The Regulation Of A New Tumor Associated Antigen BAP31Gene Expression

Background: B cell receptor associated protein31(BAP31) is a28KDintegral membrane protein of the endoplasmic reticulum. BAP31isevolutionarily conserved and expressed ubiquitously. BAP31has been shown toassociate with and promote the vesicular transport from the ER of MHC class Imolecules, cellubrevin, tetraspanins and CFTR; of CD11b/CD18from secondarygranules to the plasma membrane in neutrophils; and the retention of mIgD,cytochrome P4502C2, and PTPLB at the ER. Another function of BAP31relatedto apoptosis. The cytosolic domain of BAP31contains two identical caspaserecognition sites (AAVD.G). The p20fragment generated by BAP31cleanvagewas a downstream molecule of caspase8and was involved in many pathologyprocesses, such as ERstress, CRT turnout. Furthermore, p20causes a rapidtransmission of ER calcium signals to mitochondria, facilitate the communicationbetween ER and mitochondria. Our previous study showed that althoughubiquitously, the expression of BAP31is dramatically up-regulated in variouscarcinomas; especislly in cervical cancer. Depletion of BAP31by SiRNA in Helacell could lead to an arrest of cells in G1phase, a significant decrease in thenumber of migrating cells relative to control transfected cells; moreover, down-regulation of BAP31sensitizes cells to chemotherapeutic drugs-inducedapoptosis. All of these indicate that BAP31is a tumor associated antigen, and itmay promote the development and metastasis of tumor by interfering thebiological characteristic of cancer cells.The ETS-1gene has first been cloned and characterized as the cellularproto-oncogene of the retroviral v-ets oncogene of the avian leukaemia retrovirusE26. ETS-1has a specific DNA-binding domain, called ETS domain, whichconsists of approximately80amino acids with four tryptophane repeats andwhich binds to double-stranded DNA containing a GGAA/T core motif anddifferent flanking sequences. Transactivation as well as transrepression of manytarget genes can be mediated through ETS transcription factors. DNA-bindingspecificity is determined by the sequences flanking the GGAA/T core and bybinding of further transcriptional partners. Transactivation or transrepressioncan be regulated by interaction with other proteins, by phosphorylation ofinternal regulatory domains of specific ETS factors via protein kinases or Ca2+dependent signalling and finally by acetylation or sumoylation. Proteinsinteracting with ETS factors include other key transcription factors such as AP-1,SP-1, NF-κB, CREBP-binding protein/p300, Pax or a combination of differentETS family members. Many different functions have been attributed to ETS-1indifferent cell types and organisms at molecular and cellular levels, includingproliferation, apoptosis or the regulation of matrix degradation.Aim: study the regulation of human BAP31gene regulationMethods and results: the human genomic DNA was obtained from Hela cell.Then we predict the TSS of BAP31by webset（esemble）, and get the-5k~1.5ksequence of BAP31by PCR. Based on the obtained DNA sequence, a series oftruncated fragments were constructed and ligated into the pGL3-basic liciferase reporter vector. These vectors were transfected into Hela cell and luciferaseactivity was analyzed at48h after transfection to determine the promoter site.Data showed that the strongest luciferase activity was located at-122bp~128bp,which is the promoter. Besides this, there was still another stite that had a strongluciferase activity--4012bp~-2996bp. At the same time, there may be anegative regulation element between9bp to52bp. Bioinformatics analysisshowed that there were several ETS-1binding site in the promoter of BAP31.Then the reporter vector of BAP31promoter was co-transfected with different TFrespectively, and the luciferase activity was tested. Data showed that ETS-1enhanced the promoter activity of BAP31. Overexpression of ETS-1upregulatedthe expression of BAP31by2fold both at mRNA and protein level. Deletion ofETS-1in Hela cell by siRNA downregulated the protein leve of BAP31.Bioinformatics analysis showed that there is CpG Island in the first exon; the3’UTR of BAP31was complementary with miR-137, indicating miR-137maybeinvolved in the posttranscript regulation of BAP31.Conclusion: ETS-1regulates the expression of BAP31.