本文選題：FAAP20基因 + RAD51C基因　； 參考：《江蘇大學》2016年碩士論文

【摘要】：目的:應用si RNA干擾技術分別和同時共敲減人肺腺癌Calu-1細胞中范可尼貧血(Fanconi anemia,FA)通路FAAP20基因和RAD51C基因,觀察該細胞這兩個基因敲減后對順鉑敏感性的變化,探討抑制FA通路DNA的損傷修復功能逆轉Calu-1細胞對順鉑(cisplatin,DDP)耐藥性的可行性及其效應,并探究FA通路在Calu-1細胞株對順鉑的耐藥機制中所起的作用。方法:將針對FAAP20基因和RAD51C基因的si RNAs(FAAP20-si RNA和RAD51C-si RNA),用脂質(zhì)體轉染技術將它們分別和同時共轉染于人肺腺癌細胞Calu-1細胞。蛋白質(zhì)印跡法(Western Blotting,WB)檢測經(jīng)si RNAs轉染前后Calu-1細胞FA通路FAAP20和RAD51C蛋白的表達量的變化以證明轉染效率,并測定FANCD2蛋白的單泛素化水平;CCK-8(Cell Counting Kit-8)法測定分別和同時共轉染前后Calu-1細胞增殖率的變化;Annexin V/PI流式細胞術測定分別和同時共轉染前后Calu-1細胞早期凋亡率的變化;免疫熒光法測定分別和同時共轉染前后細胞核內(nèi)FANCD2核聚小體的形成。結果:與未轉染組比較,轉染FAAP20-si RNA組和RAD51C-si RNA組后經(jīng)順鉑處理的Calu-1細胞FA通路中FAAP20蛋白和RAD51C蛋白表達量均明顯降低(P均0.05),證明轉染FAAP20-si RNA和RAD51C-si RNA有效,這兩種基因被成功敲減。敲減FAAP20基因后,肺腺癌Calu-1細胞中經(jīng)順鉑處理誘導的FANCD2蛋白單泛素化水平顯著降低,且形成的FANCD2核聚小體明顯下降(P0.05)。而敲減RAD51C基因后,肺腺癌Calu-1細胞中經(jīng)順鉑誘導的FANCD2蛋白的單泛素化水平和核聚小體的形成并沒有明顯改變(P0.05),證實了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。無論Calu-1細胞的FAAP20基因和RAD51C基因被敲減之前或之后,順鉑處理后的Calu-1細胞增殖率均隨濃度升高而下降,呈劑量依賴性。這兩個基因敲減之后經(jīng)順鉑處理的細胞增殖率較基因敲減之前明顯下降(P0.05),細胞早期凋亡率較敲減之前明顯增高,而這兩個基因同時共敲減較單基因敲減進一步增強了Calu-1細胞對順鉑的敏感性。結論:利用si RNA干擾技術分別敲減肺腺癌細胞Calu-1細胞株FAAP20基因和RAD51C基因可抑制FA通路的DNA損傷修復功能,從而增強Calu-1細胞對順鉑的敏感性,同時敲減這兩個基因對順鉑的增敏效應可進一步提高,提示雖然FAAP20和RAD51C分別在FA通路的上下游行使功能,但對DNA損傷修復的作用可能并不完全重疊在一個相同的路徑上。本研究結果表明FAAP20和RAD51C有可能作為分子靶用于克服肺癌細胞對順鉑的耐藥以改善治療效果。
[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.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R734.2

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1 陳康;肺癌細胞FAAP20和RAD51C基因敲減對順鉑敏感性的影響[D];江蘇大學;2016年

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本文編號：1853647