CAJ | 학술논문

背景与目的：Bmi-1(B-cell specific moloneymurine leukemiavirus insertion site 1)基因是多梳基因家族的重要成员之一，主要通过调控INK4a/ARF位点来调节细胞的增殖和衰老。本研究旨在探讨Bmi-1-siRNA对具有INK4a/ARF位点的肺腺癌SPC-A1细胞生长和增殖的影响，并探讨其作用机制。方法：①本实验选用已确定有效的siRNA序列进行病毒包装，构建反转录病毒si-Bmi-1 pSUPERretro-neo，然后感染到SPC-A1细胞中，建立稳定表达Bmi-1-siRNA的肺癌细胞株。②利用RT-PCR和蛋白质印迹法(Western blot)技术分析Bmi-1基因在Bmi-1-siRNA转染后SPC-A1细胞中表达情况。③利用台盼蓝拒染法、MTT法、平板克隆形成实验和裸鼠实验，分析Bmi-1-siRNA对SPC-A1细胞体内外增殖能力的影响。④利用流式细胞术分析SPC-A1各组细胞的周期分布。⑤利用Western blot法分析增殖通路相关分子：p16INK4a、p53、Cyclin D1、PTEN和Ser473p-Akt等蛋白表达情况。结果：反转录病毒介导的Bmi-1-siRNA被转染后，有效抑制了SPC-A1细胞中Bmi-1基因的转录和表达，抑制了SPC-A1细胞的体内外增殖能力(P<0.01)，并使转染组细胞阻滞在G1期[(64.6±1.2)%，P<0.05]。沉默Bmi-1基因后，与对照组细胞相比，p16INK4a、p53和Akt蛋白表达水平无明显变化(P>0.05)，Cyclin D1和Ser473 p-Akt表达水平下降(P<0.01)，PTEN表达水平上调(P<0.01)。用PTEN抑制剂处理转染组细胞后，Bmi-1和Ser473 p-Akt蛋白表达得以重塑。结论：Bmi-1-siRNA通过将肺腺癌SPC-A1细胞周期阻滞于G1期来抑制肿瘤细胞增殖，这种抑制作用可能不依赖于p16INK4a来调控Cyclin D1的表达，进而参与调控肿瘤细胞增殖。
배경여목적：Bmi-1(B-cell specific moloneymurine leukemiavirus insertion site 1)기인시다소기인가족적중요성원지일，주요통과조공INK4a/ARF위점래조절세포적증식화쇠로。본연구지재탐토Bmi-1-siRNA대구유INK4a/ARF위점적폐선암SPC-A1세포생장화증식적영향，병탐토기작용궤제。방법：①본실험선용이학정유효적siRNA서렬진행병독포장，구건반전록병독si-Bmi-1 pSUPERretro-neo，연후감염도SPC-A1세포중，건립은정표체Bmi-1-siRNA적폐암세포주。②이용RT-PCR화단백질인적법(Western blot)기술분석Bmi-1기인재Bmi-1-siRNA전염후SPC-A1세포중표체정황。③이용태반람거염법、MTT법、평판극륭형성실험화라서실험，분석Bmi-1-siRNA대SPC-A1세포체내외증식능력적영향。④이용류식세포술분석SPC-A1각조세포적주기분포。⑤이용Western blot법분석증식통로상관분자：p16INK4a、p53、Cyclin D1、PTEN화Ser473p-Akt등단백표체정황。결과：반전록병독개도적Bmi-1-siRNA피전염후，유효억제료SPC-A1세포중Bmi-1기인적전록화표체，억제료SPC-A1세포적체내외증식능력(P<0.01)，병사전염조세포조체재G1기[(64.6±1.2)%，P<0.05]。침묵Bmi-1기인후，여대조조세포상비，p16INK4a、p53화Akt단백표체수평무명현변화(P>0.05)，Cyclin D1화Ser473 p-Akt표체수평하강(P<0.01)，PTEN표체수평상조(P<0.01)。용PTEN억제제처리전염조세포후，Bmi-1화Ser473 p-Akt단백표체득이중소。결론：Bmi-1-siRNA통과장폐선암SPC-A1세포주기조체우G1기래억제종류세포증식，저충억제작용가능불의뢰우p16INK4a래조공Cyclin D1적표체，진이삼여조공종류세포증식。
Background and purpose:The human oncogene B-cell-speciifc moloney murine leukemia virus integration site 1 (Bmi-1) is an important member of the polycomb group family, and it regulates cell proliferation and senescence via INK4a/ARF locus. This study investigated the effects of Bmi-1-siRNA on the proliferation of lung adenocarcinoma cell line SPC-A1 cells with INK4a/ARF locus and clarify the mechanism of Bmi-1-mediated effect on proliferation of lung adenocarcinoma cells. Methods:In this study, we chose the most efifcient siRNA chain the pGeneshl-2-Bmi-1 sense-1 and inserted into a pSUPER-retro-neo retroviral vector. The packaged si-Bmi-1 pSUPERret-ro-neo retroviral vector was stably transfected into lung adenocarcinoma SPC-A1 cell line. The stably transfected cells were cultured and passed. After transfection, the levels of Bmi-1 mRNA and protein expression of SPC-A1 cells were analyzed by RT-PCR and Western blot respectively. Trypan blue, MTT and plate colony forming assay were performed to observe the proliferation capibility of SPC-A1 cells and evaluate the cloning forming ability in vitro. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of SPC-A1 cells. Cell cycle distribu-tion was analyzed by lfow cytometry (FCM) in SPC-A1 cells. The expression levels of proliferation proteins including p16INK4a, p53, Cyclin D1, PTEN, Akt and Ser473p-Akt were analyzed by Western blot. Results:The mRNA and protein expression levels of Bmi-1 were signiifcantly reduced in SPC-A1-Bmi-1-siRNA cells transfected with pSUPER-retro-neo retroviral vector. Knockdown of Bmi-1 could inhibit the growth, colony formation in vitro and tumorigenesis in vivo of SPC-A1 cells (P<0.01). The transfected SPC-A1 cells were arrested in G1 phase [(64.6±1.2)%, P<0.05]. Compared with two control groups, p16INK4a, p53 and Akt were not affected (P>0.05), while Cyclin D1 and Ser473p-Akt were downregulated (P<0.01) and PTEN was up-regulated (P<0.01) in the SPC-A1-Bmi-1-siRNA cells. SPC-A1-Bmi-1-siRNA cells were treated with various concentrations of PTEN inhibitor to determine expression levels of PTEN, Bmi-1 and Ser473p-Akt protein. Ablation of PTEN rescued Bmi-1 and Ser473p-Akt expression in SPC-A1-Bmi-1-siRNA cells. Conclusion:Knockdown of Bmi-1 gene can arrest the proliferation of SPC-A1 cells through G0/G1 phase arrest by inhibiting Cyclin D1 expression indirectly, which may be not associated with p16INK4a signaling pathway.