正常肝和肝癌细胞中DHRS4L2基因选择性剪接亚型的克隆及其表达调控研究

Cloning of splice variants of DHRS4L2 in normal and malignant hepatic cell lines and regulation of its transcription

目的:鉴定正常肝细胞HL-7702和肝癌细胞Hep-G2中DHRS4L2基因的选择性剪接新亚型,并探讨其表达调控机制。方法:应用RT-PCR,3′RACE和生物信息学方法发现并鉴定DHRS4L2基因的选择性剪接新亚型;去甲基化药物5-aza-dC处理HL-7702和Hep-G2细胞,通过RT-PCR和real-time PCR方法检测DHRS4L2的表达情况。结果:发现了2个新的DHRS4L2剪接亚型DHRS4L2A和DHRS4L2A3,2个新亚型均出现了新的第1个外显子,命名为Ea,前者缺失外显子1,后者缺失外显子1和3;在正常肝细胞中去甲基化后含DHRS4L2外显子1的转录产物比处理前明显增加,在肝癌细胞中则比处理前减少。结论:获得2个新的DHRS4L2剪接亚型,并发现DHRS4L2的表达和甲基化调控相关,且甲基化的调控作用在正常肝与肝癌细胞之间存在差异,其机制尚待进一步探讨。

OBJECTIVE：Here we report the cloning and characterization of two novel splice variants of DHRS4L2 gene and potential mechanisms regulating the transcription. METHODS： RT-PCR,3＇-RACE and bioinformatics were used to clone and characterize the novel splice variant of DHRS4L2 from human hepatic immortalized cell line HL-7702. Transcription of DHRS4L2 was investigated by RT-PCR in 5-aza-dC treated HL-7702 and Hep-G2 cell lines. RESULTS： we found two novel splice variants DHRS4L2A and DHRS4L2A3,both with alternative first exon Ea. The former lacked exon 1 and the latter,exon 1 and exon 3. After 5-aza-dC treatments,DHRS4L2 transcripts with first exon E1 were increased in normal hepatic cells while the reverse trend was found in malignant hepatic cells. CONCLUSION：We found two novel splice variants of DHRS4 and suggested that the alternative starts of DHRS4L2 transcription may be regulated by methylation. The effect of methylation is different between normal and malignant cell lines and the underlying mechanism remains to be investigated.