The Preliminary Study On Regulatory Mechanism Of Human SPLUNC1 Expression

Malignant tumor is a common disease with high incidence, which can severelyendanger mankind's health. The nature of tumor is a gene disease, and thedevelopment of cancer is a consequence of a series of molecular changes that haveoccurred in cells, which lead to expressing changes of numerous genes. So thepractical therapy for tumor is indeed to be a therapeutic method whose fundamentalpoint is to reverse the genes changed in tumor.Nasopharyngeal carcinoma (NPC) is a malignant tumor with high incidence infive provinces of Southern China and Southeastern Asia. As the other tumor, NPCcarcinogenesis is a multi-cause and multi-step process. To investigate the geneexpression changes in NPC carcinogenesis, He ZW compared the differential geneexpression profiling between human normal nasopharyngeal and NPC tissue by highdensity cDNA microarray and cloned a complete sequence of EST (ExpressedSequence Tags) with relatively special expression in human nasopharyngeal tissue.This new gene was named YH1 gene with the GenBank accession number AF158745.YH1 gene is located at chromosome 20q11.2, covers approximately 7.31kb andconsists of nine exons and eight introns. YH1 cDNA contains a single open reading frame, which encodes a leucine rich (24.6%) protein of 256 amino acids. Becausediffered in splice acceptor sites in their 3'UTRs, there are two transcript variants. YH1is highly homologous to the mouse PLUNC (Palate, Lung and Nasal epitheliumClone) gene, and the size and location of its introns are remarkably conserved withother species, e.g. pig, cow and rat, so YH1 has been cataloged to PLUNC family.Due to its size is the smallest one in the PLUNC family, YH1 has been nowdesignated SPLUNC1 (short PLUNC 1).SPLUNC1 gene had the relative tissue expression specificity. SPLUNC1 usuallyis expressed in human nasopharyngeal tissues, but very low or rare in NPC tissues. Itis also expressed in human trachea but a little bit in right atrium, jejunum, lung,salivary gland and fetal lung tissue and rarely in others tissues. Furthermore, LUNX, atranscript variant of SPLUNC1, is highly expressed in non-small cell lung cancer(NSCLC), and was detected in lymph nodes diagnosed as NSCLC early-stagemetastasis by RT-PCR, so LUNX mRNA is a possible molecular marker ofmicrometastasis in NSCLCs.The function of SPLUNC1 is unknown. The results from bioinformatics analysisshows that the SPLUNC1 protein contains a signal peptide at the N terminus, and theamino acid sequence is most closely similar to the secretary proteins produced by thesalivary glands and glandular epithelium of the trachea, the von Ebner minor salivaryprotein (also known as LCN1) and the parotid secretary protein. A bactericidal/permeability-increasing protein (BPI) domain was predicted in the three-dimensionalstructure of SPLUNC1 protein. The full-length SPLUNC 1 protein was found to bestructurally related to BPI which is the bactericidal protein and capable ofneutralizing the endotoxin. Based on the structure and functional domain ofSPLUNC1 protein, it was predicated to have the bactericidal and binding function oflipopolysaccharide. Furthermore, SPLUNC1 was identified to be a genetic risk factor for NPC in the study that single nucleotide polymorphisms (SNPs) in SPLUNC1 weresystematically screened and found to be associated with susceptibility to NPC in theChinese population.The regulatory mechanism of gene expression is an important field of researchin post-genomic era. The exact gene expression depends on very complicatedregulatory mechanisms which mainly happen at transcriptional level. Promoter, aspecific DNA sequence capable of binding with the RNA polymerase, is a necessarycis-acting element for the gene transcription. Gene expression is often determined bythe initiation process of specific promoter in the regulation of gene expression.Enhancer, also a positive cis-acting element for the gene transcription, plays apromotion role for the promoter to regulate its downstream gene, and assure the exactexpression of the downstream gene accompanying with other negative cis-andtrans-acting factors (transcription factors). Different transcription factors respond todifferent stimulation by the environment outside or different signals in differentdevelopment stages, and then combine with the transcriptional acting elements, whichactivate or inhibit the gene expression, hence the gene expression is regulated. Theexact expression of a gene relies on the cooperation of cis-acting element,trans-acting factor and RNA polymerase. Nowadays, on the basis of illuminating themechanisms of transcriptional acting, it has developed into a new way for thetreatment of tumors that by selectively interfering the combination