下调βIII-微管蛋白表达可逆转A549/紫杉醇细胞系中的紫杉醇耐药性

[Down-regulated βIII-tubulin expression can reverse paclitaxel resistance in A549/taxol cells lines].

作者信息

Zhuo Yinling, Guo Qisen

机构信息

Department of Internal Medicine, Shandong Province Hospital of Occupational Diseases, Jinan 250002, China.

出版信息

Zhongguo Fei Ai Za Zhi. 2014 Aug 20;17(8):581-7. doi: 10.3779/j.issn.1009-3419.2014.08.01.

DOI:10.3779/j.issn.1009-3419.2014.08.01

PMID:25130963

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6000364/

Abstract

BACKGROUND

Chemotherapy drug resistance is the primary causes of death in patients with pulmonary carcinoma which make tumor recurrence or metastasis. β-tubulin is the main cell targets of anti-microtubule drug. Increased expression of βIII-tubulin has been implicated in non-small cell lung cancer (NSCLC) cell lines. To explore the relationship among the expression level of βIII-tubulin and the sensitivity of A549/Taxolcell lines to Taxol and cell cycles and cell apoptosis by RNA interference-mediated inhibition of βIII-tubulin in A549/Taxol cells.

METHODS

Three pairs of siRNA targetd βIII-tubulin were designed and prepared, which were transfected into A549/Taxol cells using LipofectamineTM 2000. We detected the expression of βIII-tubulin mRNA using Real-time fluorescence qRT-PCR. Tedhen we selected the most efficient siRNA by the expression of βIII-tubulin mRNA in transfected group. βIII-tubulin protein level were mesured by Western blot. The taxol sensitivity in transfected group were evaluated by MTT assay. And the cell apoptosis and cell cycles were determined by flow cytometry.

RESULTS

βIII-tubulin mRNA levels in A549/Taxol cells were significantly decreased in transfected grop by Real-time qRT-PCR than control groups. And βIII-tubulin siRNA-1 sequence showed the highest transfection efficiency, which was (87.73±4.87)% (P<0.01); Western blot results showed that the expressional level of BIII tublin protein was significantly down-reulated in the transfectant cells than thant in the control cells. By MTT assay, we showed that the inhibition ratio of Taxol to A549/Taxol cells transfeced was higher than that of control group (51.77±4.60)% (P<0.01). The early apoptosis rate of A549/Taxol cells in transfected group were significantly higher than that of control group (P<0.01); G2-M content in taxol group obviously increased than untreated samples by the cell cycle (P<0.05).

CONCLUSIONS

βIII-tubulin down-regulated significantly sensitized NSCLC A549/Taxol cells to Paclitaxel.

摘要

背景

化疗耐药是导致肺癌患者肿瘤复发或转移并最终死亡的主要原因。β-微管蛋白是抗微管药物的主要细胞靶点。βIII-微管蛋白表达增加与非小细胞肺癌（NSCLC）细胞系有关。通过RNA干扰介导抑制A549/Taxol细胞中βIII-微管蛋白的表达，探讨βIII-微管蛋白表达水平与A549/Taxol细胞系对紫杉醇的敏感性、细胞周期及细胞凋亡之间的关系。

方法

设计并制备三对靶向βIII-微管蛋白的小干扰RNA（siRNA），使用LipofectamineTM 2000将其转染至A549/Taxol细胞中。采用实时荧光定量逆转录聚合酶链反应（Real-time荧光qRT-PCR）检测βIII-微管蛋白mRNA的表达。然后根据转染组中βIII-微管蛋白mRNA的表达情况筛选出最有效的siRNA。通过蛋白质免疫印迹法检测βIII-微管蛋白的蛋白水平。采用噻唑蓝比色法（MTT法）评估转染组对紫杉醇的敏感性。通过流式细胞术检测细胞凋亡和细胞周期。

结果

实时荧光定量逆转录聚合酶链反应结果显示，转染组A549/Taxol细胞中βIII-微管蛋白mRNA水平较对照组显著降低。βIII-微管蛋白siRNA-1序列显示出最高的转染效率，为（87.73±4.87）%（P<0.01）；蛋白质免疫印迹结果显示，转染细胞中βIII-微管蛋白的表达水平较对照细胞显著下调。MTT法检测结果显示，转染后的A549/Taxol细胞对紫杉醇的抑制率高于对照组，为（51.77±4.60）%（P<0.01）。转染组A549/Taxol细胞的早期凋亡率显著高于对照组（P<0.01）；细胞周期检测显示，紫杉醇组的G2-M期含量明显高于未处理组（P<0.05）。