siRNA沉默FANCD2基因增加肺癌A549/DDP细胞对顺铂化疗的敏感性

目的: 研究siRNA沉默FANCD2基因后人肺腺癌A549/DDP耐药细胞株对顺铂(DDP)耐药性的变化。方法:合成针对FANCD2基因的siRNA(FANCD2 siRNA)，采用脂质体将其分别转染肺癌A549细胞株和A549/DDP耐药细胞株。CCK-8法测定经DDP处理的A549和A549/DDP细胞株转染siRNA前后的细胞增殖率变化；免疫印迹法测定2种细胞株转染siRNA前后FANCD2蛋白单泛素化水平；免疫荧光测定胞核中FANCD2蛋白核聚小体的形成。结果：经DDP处理的A549/DDP细胞增殖率，FANCD2蛋白单泛素化水平及核聚小体形成程度明显高于A549细胞。转染FANCD2-siRNA后，A549和A549/DDP细胞的FANCD2蛋白单泛素化水平和核聚小体形成明显降低，细胞增殖率显著下降。结论： 应用siRNA转染技术沉默FANCD2基因可抑制FA/BRCA途径的DNA修复功能，从而增加肺癌细胞对DDP的敏感性，部分逆转耐药肺癌细胞株对DDP的耐药性。

Silence of FANCD2 gene of FA/BRCA pathway reverse the resistance to cisplatin in lung cancer A549/DDP cell line

Objective: To investigate the effect of FANCD2 gene silence on the resistance to cisplatin (DDP) in lung adenocarcinoma A549/DDP cell line. Methods: FANCD2 genes of A549/DDP cells were silenced by transfection of the designed and synthesized siRNA targeted FANCD2(FANCD2-siRNA) through Lipofectamine. CCK-8 technique was used to measure the cell proliferation rate of A549 and A549/DDP cells treated with DDP before and after siRNA transfection. Western blotting was carried out to detect the expression of monoubiquitination (L) and non-monoubiquitination (S) FANCD2 protein and L/S ratio before and after siRNA transfection. Immunofluorescence assay was performed to determine the formation of FANCD2 nuclear foci. Results: The cell proliferation rate and the levels of FANCD2 monoubiquitination (L/S ratio) were markedly higher in A549/DDP cells than in A549 cells treated with DDP (P<0.05). After transfection of FANCD2-siRNA, the levels of FANCD2 monoubiquitination, nuclear foci formation of FANCD2, and the cell proliferation rate were significantly decreased in A549 and A549/DDP cells following DDP treatment (P<0.05). Conclusion: Silence of FANCD2 gene inhibited the function of FA/BRCA pathway by decreasing the monoubiquitination level and nuclear focus formation of FANCD2, resulting in the decrease of cell proliferation, and partial reversion of chemoresistance to cisplatin in lung cancer cell of DDP-resistance.