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肺癌是最常见的癌症之一,也是癌症发病率和死亡率的主要原因,预计2018年全球将有210万新发肺癌病例(占癌症总发病率为11.6%),180万人死亡(占癌症总死亡率18.4%)[1]。我国最新数据[2]显示,肺癌也是占癌症发病率和死亡率的第一位,分别为20.0%和27.3%。主要分为小细胞肺癌与非小细胞肺癌,后者约占85%[3-4]。近些年晚期肺癌的治疗进展迅速,治疗选择显著增多[5]。但肺癌标准化年龄的5年生存率为16.1%[2]。主要原因是超过50%肺癌病人在诊断时就已经是晚期[6]。且其病因十分复杂,发病机制未完全清楚,大部分的肺癌病人疗效欠佳且容易复发[7-9]。因此我们要提高对肺癌发生的分子机制的研究和理解,寻找潜在新的标志物和有效的分子治疗靶点,为临床早期诊断和治疗提供帮助。
甘油-3-磷酸脱氢酶2[glycerol-3-phosphate dehydrogenase 2,GPD2]是产生线粒体活性氧(ROS)的主要来源,ROS能促进肿瘤的进展。本实验通过RNA干扰技术沉默GPD2表达,转染肺癌细胞,检测细胞增殖、细胞凋亡的变化以及细胞侵袭能力变化的情况,分析GPD2基因敲减后对肺癌细胞生物功能学的影响。
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结果显示,相比于shCtrl组,shGPD2组的细胞明显减少,细胞感染效率达到80%以上,细胞状态正常(见图 1)。
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qPCR检测H1299细胞在成功感染慢病毒干扰载体后GPD2的mRNA表达情况,shCtrl组与shGPD2组细胞中GPD2基因表达丰度分别为1.000±0.036、0.265±0.011,差异有统计学意义(t=30.35,P < 0.05),相比于shCtrl组,shGPD2组H1299细胞中GPD2基因的mRNA表达量明显受到抑制,慢病毒敲减效率达到73.5%。Western blotting检测H1299细胞在成功感染慢病毒干扰载体后GPD2的蛋白质量的变化,相比于shCtrl组,shGPD2组H1299细胞的GPD2的蛋白含量减少(见图 2)。
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shGPD2组具有增殖活力的数目明显低于shCtrl组(P < 0.01)(见表 1),相比于shCtrl组,shGPD2组细胞增殖明显减缓。
分组 第1天 第2天 第3天 第4天 第5天 shCtr组 0.141±0.001 0.287±0.008 0.543±0.010 0.743±0.002 0.871±0.005 shGPD2组 0.132±0.003 0.181±0.004 0.251±0.007 0.321±0.008 0.372±0.007 t 15.59 15.44 74.47 112.25 96.95 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 表 1 CCK8法检测连续5 d 2组细胞的OD450值比较(x±s)
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慢病毒转染H1299细胞5 d后,用AnnexinV-APC单染法流式细胞术检测细胞凋亡,shCtrl组与shGPD2组的凋亡率分别为(4.6±1.1)%、(14.6±1.42)%(t=8.60, P < 0.01)。相比shCtrl组,shGPD2组凋亡数增多(见图 3)。
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用含Matrigel基质胶的Transwell小室检测2组细胞的侵袭能力,穿过Matrigel基质胶的shCtrl组与shGPD2组的细胞数分别为(216.00±12.74)、(68.00±21.86), 差异有统计学意义(t=16.02,P < 0.01),相比shCtrl组,shGPD2组侵袭转移率降低(见图 4)。
GPD2基因敲减对肺癌细胞H1299生物学功能的影响
Effect of GPD2 gene knockdown by shRNA on the biological function of lung cancer cells H1299
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摘要:
目的沉默甘油-3-磷酸脱氢酶2(GPD2)基因表达,验证对肺癌细胞增殖、凋亡、侵袭等生物功能及行为学的影响。 方法构建GPD2基因的RNA干扰慢病毒载体(shGPD2)和对应的对照载体(shCtrl),并感染H1299细胞,实时荧光定量(PCR)检测GPD2基因在癌细胞中的mRNA变化情况,Western blotting检测GPD2基因在癌细胞中蛋白质变化情况,验证敲减效率;CCK8法检测细胞的增殖情况,流式细胞术检测细胞凋亡情况;Transwell实验检测细胞侵袭情况。 结果成功构建慢病毒干扰载体shGPD2并感染H1299细胞,mRNA及蛋白质含量均减少,其敲减效率达到73.5%(P < 0.05);GPD2基因敲减后的H1299细胞增殖明显减缓,细胞凋亡显著增多,细胞侵袭能力明显下降(P < 0.01)。 结论GPD2基因敲减能够有效抑制肺癌细胞H1299的增殖。 -
关键词:
- 肺肿瘤 /
- 甘油-3-磷酸脱氢酶2基因 /
- RNA干扰 /
- 慢病毒载体 /
- 细胞增殖
Abstract:ObjectiveTo investigate the effects of silencing the expression of glycerol-3-phosphate dehydrogenase 2 (GPD2) gene on the biological function (proliferation, apoptosis and invasion) and behavior of lung cancer cells. Methods The RNA interference lentiviral vector containing GPD2 gene (shGPD2) and corresponding control vector (shCtrl) were constructed, and used to infect the H1299 cells.The expression levels of GPD2 gene mRNA and protein in cancer cells were detected using qPCR and Western blotting, respectively.The knockdown efficiency of GPD2 gene was verified.The proliferation, apoptosis and invasion of cells were detected using CCK8 method, flow cytometry and Transwell assay, respectively. ResultsThe lentiviral vector shGPD2 was successfully constructed, and infected H1299 cells.The mRNA and protein levels decreased, and the knockdown efficiency was 73.5% (P < 0.05).After the GPD2 gene expression decreased, the proliferation, apoptosis and invasion ability of H1299 cells significantly slowed down, increased and reduced, respectively (P < 0.01). ConclusionThe GPD2 gene knockdown can effectively inhibit the proliferation of lung cancer cells H1299. -
表 1 CCK8法检测连续5 d 2组细胞的OD450值比较(x±s)
分组 第1天 第2天 第3天 第4天 第5天 shCtr组 0.141±0.001 0.287±0.008 0.543±0.010 0.743±0.002 0.871±0.005 shGPD2组 0.132±0.003 0.181±0.004 0.251±0.007 0.321±0.008 0.372±0.007 t 15.59 15.44 74.47 112.25 96.95 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 -
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