of sometranscriptional factors with DNA, abnormal gene expression is reversed, and thenanti-cancer effect is realized.Based on the advanced research ofSPLUNC1 gene and its potential significancein the mechanism of tumorigenesis and prevention & treatment of tumor, identifyingthe exact cell types that expressed SPLUNC1 and the promoter sequences andtranscription factors essential for transcriptional regulation of SPLUNC1 gene may greatly contribute to understanding the regulatory mechanism of its expression.Furthermore, a tissue/cell-specific promoter constructed basing on the specificexpression of SPLUNC1 will offer a new way for prevention and control of tumor.We made some researches to clarify the characteristics and regulatory mechanism ofSPLUNC1 gene expression as follows:1. Identification of cell types expressed SPLUNC1 geneIn situ hybridization method was used to detect the expression of SPLUNC1gene in nearly 30 kinds of tissues. The results showed that SPLUNC1 gene wasexpressed neither in squamous epithelial cells of palate, epidermis, esophagus, andthe junction of esophagus with cardia of stomach, nor in the tumor cells ofnasopharyngeal poorly-differentiated squamous cell carcinoma, esophageal squamouscell carcinoma, and lung squamous cell carcinoma, nor in the metaplastic squamouscells in nasopharynx, trachea and uterus cervix. SPLUNC1 gene also was notexpressed in the single layer columnar epithelia cells in stomach, gallbladder, jejunum,colon, endometrium and cervix. It was expressed mainly in pseudostratified columnarepithelial cells in nasopharynx, trachea and bronchi, and interestingly, its expressionwas gradually down-regulated in respiratory tract from upper to lower end, and noexpression was found in lung tissues in the end. SPLUNC1 gene was also found notonly in duct and serous gland cells both in parotid and submandibular gland, but alsoin cells of submucosal serous gland in nasopharynx, lung and esophagus, while noexpression were found in cells of mucosal gland. The expression of SPLUNC1 wasdetected in parietal cells of gastric submucosa and epithelial cells of lobula and ductof mammary gland. In addition, SPLUNC1 gene was found strongly expressed inwell-differentiated adenocarcinoma cells in lung, stomach, colon, mammary gland,uterus endometrium and cervix. All these data suggests that SPLUNC1 is a geneprobably associated with cell differentiation, not a tumor suppressor gene in nasopharyngeal carcinoma, and its expression is highly cell specific. These results laya favorable basis for further studying the regulatory mechanisms of its expression andbiological function, and shed light on the diagnosis and treatment of tumor.2. Prediction of the regulatory elements of SPLUNC1 gene by bioinformatics toolsBy analyzing the 6kb sequence from -5000bp to 1000bp (the first nucleotide of thepublished cDNA is +1bp) of SPLUNC1 by BLAST alignment, promoter predictionsoftware and recognition of transcription factor binding sites, the results showed thetranscription start site is located at the region of -1～+10bp, a TATA box presents atthe -29bp position, and the promoter exists in the region from -490 to +89bp ofSPLUNC1. The conserved region was found in the promoter sequence betweenhuman and mouse species, especially from -245 to -1bp, with some transcriptionfactor binding sites. In addition, Enhancer element location (EEL) software predictedthat five highest scoring cis-module resulting from the alignment of 30kb human andmouse sequences was located to 5' end, interior and 3' end of SPLUNC1. Predictedresults are perhaps favorable o identify all possible regulatory regions.3. Regulatory activity assay for the 5' flanking sequence of SPLUNC1The expression of SPLUNC1 mRNA was detected by Real-time reversetranscription polymerase chain reaction (RT-PCR) in eleven cell lines includinghuman lung adenocarcinoma-derived cell line (Glc-82), NPC-derived cell lines (5-8F,6-10B, C666-1, CNE-1, CNE-2, HNE-1 and HONE-1), human embryonic kidney cellline (HEK293A), et al. The expression level of SPLUNC1 was the highest in Glc-82cells, which provided a suitable cell model to study the regulatory mechanisms ofSPLUNC1.Based on genomic sequence covering 5'end of SPLUNC1 gene, we designedfive pairs of primers that the downstream end was the first exon(+1) down stream 100bp and the end of upstream located at -3000, -2000, -1000, -500, and -240bp,respectively. The five fragments, P4 (3100bp), P3 (2100bp), P2 (1100bp), P1 (600bp)and P0 (340bp) were isolated from the human genomic DNA by PCR amplification.After the expected PCR products were cloned into T vector and confirmed by directsequencing and enzymatic digestion, the sticky-end fragments digested by KpnⅠ/XhoⅠwere inserted into PGL3-Basic, a promoter-less