2020 年第 2 期 第 15 卷

过表达富含半胱氨酸的酸性分泌蛋白对非小细胞肺癌细胞白蛋白结合型紫杉醇敏感性的影响

Effect of up-regulating secreted protein acidic and rich in cysteine on sensitivity to nanoparticle albumin-bound paclitaxel in human non-small cell lung carcinoma

作者：高萌呼群李超

英文作者：

单位：内蒙古医科大学附属医院肿瘤内科，呼和浩特010000

英文单位：

关键词：非小细胞肺癌；富含半胱氨酸的酸性分泌蛋白；白蛋白结合型紫杉醇；基因甲基化；凋亡

英文关键词：

• 摘要：

• 【摘要】目的    探讨上调富含半胱氨酸的酸性分泌蛋白（SPARC）表达对非小细胞肺癌A549白蛋白结合型紫杉醇敏感性的影响。方法    采用实时荧光定量聚合酶链反应（PCR）法和特异性甲基化PCR法检测白蛋白结合型紫杉醇对A549细胞SPARC基因表达的影响。分别设置空白对照组（未作任何处理）、阴性对照组（转染阴性对照序列）和SPARC组（转染SPARC序列），分别采用噻唑蓝、流式细胞术、Transwell小室法和划痕实验检测白蛋白结合型紫杉醇对过表达SPARC A549细胞生物学行为的影响。采用蛋白质印迹法检测过表达SPARC对A549细胞半胱氨酸蛋白酶（caspase）-3、caspase-8、剪切的多聚二磷酸腺苷核糖聚合酶（PARP）蛋白表达的影响。结果    经特异性甲基化PCR法检测，白蛋白结合型紫杉醇可逆转A549细胞SPARC基因启动子区高甲基化状态，并增加SPARC mRNA表达量（1.44±0.23），较处理前（1.00±0.00）明显升高（t=4.962，P<0.001）。经不同浓度白蛋白结合型紫杉醇（0.01、0.1、1、10、100、1 000 mg/L）作用于SPARC组A549细胞后，增殖抑制率分别为（2.88±0.18）%、（10.96±1.23）%、（19.35±2.14）%、（48.97±5.31）%、（72.73±8.91）%、（75.46±8.26）%，从1 mg/L起SPARC组高于空白对照组和阴性对照组（均P<0.05）。白蛋白结合型紫杉醇作用于SPARC组细胞，其凋亡率、侵袭率和迁移率分别为（23±3）%、（33±12）%、（51±15）%，与空白对照组和阴性对照组相比，SPARC组A549细胞凋亡率更高，侵袭率和迁移率降低（均P<0.05）。另外，上调SPARC蛋白表达后，SPARC组A549细胞caspase-2、caspase-8、剪切的PARP蛋白表达量分别为（0.93±0.15）、（2.11±0.34）、（1.08±0.17），明显高于空白对照组和阴性对照组（均P<0.05）。结论    上调SPARC可增加A549对白蛋白结合型紫杉醇的敏感性，可能与降低SPARC基因启动子甲基化状态有关。

• 【Abstract】Objective    To discuss the effect of up-regulating secreted protein acidic and rich in cysteine(SPARC) on nanoparticle albumin-bound（nab）-paclitaxel sensitivity in human non-small cell lung carcinoma A549 cells. Methods    Quantitative real-time fluorescent polymerase chain reaction(PCR) and methylation specific PCR（MSP） were used to detect expression and methylation of SPARC mRNA in A549 cells affected by nab-paclitaxel. SPARC was transfected into A549 cells. The blank control group(without any treatment), negative control group(transfected with negative control sequence) and SPARC group(transfected with SPARC sequence) were prepared. Methyl thiazolyl tetrazolium assay, flow cytometry, transwell assay and scratching assay were used to detect proliferation, migration and invasion of A549 cells affected by nab-paclitaxel. Expression levels of caspase-3, caspase-8 and cleaved poly ADP-ribose poly-merase(PARP) in A549 cells with SPARC overexpression. Results    MSP results showed that nab-paclitaxel down-regulated methylation of SPARC mRNA and increased expression of SPARC mRNA［(1.44±0.23) vs （1.00±0.00）, t=4.962, P<0.001］ in A549 cells. After treatment with gradient concentrations of nab-paclitaxel(0.01, 0.1, 1, 10, 100, 1 000 mg/L), proliferation inhibition rates of A549 cells in SPARC group were (2.88±0.18)%, (10.96±1.23)%, (19.35±2.14)%, (48.97±5.31)%, (72.73±8.91)% and (75.46±8.26)%, respectively, and the latter four rates were significantly higher than those in blank control and negative control groups(all P<0.05). Apoptosis rate induced by nab-paclitaxel was higher, but migration rate and invasion rate were lower in SPARC group［（23±3）%, （33±12）%, （51±15）%］ than those in blank control and negative control groups（all P<0.05）. After treatment with SPARC overexpression, levels of caspase-3, caspase-8 and cleaved PARP proteins of A549 cells in SPARC group were (0.93±0.15), (2.11±0.34) and (1.08±0.17), respectively, which were significantly higher than those in blank control and negative control groups(all P<0.05). Conclusion    Up-regulation of SPARC can increase the sensitivity to nab-paclitaxel of A549 cells, which may be related to the decrease of SPARC gene methylation.