肺癌细胞FAAP20和RAD51C基因敲减对顺铂敏感性的影响
发布时间:2018-05-06 19:52
本文选题:FAAP20基因 + RAD51C基因 ; 参考:《江苏大学》2016年硕士论文
【摘要】:目的:应用si RNA干扰技术分别和同时共敲减人肺腺癌Calu-1细胞中范可尼贫血(Fanconi anemia,FA)通路FAAP20基因和RAD51C基因,观察该细胞这两个基因敲减后对顺铂敏感性的变化,探讨抑制FA通路DNA的损伤修复功能逆转Calu-1细胞对顺铂(cisplatin,DDP)耐药性的可行性及其效应,并探究FA通路在Calu-1细胞株对顺铂的耐药机制中所起的作用。方法:将针对FAAP20基因和RAD51C基因的si RNAs(FAAP20-si RNA和RAD51C-si RNA),用脂质体转染技术将它们分别和同时共转染于人肺腺癌细胞Calu-1细胞。蛋白质印迹法(Western Blotting,WB)检测经si RNAs转染前后Calu-1细胞FA通路FAAP20和RAD51C蛋白的表达量的变化以证明转染效率,并测定FANCD2蛋白的单泛素化水平;CCK-8(Cell Counting Kit-8)法测定分别和同时共转染前后Calu-1细胞增殖率的变化;Annexin V/PI流式细胞术测定分别和同时共转染前后Calu-1细胞早期凋亡率的变化;免疫荧光法测定分别和同时共转染前后细胞核内FANCD2核聚小体的形成。结果:与未转染组比较,转染FAAP20-si RNA组和RAD51C-si RNA组后经顺铂处理的Calu-1细胞FA通路中FAAP20蛋白和RAD51C蛋白表达量均明显降低(P均0.05),证明转染FAAP20-si RNA和RAD51C-si RNA有效,这两种基因被成功敲减。敲减FAAP20基因后,肺腺癌Calu-1细胞中经顺铂处理诱导的FANCD2蛋白单泛素化水平显著降低,且形成的FANCD2核聚小体明显下降(P0.05)。而敲减RAD51C基因后,肺腺癌Calu-1细胞中经顺铂诱导的FANCD2蛋白的单泛素化水平和核聚小体的形成并没有明显改变(P0.05),证实了FAAP20蛋白作用在FA通路上游,而RAD51C蛋白在FA通路下游行使功能。无论Calu-1细胞的FAAP20基因和RAD51C基因被敲减之前或之后,顺铂处理后的Calu-1细胞增殖率均随浓度升高而下降,呈剂量依赖性。这两个基因敲减之后经顺铂处理的细胞增殖率较基因敲减之前明显下降(P0.05),细胞早期凋亡率较敲减之前明显增高,而这两个基因同时共敲减较单基因敲减进一步增强了Calu-1细胞对顺铂的敏感性。结论:利用si RNA干扰技术分别敲减肺腺癌细胞Calu-1细胞株FAAP20基因和RAD51C基因可抑制FA通路的DNA损伤修复功能,从而增强Calu-1细胞对顺铂的敏感性,同时敲减这两个基因对顺铂的增敏效应可进一步提高,提示虽然FAAP20和RAD51C分别在FA通路的上下游行使功能,但对DNA损伤修复的作用可能并不完全重叠在一个相同的路径上。本研究结果表明FAAP20和RAD51C有可能作为分子靶用于克服肺癌细胞对顺铂的耐药以改善治疗效果。
[Abstract]:Aim: to investigate the changes of the sensitivity to cisplatin by using si RNA interference technique to reduce the FAAP20 gene and RAD51C gene in the Fanconi anemiaFAA pathway of human lung adenocarcinoma Calu-1 cells, respectively, and to observe the sensitivity of these two genes to cisplatin after knockout. To investigate the feasibility and effect of inhibiting the damage and repair function of FA pathway DNA in reversing the drug resistance of Calu-1 cells to cisplatin, and to explore the role of FA pathway in the mechanism of cisplatin resistance in Calu-1 cell lines. Methods: si RNAs(FAAP20-si RNA and RAD51C-si RNAs targeting FAAP20 gene and RAD51C gene were co-transfected into human lung adenocarcinoma Calu-1 cells by liposome transfection technique. Western blotting method was used to detect the changes of FAAP20 and RAD51C protein expression in FA pathway of Calu-1 cells before and after transfection with si RNAs to prove the transfection efficiency. The changes of proliferation rate of FANCD2 cells before and after co-transfection were measured by CCK-8 cell Counting Kit-8 method. Annexin V/PI flow cytometry was used to detect the early apoptosis rate of Calu-1 cells before and after co-transfection. The formation of FANCD2 nucleopolymer in the nucleus before and after co-transfection was determined by immunofluorescence assay. Results: compared with the non-transfection group, the expression of FAAP20 protein and RAD51C protein in the FA pathway of Calu-1 cells treated with cisplatin after transfection of FAAP20-si RNA and RAD51C-si RNA decreased significantly (P 0.05), which proved that the transfection of FAAP20-si RNA and RAD51C-si RNA was effective and the two genes were successfully knocked down. After knockout of FAAP20 gene, the level of monoubiquitin of FANCD2 protein induced by cisplatin treatment in Calu-1 cells of lung adenocarcinoma decreased significantly, and the formation of FANCD2 nucleopolymer decreased significantly (P 0.05). However, after knockout of RAD51C gene, the level of monoubiquitin of FANCD2 protein induced by cisplatin and the formation of nucleopolymer in Calu-1 cells of lung adenocarcinoma did not significantly change P0.05, which proved that FAAP20 protein acted on the upstream of FA pathway, while RAD51C protein acted on the downstream of FA pathway. No matter before or after the FAAP20 gene and RAD51C gene of Calu-1 cells were knocked down, the proliferation rate of Calu-1 cells after cisplatin treatment decreased with the increase of concentration in a dose-dependent manner. The proliferation rate of the cells treated with cisplatin was significantly lower than that before the gene knockout, and the early apoptosis rate was significantly higher than that before the knockout. The co-knockout of these two genes further enhanced the sensitivity of Calu-1 cells to cisplatin compared with that of single gene knockout. Conclusion: using si RNA interference technique to knockout FAAP20 gene and RAD51C gene of lung adenocarcinoma Calu-1 cell line respectively can inhibit DNA damage and repair function of FA pathway, thus enhancing the sensitivity of Calu-1 cell to cisplatin. At the same time, knockout of these two genes can further enhance the sensitivity of cisplatin, suggesting that although FAAP20 and RAD51C function in the upstream and downstream of FA pathway, the function of DNA damage repair may not overlap completely on the same pathway. Our results suggest that FAAP20 and RAD51C may be used as molecular targets to overcome cisplatin resistance in lung cancer cells.
【学位授予单位】:江苏大学
【学位级别】:硕士
【学位授予年份】:2016
【分类号】:R734.2
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1 陈康;肺癌细胞FAAP20和RAD51C基因敲减对顺铂敏感性的影响[D];江苏大学;2016年
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