Cloning Of NR6A1 Gene Promoter And Its Transcriptional Regulation In Oxidative Damage

NR6A1,also known as germ cell nuclear factor,is the nuclear receptor subfamily 6 group member 1.Its ligand is unknown and belongs to orphan nuclear receptor family.Previous studies have shown that NR6A1 is expressed in the developing placenta,nervous system,adult human and animal testis,ovary and most tumor tissues,and its function mainly involves in cell differentiation and tumor formation,but the molecular mechanism of its transcriptional regulation is not clear.In this study,the full-length and truncated sequences of human and mouse NR6A1 gene promoter were cloned respectively.Then,the luciferase reporter gene expression system of different truncated NR6A1 promoter were constructed respectively,and transfected into mouse GC-1 cell line(B spermatogonial cells),TM4 cell line(sertoli cells),human testicular teratoma cell line NT-2 and human embryonic kidney cell line 293,to detect the transcriptional activity of each promoter fragment.The experimental results showed that the core region of the mouse Nr6al promoter is located at-169bp+78bp while that of human NR6A1 promoter gene is located at-280 bp?+168 bp,and both of them have the strongest transcriptional activity.Bioinformatics analysis showed that binding sites of 6 transcription factors such as SP1,KLF5,KLF4,CREB1,E2F6 and Mafb,are common sites in the core promoter region of human and mouse Nr6a1.SP1,KLF5 and KLF4 are known to belong to KLF family members,while 3 of the 6 different transcription factors are KLF family factors.Binding sites analysis indicated that transcription factor SP1 site is enriched in both human and mouse NR6A1 promoter,and plays an important role in the transcriptional regulation of NR6A1.Further,combining with the methods of SP1 ORF overexpression experiment and the luciferase reporter gene analysis,it was found that in TM4,GC-1 and 293 cell lines,the transcriptional activity of NR6A1 promoter was enhanced under the action of SP1,and it was found that the activity of the different promoter fragments was independent of the number of the SP1 binding sites.At the same time,RNA interference of SP1 was performed to verify the decrease of NR6A1 promoter activity after knocking down SP1 in 293 and NT-2 cells.These experiments confirmed that SP1 has a positive regulation on the transcriptional activity of NR6A1 promoter.SP1 binding site of NR6A1 promoter near the start codon may play a stronger role in transcriptional regulation.Finally,we discussed the regulatory role of SP1 in the process of NR6A1 response to oxidative damage.An oxidative damage model of NT-2 cells was established by using H2O2 as oxidant.The cells were treated with the H2O2 of O?70 M for 12h.The cell morphology observation and biochemical test showed that with the increase of H2O2 concentration,the cells were gradually shrinking and flattened,gradually lost contact,the growth was inhibited.The decrease of superoxide dismutase(SOD)activity and glutathione(GSH)content,and the increase of lipid oxidation products malondialdehyde(MDA)indicated that H2O2 causes oxidative damage to cells.Quantitative PCR and Western Blot analysis showed that the mRNA and protein levels of SP1 and NR6A1 were significantly increased under the action of H2O2.In combination with the luciferase reporter gene experiment,it was further verified that the activity of both full length and truncated fragment of NR6A1 promoter was significantly enhanced under H2O2,suggesting that the expression of NR6A1 gene in oxidative stress is closely related to its promoter activity.After knocking down the SP1 gene in oxidative damaged NT-2 cells,the mRNA of NR6A1 gene was also reduced,indicating that SP1 was involved in the response of NR6A1 gene to the H2O2 damage signal.In summary,both human and mouse NR6A1 gene promoter were cloned,and their core promoter region was determined in this study.It also confirmed that SP1 had a positive regulatory effect on transcriptional activity of NR6A1 promoter,and NR6A1 could respond the damage signal through SP1 binding site.These findings lay a foundation for understanding the transcriptional regulation of NR6A1 gene.