A549세포적체내외증식능력，사간우조세포적세포주기조체우G1기；침묵Bmi-1기인적표체후，간우조세포중PTEN、P21、P27단백증가，p-AKT、cyclin D1단백표체강저。결론：Bmi-1-siRNA통과사세포주기조체우G1기래억제폐선암A549적체내외증식능력，저충억제작용섭급cyclin D1화p-AKT표체하강이급P21/P27화PTEN적표체상조。
Background and purpose:The pro-oncogene Bmi-1 is a member of the polycomb-group family, can regulation of the proliferation and self-renewal of normal and tumor stem cells. In recent years, Bmi-1 has been found that it is overexpressed in varieties of human malignant tumors. The study aimed to observe the effects of Bmi-1-siRNA on the growth capacity of lung cancer cell line A549 in vivo and in vivo, and explore its mechanism. Methods:The most effective one as a target sequence was chosen from four Bmi-1 siRNA sequences which were designed by our lab, and one random sequence was chosen as a negative control. In short, the chemically synthesized siRNA and control sequences were connected to a retrovirus expressing vector, pSUPERretro-Neo plasmid, and then transfected into A549 cells. The stably transfected cells were cultured and passed. The level of mRNA and protein of Bmi-1 in A549 cells were assessed by RT-PCR and Western blot respectively. The proliferations of A549 cells in vivo was analyzed with MTT, trypan blue exclusion and plate colony forming methods. Flow cytometry was used for cell cycle analysis. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of A549 cells. The expressions of cyclin D, p21/27, p-AKT and PTEN were analyzed by Western blot. Results:Compared to A549-ctr and A549-wt cells, Bmi-1 mRNA and protein levels all signiifcantly reduced in A549-Bmi-1-siRNA cells. Bmi-1-siRNA inhibited the growth, colony formation in vitro and tumorigenesis in vitro of A549 cells, and the interference cells cell cycle arrested in G1 phase. In A549-Bmi-1-siRNA cells, p-AKT and cyclinD1 expression were down-regulated while p21/p27 and PTEN were up-regulated. Conclusion:Silencing Bmi-1 gene inhibits the proliferation of A549 cells through G1 phase arrest, which involves the downregulation of cyclin D/p-AKT and upregulation of p21/p27/PTEN.