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The Effect of RNA Interference-mediated ERCC1 Gene on the Chemo-treatment Sensitivity of NSCLC in Vitro

Author: SongZhiYu
Tutor: WangJiaHui;ZhouHang
School: Zunyi Medical College,
Course: Oncology
Keywords: RNA interference ERCC1 gene Non - small cell lung cancer A549/DDP cells Cisplatin-resistant
CLC: R734.2
Type: Master's thesis
Year: 2011
Downloads: 7
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Abstract


Objective:To investigate changes of platinum-based chemotherapy sensitivity of silencing ERCC1 gene expression by using RNA interference in NSCLC (non-small-cell lung cancer) drug resistance cell lines; It provides a experimental evidence to overcome non-small cell lung cancer resistant to cisplatin, And provides new ideas and methods to improve the prognosis of clinical NSCLC patients.Methods:NSCLC A549/DDP cells were routinely cultured of RPMI-1640 medium; ERCC1-siRNA1、ERCC1-siRNA2、ERCC1-siRNA3、NC ERCC1-siRNA targeting ERCC1 gene were designed and synthesized. Recombinant plasmid expression vector (PERCC1-shRNA) which carrying ERCC1- shRNA was constructed and transfected into A549/DDP cells with Lipofectamine 2000, Transfection efficiency was measured in the fluorescent microscope；The expression of ERCC1mRNA was detected by Reverse Transcription-polymerase chain reaction(RT-PCR);2.The change of cisplatin sensitivity after interference was test by MTT assay.Results: 1.The A549/DDP cells were harvestd after transfection with in 24,36,48hours, after treated with siRNA 24 hours, The ERCC1mRNA relative expression value of ERCC1-siRNA1 group is (0.580±0.015), ERCC1-siRNA2 group mRNA relative expression value is (0.732±0.365), ERCC1-siRNA3 group mRNA relative expression value is (0.441±0.563), compared with blank control and NC ERCC1-siRNA group, the ERCC1mRNA expressions level in A549/DDP cells were all reduced(P<0.05), the most obviously of them is ERCC1-siRNA3(P<0.01); after treated with siRNA 36 hours,ERCC1-siRNA1 group mRNA relative expression value is (0.576±0.468), ERCC1-siRNA2 group mRNA relative expression value is (0.652±0.273), ERCC1-siRNA3 group mRNA relative expression value is (0.212±0.236), compared with blank control and NC ERCC1-siRNA group, the ERCC1mRNA expressions level in A549/DDP cells were all reduced(P<0.05), the most obviously of them is ERCC1-siRNA3(P<0.01); after treated with siRNA 48 hours,ERCC1-siRNA1 group mRNA relative expression value is (0.556±0.134), ERCC1-siRNA2 group mRNA relative expression value is (0.618±0.025), ERCC1-siRNA3 group mRNA relative expression value is (0.206±0.089), compared with blank control and NC ERCC1-siRNA group, the ERCC1mRNA expressions level in A549/DDP cells were all reduced(P<0.05), the most obviously of them is ERCC1-siRNA3(P< 0.01); compared within each group, no significant difference.2. MTT assay results show that the IC50 value of A549/DDP cells line was decreased after transfection. The IC50 value of blank control group is 19.72±2.98ug/ml, of which,ERCC1-siRNA1 group is 4.59±1.56ug/ml, Transfected with ERCC1-siRNA1, ERCC1-siRNA2 and ERCC1-siRNA3,IC50 value were 4.59±1.56ug/ml、5.61±2.11ug/ml、0.88±3.Olug/ml,The diffenerces were significan compare to the controls (P<0.05);But IC50 value was 18.26±2.74ug/mL transfected with NC ERCC1-siRNA,there was no statistical significance compare to the controls (P> 0.05).Conclusion:Highly effective and specific siRNA targeting ERCC1 gene can be successfully screened by RNA interference technique. Selective silencing of ERCC1 gene promotes the sensitivity to cisplatin in A549/DDP cell. And can partly reverse the cisplatin resistance in human cisplatin resistant lung adeno-carcinoma cell lines A549/DDP in vitro.

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