沉默三联基序蛋白25基因对A549/DDP细胞株顺铂耐药性及凋亡的影响

Effects of tripartite motif containing 25 gene silencing on cisplatin resistance and apoptosis in A549/DDP cells

摘要:

目的：三联基序蛋白２５（tripartite motif containing 25，TRIM25）为泛素化E3连接酶，通过抑制TRIM25表达，研究其对肺癌顺铂（cis-dichlorodiamine platinum，DDP）耐药细胞株A549/DDP耐药性及凋亡的影响。方法：针对TRIM25基因设计4条不同序列shRNA，分别构建重组质粒载体pt1、pt2、pt3、pt4。利用脂质体将质粒转染至A549/DDP细胞，采用real-time PCR、Western blot检测抑制效果，MTT实验和流式细胞仪（flow cytometry，FCM）实验分别检测在DDP作用下细胞耐药性改变和凋亡情况。结果：干扰质粒pt1在mRNA和蛋白质水平上均能特异性地抑制TRIM25的表达；MTT法结果显示，抑制TRIM25表达后，干扰组pt1细胞IC50值低于pNC组（P=0.001）及对照组（P=0.000）；FCM结果显示，干扰组pt1的细胞凋亡率明显低于pNC组（P=0.005）、对照组（P=0.004）。结论：抑制 TRIM25表达后，能有效地降低 A549/DDP细胞株的DDP耐药性并促进其凋亡。

Abstract:

Objective：To research the effects of silencing tripartite motif containing 25（TRIM25） gene on cisplatin resistance and apop－tosis in cisplatin resistant lung cancer cell lines A549/DDP. Methods：Four types of short hairpin RNA were designed and prepared. The recombinant plasmids（pt1，pt2，pt3，pt4） were constructed respectively，which were transfected into A549/DDP cells using lipofectamine. Real-time PCR and Western blot were used to detect the inhibitory effects. The resistance of cisplatin to A549/DDP cells was detected by MTT assay. Cell apoptosis was tested by flow eytometry（FCM）. Results：The recombinant plasmid pt1 can inhibit mRNA and protein expression of TRIM25 specifically. MTT data showed that the cisplatin IC50 was decreased significantly in pt1 group than in pNC group（P=0.000） and control group（P=0.000） after the TRIM25 gene inhibition. According to the results of FCM，apoptotic cells was significantly decreased in pt1 group than in pNC group（P=0.005） and control group（P=0.004）. Conclusion：TRIM25 gene silencing in A549/DDP cells can effectively reduce cisplatin resistance and promote cells apoptosis.