融合基因MSH3-RASGRF2 在MTX诱导DHFR扩增耐药中的功能

The main mechanism of methotrexate (MTX)-resistance is dihydrofolate reductase (DHFR) gene amplification, and amplicons exist in two forms of Homogeneous Staining Region (HSRs) and / or Double Minute (DMs). Gene amplification begin from chromosome break, then rejection created gene rearrangement, and hot gene rearrangement may affect the stability of gene amplification and drug-resistance. Gene rearrangements in DHFR amplicon were analyzed in 10-5M (HSRs)、10-4M (HSRs/DMs)、3×10-4M (DMs) MTX-resistant HT 29 cell strain, and parental HT 29 as a control. MSH3-RASGRF2 fusion transcripts were identified in all gradient-resistant cell strains. Based on previous work, we want identify the DNA linker sequence of the fusion gene and the epression of the fusion protein; To clarify the relationship between the gene and drug-resistance further, vitro and vivo experiments would be performed to study its effect on tumor cell phenotype and susceptibility to MTX; DHFR Amplicons generation (DSBs, DSB), the amplification level, the amplified form (HSRs / DMs) and efflux (micronucleus) were detected to reveal the relationship between MSH3-RASGRF2 and generation and stability of amplicons. The project will provide xperimental basis for in-depth understanding the molecular mechanisms of MTX-resistance, and new ideas in folic acid antagonist drug-resistance studies.

甲氨蝶呤（MTX）耐药的主要分子机制是DHFR扩增，扩增以均质染色区（HSRs）和/或双微体（DMs）形式存在。基因扩增始于染色体断裂，断裂重接产生基因重排，鉴于热点重排可能影响基因扩增和细胞耐药。前期以HT29亲本为对照，分析了耐10-5M (HSRs)、10-4M (HSRs/DMs)、3×10-4M (DMs) MTX的HT29细胞株中DHFR扩增区域的基因重排，所有梯度中均鉴定到融合转录本MSH3-RASGRF2。本项目拟在前期工作基础上，鉴定融合基因的DNA断裂连接序列及融合蛋白的表达；通过体外和体内实验，研究其对肿瘤细胞表型的影响及对MTX敏感性的变化，进一步明确该基因与耐药的关系；通过检测DHFR扩增产生、扩增水平、扩增形式以及外排的变化，揭示融合基因与DHFR扩增产生和稳定的关系。本项目将为深入理解MTX耐药分子机制提供实验基础，同时为叶酸拮抗类药物的耐药研究提供新思路。

前期我们从耐MTX的HT29结肠癌细胞中特异筛选出融合转录本MSH3-RASGRF2。本研究确定了MSH3-RASGRF2基因的序列、CDS序列并且在耐药细胞中检测到融合蛋白的表达；利用过表达和干扰技术，我们发现MSH3-RASGRF2基因影响肿瘤细胞的耐药性、促进细胞生长、抑制细胞凋亡。我们也发现干扰MSH3-RASGRF2基因引起DNA的双链断裂增加，DHFR扩增和表达降低，DMS的发生减少。研究表明MSH3-RASGRF2基因在MTX耐药中起作用，并且是通过影响基因扩增发生作用。本研究为深入理解MTX耐药提供了基础，同时也为叶酸拮抗类药物研究提供了新的思路。