目的:探讨不同梯度浓度顺铂(CDDP)处理不同肺癌细胞后新发现的剪接因子SRSF7 mRNA异构体在同浓度CDDP处理后不同的细胞中的水平变化.方法:采用梯度浓度的CDDP处理肺癌细胞A549(8、18、24、32、40、48及56 μmol/L)和H1299(12、24、36、48、60、72及84 μmol/L),处理24 h后提取总RNA,进行RT-PCR检测SRSF7 mRNA剪接异构体,并选取NCBI中未确定的异构体进行TA克隆后测序并分析鉴定异构体类型;用56 μmol/L的CDDP处理人胚肾细胞293FT,宫颈癌细胞CaSki、SiHa、C33A细胞24 h,提取总RNA后验证新发现的SRSF7 mRNA剪切异构体是否存在这些细胞中.结果:发现3种新的SRSF7mRNA剪接异构体(分别标记为New1、New2及New3),在肺癌细胞A549、H1299,人胚肾细胞293FT,宫颈癌细胞CaSki、SiHa、C33A细胞等多种肿瘤细胞中均存在,根据NCBI的预测New1,New2,New3异构体均为非编码蛋白的mRNA异构体,随着处理CDDP浓度的增加,其比例明显上升;而编码蛋白的mRNA异构体a和(或)b所占比例明显下降;同时SRSF7的总mRNA水平逐渐降低.结论:新发现的3种非编码的mRNA异构体的变化揭示细胞可能通过SRSF7自身的mRNA 选择性剪接来应对DNA损伤.%Objective:To explore the changes of mRNA isoforms by gradient dose of cisplatin and analyze the reasons in A549 and H1299.Methods:Total RNA of the cells were abstracted 24h after the treatment by cisplatin so as to detect mRNA isoforms of SRSF7 gene by RT-PCR and analyze the ratio of the newly discovered mRNA isoforms.The undetermined isomers of NCBI were analyzed.Resuits:Three new mRNA splicing isoforms (New1,New2,New3) of SRSF7 were found,which were ubiquitous in tumor cells and were both non-coding mRNA isoforms.With the increase of the concentration of cisplatin,their proportion significantly increased,but the ratio of mRNA isoforms that could encode protein was significantly declined.Simultaneously,the total mRNA level decreased.Conclusion:The newly discovered changes in three non-coding mRNA isoforms (New1,New2,New3) reveals that the cells may respond to DNA damage by alternative splicing of SRSF7's own mRNA.
展开▼
