| Dax1(Dosage-sensitive sex reversal-AHC-critical region on the X-chromosome gene 1,NR0B1),a nuclear receptor protein whose gene structure and function is conserved in human and mouse,plays important roles in reproductive development,sex determination and homonogenesis.Recent studies showed that the expression level of Dax1 is upregulated in several cancers(e.g.lung cancer,ovarian cancer,prostate cancer),and closely associated with tumor grade and prognosis.However,the underlying mechanisms are not well understood.Tumor cells,similar to embryonic stem cell(ESC),can proliferate rapidly,maintain self-renewal and pluripotency.Tumorigenesis is caused by oncogenic transformation of normal cells with the altered expression of many oncogenes.There are many similarities between oncogenic transformation and production of induced pluripontency stem cell(i PSC)with somatic cells.In addition,ESC,iPSC and high grade tumors share many transcript features.Stemness genes and reprogramming factors(e.g.Oct4,Nanog,Lin28,Klf4)are usually expressed aberrantly in tumor cells,inducing oncogenic transformation of normal cells and facilitating cancer occurrence and development.Therefore,studies of gene function in ESC and iPSC provide valuable references for understanding the mechanisms of tumorigenesis.Dax1 has been identified as a major component in both Oct4-centred and Nanog-centred protein interaction networks by proteomics and genomics studies.Our previous study showed that Dax1 acts in parallel with Nanog to inhibit the differentiation,and is indispensable in maintaining stable pluripotent state of mouse ESC(m ESCs).Moreover,it is required for full reprogramming to induce pluripotency.Considering the close relationship between Dax1 and cancer,we suggest that Dax1 play an important role in oncogenic transformation.Alternative splicing(AS)of pre-messenger RNAs is a key step in the gene expression process,which allows the synthesis of different products from the same gene and increases the complexity of the proteome coded by a limited number of genes.Genome-wide approaches have revealed that tumorigenesis often involves large-scale alterations in AS.The expression of Different alternative splicing variants may induced the tumorigenesis transformation.It have been reported that hDax1 have a different variants in testis,ovary and pancreas tissues,but the new alternative splicing variants and their roles in oncogenic transformation need more study.In this study,we identified several new alternative splicing variants of hDax1,and analyzed the correlation between their expression and tumor tissues.Furthermore,we studied their roles in inducing oncogenic transformation and the underlying mechanisms.The main results are summarized as below:1.The characterization of new hDax1 alternative splicing variants.1.1 Nests-PCR was used to detect the expression of hDax1 in 16 different human tissues,the PCR products were sequenced and we found there are 8 different hDax1 splicing variants: hDax1L-1413,hDax1L-729,hDax1L-549,hDax1L-453,hDax1A-1203,hDax1A-603,hDax1A-519 and hDax1A2-189.Each variants have different m RNA and amino acid sequence,and can be translated to proteins with different functional domains.These variants express tissue specificity.Western blots were used to verify the endogenous expression of new hDax1 variants.1.2 The cellular localization of hDax1 variants was detected by Immunofluorescence;we found the variants have distinct cellular localization and were cell specific.Luciferase reporter and ChIP assay were carried out,and the results showed that these variants have different transcriptional repression and DNA binding ability.co-IP results showed hDax1 variants have different protein binding abilities.These results indicate that the hDax1 variants have different functions.2.The correlation between hDax1 alternative splicing variants expression and tumor2.1 We detected the expression of hDax1 splicing variants in different cancer cell lines,the results showed that there are 4 variants exist in cancer cell at least,they are hDax1L-1413,hDax1L-729,hDax1A-1203,and hDax1A-519.The hDax1 variants have cell specificity expression.2.2 The gene expression profile data in GEO database was used to analysis the hDax1 expression in normal and tumor tissues,the results showed that hDax1 was upregulated in many tumors vs normal tissues,especially in lung cancer and skin melanoma,the high expression level of hDax1 indicate poor prognosis.We proceeded to use the RNA-seq data in TCGA database to analysis the expression of hDax1 splicing variants in tumor tissues,the results showed a distinct expression profile of these variants in different types of tumor tissues.We next detected the hDax1 variants in lung cancer and skin melanoma tissues,the results showed that these variants have different expression profile in the same type of tumor tissues either.Together,these results indicate hDax1 splicing variants may participate in different tumorigenesis process.2.3 Single cell RT-PCR was used to detect the hDax1 variants expression in the same cell line,we found hDax1 splicing variants show heterogeneous expression patterns in single cell,which indicates the variants have distinct cellular functions.3.The function of hDax1 splicing variants in induced oncogenic transformation of normal cell.hDax1 splicing variant was ectopically overexpressed in NIH3T3 by stable transfection,and its effect on the cellular phenotype was determined by in vitro and in vivo assays,such as anchorage-independent growth assay,proliferation assay and xenograft tumor formation.The results showed that only hDax1L-1413,hDax1A-1203,hDax1A-519 and hDax1A2-189 can induce the transformation of NIH3T3,they were able to form spheres in vitro and initiate xenograft tumors in nude mice.Furthermore,we found that h Dax A-1203/NIH3T3 and hDax1A2-189/NIH3T3 induce the xenograft tumors grow more rapidly,but hDax1L-1413/NIH3T3 and hDax1A2-189/NIH3T3 were shown to possess increased tumor-initiating ability,which indicate hDax1 splicing variants have different oncogenicity,and may via distinct mechanisms.The proliferation assay showed that hDax1L-1413,hDax1A-1203,hDax1A-519 and hDax1A2-189 cannot promote the proliferation of NIH3T3,indicate the transformation is proliferation-independent.Other variants hDax1L-729 、 hDax1L-549 、 hDax1L-453,hDax1A-603 cannot induce the transformation of NIH3T3.4.The mechanisms of hDax1 splicing variants mediated cellular transformation of NIH3T3Gene expression microarray was used to identify genes regulated by hDax1 variants and potentially responsible for tumorigenic potential of hDax1 variants expressing NIH3T3.The results showed that hDax1L-1413,hDax1A-1203,hDax1A-519 and hDax1A2-189 variants induced transformation of NIH3T3 through distinct mechanisms.hDax1L-1413 upregulate the factors of small GTPase mediate signal pathway;hDax1A-1203 could activate the HIF-1,SHH,MAPK pathway;hDax1A-519 could activate the HIF-1 pathway;hDax1A2-189 could activate the HIF-1,SHH,WNT pathway.To validate our microarray data,expression of the genes involved in the signal pathways was examined by q RT-PCR,the results were in complete agreement with the microarray data.In summary,we identified new hDax1 splicing variants which have distinct protein structure and functions.These splicing variants have tissue specificity expression in normal and tumor tissues.Furthermore,hDax1-expressing NIH3T3 cells spontaneously formed tumors in nude mice,which demonstrate,for the first time,hDax1 functions as a bona fide oncogene in cell transformation and tumorigenesis.Additionally,different hDax1 variants may function through different regulation mechanisms,indicating each variant may participate in different tumorigenesis process with distinct functions.The identification of new hDax1 splicing variants re-annotations this gene.The distinct functions of different hDax1 variant in cell transformation and mechanisms of hDax1 splicing variants mediated transformation may provide new insight into the tumorigenesis research,and offer possible targets for the specific clinical diagnose and treatment of cancer. |
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