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  • Title: [Down-regulated βIII-tubulin expression can reverse paclitaxel resistance in A549/taxol cells lines].
    Author: Zhuo Y, Guo Q.
    Journal: Zhongguo Fei Ai Za Zhi; 2014 Aug 20; 17(8):581-7. PubMed ID: 25130963.
    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. 背景与目的 化疗耐药导致肿瘤很快复发和/或转移，是目前肺癌死亡的主要原因之一。β-tubulin是抗微管药物的主要细胞靶点。已有的研究证明：βIII-tubulin高表达与非小细胞肺癌（non-small cell lung cancer, NSCLC）耐药有关。利用RNA干扰技术沉默耐紫杉醇A549细胞（A549/Taxol）中βIII-tubulin基因表达，探讨靶基因下调后对化疗药物紫杉醇的敏感性的变化以及细胞周期和细胞凋亡情况。方法 构建靶向βIII-tubulin的siRNA，以脂质体为载体介导βIII-tubulin siRNA转染A549/Taxol细胞，利用qRT-PCR检测细胞内βIII-tubulin mRNA的变化情况，并筛选出最佳干扰序列；Western blot法检测A549/Taxol细胞内βIII-tubulin蛋白表达的变化；MTT法检测转染后细胞株对紫杉醇敏感性的变化；流式细胞仪检测细胞周期和细胞凋亡的变化。结果 实时荧光qRT-PCR法显示转染后细胞株靶基因水平较对照组降低，其中βIII-tubulin siRNA-1序列抑制率最高为（87.73±4.87）%（P<0.01）；Western blot显示转染后靶蛋白水平较对照组明显降低；MTT法表明紫杉醇处理转染后细胞株的细胞抑制率较对照组明显增加（51.77±4.60）%（P<0.01）；细胞凋亡显示βIII-tubulin siRNA+Taxol组细胞早期凋亡率较对照组明显增加（P<0.01），两者的差异有统计学意义；细胞周期检测结果显示紫杉醇处理组的G2/M期细胞百分率高于对照组，且转染后紫杉醇处理组的细胞晚期凋亡率较对照组增加。结论 βIII-tubulin表达下调明显提高A549/Taxol细胞株对Taxol的敏感性。
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