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胃癌是最常见的恶性疾病之一,是全世界癌症相关死亡的第三大原因[1-2]。晚期胃癌病人的预后较差,5年总生存率低至约25%[3]。因此,胃癌的早期诊断和有效治疗对人体健康具有重要意义。目前,肿瘤已被广泛认为起源于癌症干细胞。肿瘤干细胞是肿瘤体内的一小部分细胞,具有很高的球形成能力,自我更新、迁移、侵袭以及对化疗和放疗的抵抗力[4]。它们产生不同表型的非致瘤性肿瘤细胞,甚至在治疗结束后导致肿瘤复发。因此,癌症干细胞有助于不同类型肿瘤的发生、发展和复发[5-6]。据报道[7-9],肿瘤干细胞已在多种癌症中成功鉴定,包括黑素瘤、乳腺癌、脑癌、结肠癌和胃癌。新的证据表明[10],肿瘤干细胞是胃癌复发的主要原因,肿瘤干细胞的存在在胃癌进展和治疗抵抗中起关键作用。这些发现表明肿瘤干细胞是胃癌治疗的重要治疗靶点。近年来,星形胶质细胞上调基因-1 (AEG-1)已成为肿瘤进展的几个方面的潜在关键介质[11]。然而,AEG-1在人胃癌干细胞致瘤性中的作用仍不清楚。本文就AEG-1在调节胃癌干细胞中过程中的生物学功能作一探讨。
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ALDH1阳性细胞是肿瘤干细胞,而ALDH1阴性细胞是非干细胞(见图 1)。ALDH1阳性细胞均能形成大的细胞球(见图 2)。注射ALDH1阳性细胞的裸鼠中都能长成肿瘤(见图 3)。定量实时PCR的结果显示,与癌症非干细胞相比,AEG-1表达在胃癌干细胞MKN-45中显著上调(P < 0.01)(见表 1)。Western blotting基本上也显示了类似的结果(见图 4)。
分组 n HGC-27 MKN-45 干细胞 3 1.083±0.055 1.100±0.089 非干细胞 3 0.620±0.046 0.753±0.055 t — 1.45* 5.74 P — >0.05 < 0.01 *示t′值 表 1 定量PCR分析AEG-1的mRNA水平(x±s)
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当将AEG-1特异性siRNA转染到胃癌干细胞中时,mRNA表达非常低(P < 0.01)(见表 2)。进一步通过Western blotting分析检测蛋白质表达,与对照相比,低mRNA表达与低蛋白质表达相关(见图 5)。AEG-1 siRNA转染的胃癌干细胞增殖明显减少(P < 0.01),而AEG-1敲低后对胃癌非干细胞的生长没有显著影响(P>0.05)(见表 3)。AEG-1-siRNA处理的细胞在G1期中的百分比远高于对照组(P < 0.01)(见表 4)。
分组 n HGC-27 MKN-45 干细胞 非干细胞 干细胞 非干细胞 AEG-1-siRNA 3 0.197±0.019 0.200±0.090 0.155±0.043 0.189±0.065 AEG-1-siRNA-scrambled 3 1.045± 0.059 0.616±0.045 1.105±0.085 0.765±0.059 t — 23.68 7.16 17.27 11.37 P — < 0.01 < 0.01 < 0.01 < 0.01 表 2 AEG-1-siRNA转染HGC-27或MKN-45干细胞和非干细胞中的AEG-1 mRNA水平(x±s)
分组 n HGC-27 MKN-45 干细胞 非干细胞 干细胞 非干细胞 AEG-1-siRNA 3 0.669±0.054 1.108±0.056 0.740±0.075 1.097±0.075 AEG-1-siRNA-scrambled 3 1.288±0.115 1.097±0.049 1.382±0.019 1.118±0.083 t — 8.44 0.26 13.52 0.33 P — < 0.01 >0.05 < 0.01 >0.05 表 3 AEG-1敲低对HGC-27或MKN-45干细胞及非干细胞增殖的影响(x±s)
分组 n HGC-27 MKN-45 干细胞 非干细胞 干细胞 非干细胞 AEG-1-siRNA 3 0.510±0.023 0.381±0.020 0.412±0.032 0.408±0.026 AEG-1-siRNA-scrambled 3 0.379±0.026 0.403±0.0.007 0.274±0.021 0.428±0.026 t — 6.52 1.79 6.24 0.94 P — < 0.01 >0.05 < 0.01 >0.05 表 4 AEG-1敲低对HGC-27或MKN-45干细胞及非干细胞细胞周期的影响(x±s)
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Caspase-3/7活性检测显示,与对照相比,胃癌干细胞中AEG-1基因敲除后的细胞凋亡比例显著上升(P < 0.01)(见表 5)。Annexin V实验发现,AEG-1敲低可诱导胃干细胞凋亡(P < 0.01)(见图 6、表 6)。
分组 n HGC-27 MKN-45 干细胞 非干细胞 干细胞 非干细胞 AEG-1-siRNA 3 9 385.7±520.5 6 126.0±788.0 8 513.3±575.0 6 019.7±934.1 AEG-1-siRNA-scrambled 3 5 775.0±549.2 5 964.7±788.3 5 619.0±1 252.5 5 664.3±414.2 t — 8.27 0.25 3.64 0.60 P — < 0.01 >0.05 < 0.05 >0.05 表 5 AEG-1基因敲低对胃癌细胞Caspase-3/7酶活的影响(x±s)
