Cloning Of Splice Variants Of DHRS4L2 In Normal And Tumor Hepatic Cell Lines And Regulation Of DHRS4L2 Transcription

Objective:All-trans retinoic acid (atRA) is a biologically active fat-soluble compound found naturally in many organisms. It is a morphogen for organogenesis during embryonic development. In addition, atRA plays a vital and extensive role in biological differentiation, immunomodulation, anti-infection, tumor growth inhibition and induced differentiation, and apoptosis of tumor cells. atRA is derived from vitamin A through a two-step oxidative mechanism.NADP(H)-dependent retinol dehydrogenase/reductase (NRDR) is an important retinol dehydrogenase/reductase enzyme required for the synthesis of atRA and is found in a wide range of mammalian tissues. The NRDR gene is localized on chromosome 14q11.2, within a DHRS4 gene cluster consisting of three homologous genes, namely DHRS4, DHRS4L2, and DHRS4L1. DHRS4 and DHRS4L2 exhibit 97.5% identity at the nucleotide level. There is an insertion fragment with a length of about 20000 bp between DHRS4 and DHRS4L2, and also between DHRS4L2 and DHRS4L1 (insertion 1 and insertion 2). Previous studies have found many alternative splice variants of DHRS4L2 in tumor cells, in particular neuroblastoma cells. Examination of existing transcripts of DHRS4L2 revealed mRNAs in which the first exon(E1) was replaced by Exon a (Ea) whose genomic sequence in located approximately 19 kb upstream from the DHRS4L2 exon (E1). Exon a (Ea) is known to be present in DHRS4L2 mRNA from many tissues and organs, such as human brain, testis, placenta, and esophagus. However, few studies address the alternative transcription pattern of DHRS4L2 in human liver. Sequence analysis of the DHRS4 gene clusters revealed the CpG islands upstream of both of the Ea and DHRS4L1 E1 exons. Determination of methylation status revealed that the CpG island upstream of DHRS4L2E1 is completely methylated, whereas the CpG island upstream of Ea is unmethylated, pointing to a potential regulatory role of methylation in DHRS4L2 transcription. This study clones and characterizes two novel splice variants of the DHRS4L2 gene and investigates the role of DNA methylation in regulating DHRS4L2 gene transcription.Methods:RT-PCR, 3'-RACE, and bioinformatics were used to clone and characterize the novel splice variants of DHRS4L2 in the immortal HL-7702 human hepatic cell line and the Hep-G2 human hepatocellular carcinoma cell line. Transcription of DHRS4L2 was determined by RT-PCR in 5-aza-dC-treated HL-7702 and Hep-G2 cell lines.Results:We identified two novel splice variants, denoted DHRS4L2A and DHRS4L2A3,both containing an alternative first exon Ea. Following 5-aza-dC treatment, Ea-containing transcripts of DHRS4L2A and DHRS4L2A3 decreased while E1-containing DHRS4L2 transcripts increased.Conclusions:We found two novel splice variants of DHRS4L2. Our findings suggest that DNA methylation regulates transcription from alternative start sites to govern DHRS4L2 expression. The effect of methylation may depend on the nature of the cell line (i.e. normal or tumor cell lines). However, the underlying mechanism remains unclear and thus requires further study.