宫颈鳞癌组织中NRDR选择性剪接新亚型的鉴定及意义

Identification of a novel alternatively spliced variant of NADP(H)-dependent retinol dehydrogenase/reductase (NRDR) in cervical squamous carcinoma tissues and clinical significance

目的:探讨NRDR选择性剪接新亚型NRDRB1 mRNA和蛋白在宫颈鳞癌组织中的表达及其临床意义。方法:采用RT-PCR、免疫荧光、免疫沉淀、免疫印迹、MALDI-TOF质谱分析等技术,检测正常宫颈上皮及宫颈鳞癌组织中NRDRB1mRNA及蛋白表达。结果:NRDRB1亚型全长1 171 bp,其蛋白保留了SDR家族的N末端辅酶结合模序、C末端底物结合模序以及过氧化物酶体定位信号。因第3外显子的缺失,导致SDR家族典型的“LVSNAA”折叠的丢失,使NRDRB1蛋白空间构象发生改变,从而影响该蛋白的细胞内定位和功能,NRDRB1蛋白的亚细胞定位明显不同于NRDR,酶活性亦明显低于NRDR。NRDRB1 mRNA及蛋白在宫颈鳞癌组织中表达(15/27,55.6%),明显高于正常宫颈上皮组织(P<0.05),正常宫颈上皮除NRDR外,未检测到NRDRB1 mRNA及其蛋白。结论:NRDR选择性剪接的异常,以及新的剪接亚型NRDRB1细胞内定位及蛋白酶活性的改变可能与宫颈鳞癌的发生发展密切相关。

Objective： To determine the mRNA and protein expression of NRDRB1, a novel alternatively spliced subtype of NADP（H）-dependent retinol dehydrogenase/reductase （NRDR）, in cervical squamous carcinoma tissues and discuss its clinical significance. Methods..RT-PCR, immunofluorescence, immuno-precipitation, immunoblotting, MALDI-TOF mass spectrometry analysis were used to detect NRDRB1 mRNA and protein expression in normal cervical epithelium and cervical squamous carcinoma tissues. Results： The full-length of NRDRB1 subtype was 1171 bp. NRDRB1 protein conserved the coenzyme binding motifs of SDR family at N terminus, substrate binding motifs at C terminus, and peroxisome-targeting signal. NRDRB1 was characterized by a complete deletion of exon 3 which caused the typical loss of LVSNAA-sheet of SDR family and changed the space conformation of NRDRB1 protein, therefore, influenced the cellular localization and function of NRDRB1 protein. The subcellular localization of NRDRB1 was significantly different compared with NRDR. The activity of NRDRB1 was significantly lower than NRDR. NRDRB1 mRNA was detected in 15 of 27 （55.6%） cervical squamous carcinoma tissues, significantly higher than that in normal cervical epithelial tissues （P〈0.05）. NRDR were detected in normal cervical epithelium but NRDRB1 mRNA and protein were not. Conclusion： Abnormal alternative splicing of NRDR and the changes of subcellular localization and enzyme activity of a novel spliced subtype NRDRB1 had close association with the initiation and progression of cervical squamous carcinoma.