分组 n HGC-27 MKN-45 干细胞 非干细胞 干细胞 非干细胞 AEG-1-siRNA 3 0.163±0.012 0.043±0.006 0.173±0.006 0.067±0.015 AEG-1-siRNA-scrambled 3 0.047±0.006 0.033±0.006 0.037±0.006 0.047±0.006 t — 14.98 2.04 27.76 2.14 P — < 0.01 >0.05 < 0.01 >0.05 表 6 Annexin V检测AEG-1基因敲低胃癌细胞的细胞凋亡情况(x±s)
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当AEG-1表达被敲低时,显著抑制了胃癌干细胞的成球能力,其长成的细胞球明显小于对照组(见图 7)。
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2组处理的瘤块大小及瘤块质量存在显著差异(见图 8),注射AEG-1-siRNA处理的小鼠肿瘤瘤块体积和质量(0.167±0.042)g明显小于对照组(0.480±0.030)g(t=10.50,P < 0.01)(见图 9)。采用Western blotting检测瘤块内AEG-1的表达情况,与对照组相比,AEG-1敲低显著降低了小鼠实体瘤中AEG-1的表达水平(见图 10)。
星形胶质细胞上调基因-1对胃癌干细胞增殖、凋亡及干性的影响
Effect of astrocyte elevated gene-1 on the proliferation, apoptosis and stemness of gastric cancer stem cells
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摘要:
目的探讨星形胶质细胞上调基因-1(AEG-1)在胃癌干细胞中的表达及其作用。 方法采用实时荧光定量技术(qRT-PCR)及Western blotting检测胃癌干细胞(HGC-27、MKN-45)及相应非干细胞mRNA及蛋白表达水平,采用瞬时转染法对AEG-1进行处理,使用MTS、细胞周期检测细胞增殖情况及作用机制,Annexin Ⅴ及Caspase-3/7活性检测细胞凋亡情况,干细胞成球实验评估AEG-1沉默对胃干细胞干性的影响,裸鼠体内成瘤实验评估AEG-1对裸鼠体内胃干细胞肿瘤发生的影响。Ⅴ 结果胃癌干细胞MKN-45中AEG-1表达水平高于非干细胞组(P < 0.01);AEG-1-siRNA转染的癌干细胞导致癌干细胞周期停滞在G1期,明显抑制了细胞增殖(P < 0.01);Annexin Ⅴ及Caspase 3/7活性检测AEG-1敲低可诱导胃干细胞凋亡;AEG-1表达被敲低时显著抑制了胃癌干细胞的成球能力;AEG-1缺失抑制了肿瘤干细胞的体内成瘤能力。 结论AEG-1沉默抑制细胞增殖,诱导胃癌干细胞G1期细胞周期停滞和细胞凋亡,抑制肿瘤发生,在胃癌干细胞的肿瘤发生中起着积极作用。 -
关键词:
- 胃肿瘤 /
- 星形胶质细胞上调基因-1 /
- 肿瘤干细胞
Abstract:ObjectiveTo investigate the expression level and effects of astrocyte elevated gene-1(AEG-1) in gastric cancer stem cells. MethodsThe expression levels of AEG-1 mRNA and protein in gastric stem cell lines(HGC-27 and MKN-45) were detected using RT-PCR and Western blotting, respectively.The AEG-1 was processed using transient transfection, and the cells proliferation and its mechanism were investigated using the MTS assay and cell-cycle analysis.The apoptosis was examined using the activities of Caspase-3/7 and annexin V, the effect of silencing AEG-1 on the stemness of gastric stem cells was evaluated using the tumorsphere formation assay, and the tumor formation experiment was conducted to evaluate the effects of AEG-1 on the tumorigenesis of gastric stem cells in nude mice. ResultsThe expression level of AEG-1 in gastric stem cells was higher than that in non-stem cells(P < 0.01).Cancer stem cells transfected with AEG1-siRNA resulted in the cancer stem cell