luciferase reporter vector to form fiveplasmid constructs, pGL3-P0～pGL3-P4. Transient transfections included reporterplasmids, pGL3-Basic, pGL3-Control as positive control and pSV-β-Galactosidasereporter vector as an internal control. The promoter activity of each construct wasevaluated by luciferase assay of cell extracts from Glc-82 and 293A transfected 48hours later. We determined that the differences among the transcriptive activity of fivepromoter fragments in Glc-82 and 293A cells, and found that the transcriptiveactivities of P0 in both cell lines were higher than that of other fragments. Thetransfected reporter genes pGL3-P0 displayed an enhanced expression of luciferaseactivity by 5.10-fold and 2.69-fold compared with the blank vector pGL3-Basic inGlc-82 and 293A cells, respectively. These results indicated that the 5' flankingregion from -240 to +100bp manifested relatively low but functional promoteractivity, and the promoter of SPLUNC1 gene might be located at this region.Furthermore, taking the luciferase activity of pGL3-Control as 100%, the luciferaseactivity of the five fragments in Glc-82 cells were higher than that in 293A,suggesting the promoter of SPLUNC1 is tissue specific.4. Identification of SPLUNC1 enhancersBased on the human genomic sequence, five pairs of primers targetingenhancers' fragments (E0-E4) predicted by bioinformatics were designed and thenisolated from the human genomic DNA by PCR amplification. After the expectedPCR products were confirmed by direct sequencing, the sticky-end fragments, digested by KpnⅠ/XhoⅠand by BamHⅠ/SalⅠrespectively, were inserted intoPGL3-Promoter, a reporter vector, to form five reporter plasmid constructs,pGL3P-E0～4-up, and five plasmid constructs, pGL3P-EO～4-down. Transienttransfections to 293T cells included all reporter plasmids, pGL3-Promoter,pGL3-Control as positive control and pSV-β-Galactosidase reporter vector as aninternal control. The enhancer activity of each construct was evaluated by luciferaseassay of cell extracts transfected 48 hours lated. The results showed that when theenhancer fragment was located at the upstream of reporter gene promoter, theregulatory activity of enhancer E0 and E2 were highly increased, which was 2.92-foldand 3.03-fold higher compared with that of pGL3-Promoter, respectively, whileenhancer fragments were located at the downstream of reporter gene, the regulatoryactivity of enhance E0, E1, and E2 were highly increased, which was significantlyhigher than that of pGL3-Promoter (the difference was 2.65-fold, 2.83-fold, 3.13-foldrespectively). In addition, in comparison of regulatory activity of enhancer fragmentsin different reporter gene locations, the activity of fragment E1 located at thedownstream of reporter gene was obviously higher than that at the upstream ofreporter gene.5. Identification of trans-acting factors in the promoter of SPLUNC1The 3' end of 22～30bp oligonucleotide DNA sequences derived from thepromoter of SPLUNC1 containing the consensus motifs of Snail/YY1/n-MYC/HNF-3beta were labeled with biotin. The biotin-labeled complementaryoligos were annealed to form double-stranded probe for elcetrophoretic mobility shiftassays. The nucleic protein was extracted from Glc-82 cells and bound to the labeledprobe with unlabeled specific-competitive and unspecific-competitiveoligonucleotides. A specific band was observed in the gel with probes containing aputative Snail site and unaffected by excessive mutant probe, while no specific band was observed with excessive non-labeled probe. Similar band patterns were observedusing probes containing the binding sites of YY1 and n-MYC, while no specificbinding was found in the HNF-3beta reaction. These results suggest that Snail, YY1and n-MYC proteins bind to the binding sites located at the proximal region ofSPLUNC1 promoter. To further confirm that Snail could bind to the SPLUNC1promoter, chromatin immunoprecipitation assays (CHIP) was carried out. AfterGlc-82 cells were cross-linked with formaldehyde and incubated with Snail antibody,DNA-protein-antibody complexes were precipitated. DNAs interacted with Snailprotein were purified and were determined to have the -240 to +100bp and -157 to+100bp region of human SPLUNC1 promoter.Conclusions:1. SPLUNC1 is a gene probably associated with cell differentiation, not a tumorsuppressor gene in nasopharyngeal carcinoma, and its expression is highly cellspecific.2. The 5' flanking region from -240 to +100bp of SPLUNC1 gene manifestedfunctional promoter activity and cell specificity.3. The DNA sequences from -158 to -9bp, +3770 to +3959b and +2061 to +2201bphad the activity promoted transcription.4. The transcription factors, Snail, YY1 and n-MYC, maybe participate in theregulation process of transcription of SPLUNC1 gene.