剪接因子对雌激素受体α7号外显子选择性剪接调控作用的研究

Splicing factors in regulation of alternative splicing of estrogen receptor α exon 7

关键词:  ERα  剪接异构体  剪接因子

Key words:  ERα  splice isoforms  splicing factors

摘要:

目的通过上调或下调肿瘤细胞内hnRNP G、hnRNP I、h Tra2-beta1的表达,探索细胞内三种剪接因子对内源性ERaΔ7形成的作用。方法 Real-Time PCR和Western blot筛选ERα阳性表达细胞株,对细胞株转染hnRNP G、hnRNP I、hTra2-beta1表达质粒和shRNA干扰质粒以上调或下调三种剪接因子的表达,Real-time PCR检测ERaΔ7和ERαexon7的表达量。结果筛选出M CF-7为ERα阳性表达细胞株。在hnRNP G表达上调组中,ERαΔ7和ERαexon7的相对表达量与上调空载体对照组存在统计学差异（P〈0.05,P〈0.05）,在hnRNP G表达下调组中,ERαΔ7和ERαexon7的相对表达量与下调空载体对照组显著差异（P〈0.05,P〈0.05）;在hnRNP I表达上调组中,ERαexon7的相对表达量与上调空载体对照组显著差异（P〈0.05）;在h Tra2-beta1表达上调组中,ERαΔ7和ERαexon7的相对表达量与上调空载体对照组显著差异（P〈0.05,P〈0.05）,在h Tra2-beta1表达下调组中,ERαΔ7和ERαexon7的相对表达量与下调空载体对照组显著差异（P〈0.05,P〈0.05）,其中hnRNP G和h Tra2-beta1对ERα7号外显子的作用为相反趋势。结论 hnRNP G和h Tra2-beta1这两种蛋白可能在ERαexon7的选择性剪接方面存在相互拮抗的作用,hnRNP I对于ERαexon7的纳入存在拮抗作用,据此推测hnRNP G和hnRNP I在ERαexon7的选择性剪接存在协同作用。

Abstract:

Objective To investigate the effect of splicing factors hnRNP G,hnRNP I and h Tra2-beta1 in regulation of alternative splicing of estrogen receptor α exon 7 deletion（ ERαΔ7）.Methods Cell lines expressing ERα were were screened by Real-Time PCR and Western blotting.The screened cell lines were transfected with hnRNP G,hnRNP I and h Tra2-beta1 expression plasmid or shRNA interference plasmid to over-express or dow n-regulate these three splicing factors. Real-Time PCR was applied to detect ERαΔ7 and ERα exon7 expression levels. Results M CF-7 was selected as ERα positive cell line. In hnRNP G up-regulated group,the expression of ERαΔ7 and ERα exon7 was statistically different compared with control group（ P〈0. 05） and hnRNP G dow n-regulation group（ P〈0. 05）. In hnRNP I up-regulated group,the expression of ERα exon7 was statistically different compared with control group（ P〈0. 05, P〈0. 05）. In h Tra2-beta1 up-regulated group, the expression of ERαΔ7 and ERα exon7 was statistically different compared with control group（ P〈0. 05） and h Tra2-beta1 dow n-regulation group（ P〈0. 05）. The regulation of hnRNP G and h Tra2-beta1 to ERαΔ7 displayed opposite trend. Conclusion hnRNP G and h Tra2-beta1 might play antagonistic roles in ERαexon7 alternative splicing. Besides,hnRNP I might antagonize the inclusion of ERα exon7. Thereby,w e hypothesized that hnRNP G and hnRNP I might have synergistic effect in ERα exon7 alternative splicing.