cycle arrest in G1 phase, and could significantly inhibit the cell proliferation(P < 0.01).The results of Annexin V and Caspase-3/7 activities detection showed that the knockdown of AEG-1 could induce the apoptosis of gastric stem cells.The low level of AEG-1 could significantly inhibit the ability of gastric cancer stem cells to form into balls.The loss of AEG-1 inhibited the tumorigenesis of tumor stem cells in vivo. ConclusionsThe silencing expression of AEG-1 can suppress the cell proliferation, induce the cell cycle arrest in G1 phase and apoptosis of gastric cancer stem cells, inhibit the tumorigenesis, which plays a positive role in tumorigenesis of gastric cancer stem cells. -
Key words:
- gastric neoplasms /
- astrocyte elevated gene-1 /
- cancer stem cell
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表 1 定量PCR分析AEG-1的mRNA水平(x±s)
分组 n HGC-27 MKN-45 干细胞 3 1.083±0.055 1.100±0.089 非干细胞 3 0.620±0.046 0.753±0.055 t — 1.45* 5.74 P — >0.05 < 0.01 *示t′值 表 2 AEG-1-siRNA转染HGC-27或MKN-45干细胞和非干细胞中的AEG-1 mRNA水平(x±s)
分组 n HGC-27 MKN-45 干细胞 非干细胞 干细胞 非干细胞 AEG-1-siRNA 3 0.197±0.019 0.200±0.090 0.155±0.043 0.189±0.065 AEG-1-siRNA-scrambled 3 1.045± 0.059 0.616±0.045 1.105±0.085 0.765±0.059 t — 23.68 7.16 17.27 11.37 P — < 0.01 < 0.01 < 0.01 < 0.01 表 3 AEG-1敲低对HGC-27或MKN-45干细胞及非干细胞增殖的影响(x±s)
分组 n HGC-27 MKN-45 干细胞 非干细胞 干细胞 非干细胞 AEG-1-siRNA 3 0.669±0.054 1.108±0.056 0.740±0.075 1.097±0.075 AEG-1-siRNA-scrambled 3 1.288±0.115 1.097±0.049 1.382±0.019 1.118±0.083 t — 8.44 0.26 13.52 0.33 P — < 0.01 >0.05 < 0.01 >0.05 表 4 AEG-1敲低对HGC-27或MKN-45干细胞及非干细胞细胞周期的影响(x±s)
分组 n HGC-27 MKN-45 干细胞 非干细胞 干细胞 非干细胞 AEG-1-siRNA 3 0.510±0.023 0.381±0.020 0.412±0.032 0.408±0.026 AEG-1-siRNA-scrambled 3 0.379±0.026 0.403±0.0.007 0.274±0.021 0.428±0.026 t — 6.52 1.79 6.24 0.94 P — < 0.01 >0.05 < 0.01 >0.05 表 5 AEG-1基因敲低对胃癌细胞Caspase-3/7酶活的影响(x±s)
分组 n HGC-27 MKN-45 干细胞 非干细胞 干细胞 非干细胞 AEG-1-siRNA 3 9 385.7±520.5 6 126.0±788.0 8 513.3±575.0 6 019.7±934.1 AEG-1-siRNA-scrambled 3 5 775.0±549.2 5 964.7±788.3 5 619.0±1 252.5 5 664.3±414.2 t — 8.27 0.25 3.64 0.60 P — < 0.01 >0.05 < 0.05 >0.05 表 6 Annexin V检测AEG-1基因敲低胃癌细胞的细胞凋亡情况(x±s)
分组 n HGC-27 MKN-45 干细胞 非干细胞 干细胞 非干细胞 AEG-1-siRNA 3 0.163±0.012 0.043±0.006 0.173±0.006 0.067±0.015 AEG-1-siRNA-scrambled 3 0.047±0.006 0.033±0.006 0.037±0.006 0.047±0.006 t — 14.98 2.04 27.76 2.14 P — < 0.01 >0.05 < 0.01 >0.05 -
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