The Research Of Alternative Splicing Variants Of NADP(H)-Dependent Retinol Dehydrogenase/Reductase In Human Neuroblastoma

All-trans Retinoic Acid is a hormonelike substance with important physiological functions. It maintains a low physiological concentration in vivo. Ml-trans Retinoic Acid can be produced via a two steps metabolism from vitamin A, which is also called retinol, with an intermediate product of retinal.NADP(H)-dependent Retinol dehydrogenases /reductases (NRDR) of the short chain dehydrogenase/reductase family was a critical enzyme for vitamin A metabolism. It was first found, purified and identified from the rabbit liver by Prof. DongYang Huang(Huang and Ichikawa, 1997). It is an important member of the short chain dehydrogenase/reductase family.NRDR is broadly expressed in various tissues of mammal. Its cDNAs of human and some other major mammal species have been cloned one after the other. Recent report shows that an alternative splicing variant, NRDRiso (AY071856), was cloned from human liver (Du et al,2004) , suggesting that alternative splicing probably play an important role in the functional regulation of NRDR gene, and that it is possible for other forms of alternative splicing of this gene to exist.Neuroblastoma is one of the most familiar children' s malign tumors, which originates from the sympathetic nerve adrenal gland system of the neural crest during embryonic life. ATRA can induce the differentiation of human neuroblastoma cell line in vitro. This suggests that it is reasonable to explore the possibility of endogenase (physiological) ATRA generation obstruction. Recent research has shown that abnormal alternative splicing correlates with the tumor generation and progress. If the abnormal NRDR alternative splicing variants and their protein were produced in human neuroblastoma, the regular NRDR expression will decrease and the generation of endogenase ATRA will be retarded. Concequently, investigation of the abnormal neuroblastoma NRDR alternative splicing may be helpful our exploration of the occurance and development of human neuroblastomaMethodsWe have cloned 23 new alternative splicing variants from human neuroblastoma SK-N-SH by PCR and RACE and tried to determine the subcellular location of humNRDR A2 by GFP. We have also analyzed the 23 new NRDR alternative splicing variants and the NRDR gene structure, as well.ResultsThere are three copies for human NRDR genes, including DHRS4, DHRS4L2 and DHRS4X. The identity between the former two genes is 97.5%, suggesting that they are segmental duplication (90% -98%; ^1 kb). Inserting sequence among the three copies genes are named truncated INSERTION, INSERTION1 and INSERTION2, respectively.The new alternative splicing variants of human neuroblastoma SK-N-SH include AY616183, AY920361, AY920362 AY943857, DN237879, DN237881, DN237882, DN237883, DN237884, DN237885, DN237886, DN237887, DN237888, DN237890, DN237891, DN237892, DN237893, DN237895, DN237896. Among the above sequences, we have obtained the cDNA full sequence of the following forms: AY616182, AY616183, AY920361, AY920362, AY943857.The coding region of human neuroblastoma alternative splicing variant NRDR A2 contains framshift and nuclear targeting sequence. The failure of the experiment of its subcellular location suggests that it might encode a protein that is toxic to cells. mRNA of AY616183 and AY943857 encodes 5' terminal truncated proteins. And the open reading frames for AY920361 and AY920362 are still not determined.There are several transcription start sites for DHRS4L2, including the normal one before exon 1, the one before exon (a) on INSERTION1 and the one at the middle of exon2. This suggests that the DHRS4L2 gene boundary should be extended to exon (a). We found the the transcription start site before exon (a) only presented athuman neuroblastoma SK-N-SK, normal brain hypothalamus, testis and placenta, indicating that this transcription start site probably is linked to some special metablisms or functions of human nerve system and procreation system.No expression of DHRS4X, the third copy of human DHRS4, was observed at human neuroblastoma SK-N-SH. It also rarely presents in the ESTs pool.ConclusionsWe have cloned 23 new human neuroblastoma NRDR cDNAs. We have also analyzed and elucidated the structure characteristics of NRDR gene by bioinformatics. We have found the multiple transcription start sites of DHRS4L2 gene. Further, we have carried out the experiment of subcellular location of the special splicing variant NRDR A2, which has a framshift and nuclear targeting sequence.DHRS4 and DHRS4L2 express large amout of abnormal alternative splicing variants in human neuroblastoma. We believe that there are some correlation between the abnormal splicing and the tumor. These abnormal splicing in neuroblastoma may take part in the occurance or development of tumor. It is also possible that they are the results of tumor itself. This investigation contributes to the future research about the correlation.