RNA干扰血管内皮钙黏蛋白表达对Ph＋急性淋巴细胞白血病细胞株Sup—B15甲磺酸伊马替尼敏感性的影响

Effect of VE-cadherin on sentitivity to Imatinib in Sup-B15 Philadelphia chromosome positive acute lymphoblastic leukemia cells

目的探讨下调Ph＋急性淋巴细胞白血病细胞血管内皮钙黏蛋白（CD144）表达对甲磺酸伊马替尼敏感性的影响及可能的机制。方法通过慢病毒载体介导的RNA干扰（RNAi）沉默急性淋巴细胞白血病株Sup—B15细胞的CD144表达，建立CD144表达稳定下调的细胞株Sup—B15／shVEC。用CCK-8法检测甲磺酸伊马替尼对细胞增殖的影响；用AnnexinV／7一AAD标记，流式细胞术检测细胞凋亡；用流式细胞术检测CD34＋CD38-细胞比例变化；荧光定量PCR检测细胞ALDHImRNA水平；Westernb1ot法检测细胞CD144、CD133、Bcr—ab1、B—catenin表达水平。结果不同浓度甲磺酸伊马替尼对Sup—B15、Sup—B15／shVEC细胞均有增殖抑制作用，其半数抑制浓度（IC50值）分别为25．1、18．7txmo1／L，两组细胞相比差异有统计学意义（P〈0．05）。20txmo1／L甲磺酸伊马替尼作用48h，Sup—B15及Sup—B15／shVEC细胞凋亡率分别为（3．03±0．72）％、（13．52±2．06）％，两组细胞相比差异有统计学意义（P〈0．05）。流式细胞术检测结果显示Sup—B15细胞CD34＋CD38-细胞率明显高于Sup—B15／shVEC细胞[（2．39±0．28）％对（0．96±0．07）％]，两组相比差异有统计学意义（P〈0．05）。Sup—B15／shVEC细胞与Sup—B15细胞相比，ALDH1转录水平明显下降（0．043±0．008对0．016±0．003），CD133蛋白、B—catenin总蛋白及核蛋白表达水平也明显下降，而bcr—ab1融合蛋白表达水平无明显变化。结论Sup—B15细胞CD144表达稳定下调后其对甲磺酸伊马替尼敏感性明显增高，该作用可能是通过降低p—catenin蛋白稳定性、减少p—catenin蛋白核转位实现的。

Objective To investigate the sensitivity of imatinib mesylate （IM） on Sup-B15 Ph＋ acute lymphoblastic leukemia （ALL） cells knockdown of VE-cadherin （CD144） , and to further explore its mechanism. Methods CD144 in Sup-B15 leukemia cells was stably knockdowned via lentivirus-mediated RNA interference （named as Sup-B15/shVEC）. The inhibitory effects of IM on Sup-B15/shVEC and Sup- B15 leukemia cells were measured by CCK-8 test, and the apoptosis of those cells was determined by Annexin V/7-AAD dyeing using flow cytomery, the percentage of CD34 ＋ CD38- leukemia cells also by flow cy- tomtery. ALDH1 mRNA levels were detected by real-time RT-PCR, and protein levels of CD144, CD133, Bcr-abl and β-catenin by Western blot. Results IM treatment presented inhibitory effects on Sup-B15/sh- VEC and Sup-B15 leukemia cells at multiple concentrations of IM. The IC50 of IM on Sup-B15/shVEC and Sup-B15 leukemia cells were 25. 1μmol/L and 18.7μmoL/L, respectively （P 〈 0.05 ）. After 48h of 20 μmol/L IM treatment, the percentages of apoptosis cell in Sup-B15/shVEC cells and Sup-B15 cell were （ 13.52±2.06） % and （3.03±0.72） %, respectively （P 〈 0.05 ）. The percentage of CD34 ＋ CD38 - cells in Sup-B15 cells was significantly higher than in Sup-B15/shVEC cells [ （2.39±0.28）% vs （0.96 ±0.07） %, P 〈 0.05）. As compared to Sup-B15 cells, the transcription of ALDH1 in Sup-B15/shVEC was remarkably downregulated, and the CD133 protein level was also downregulated in Sup-B15/shVEC ceils. Both cytoplamic and nucleic β-catenin protein levels （but not for Bcr-abl levels ） decreased in Sup-B15/ shVEC cells as compare to Sup-B15 cells. Conclusion Knockdown of CD144 sensitized Sup-B15 Ph＋ ALL cells to IM. The possible mechanisms underlying this phenomenon might be via inhibiting β-catenin nucleic translocation and facilitating β-catenin degradation.