The Construction And Expression Of HOXA11 Promoter Reconbinant Plasimid

Objective: Homeobox genes,a family of regulatory genes controlling embryonic development and cell differentiation, contain a common 183-nucleotic sequence and altitude consevertive homeobox. As a big group of Homeobox, HOX genes can code for special nuclear proteins that act as transcriptional regulators and have a 61-amino-acid.the homeodomain, responsible for reconition and binding of sequence-specific DNA motifs to regulate target gene. HOXA11 as a member of HOX genes family is expressed in the prosterior region of embryo and in adult uterus, and controls the form and the development of the uterus. Recent evidence has demonstrated that HOX genes, the principle regulators of tissue differentiation in the embryo, are also essential for endometrial development and differentiation.In recent years, the relationship between ovarian hormones and HOX gene expression has become hot spot in the field of reproductive research. Now it is considered that HOX gene is the important mediation by which sex hormones regulates the changes of endometrium.By previous study, we have found that HOXA11 gene expression in human endometrium is regulated by sex steroid and changed obviously at mid-secretory phase of endometrial glandular epithelium; and we firstly discovered that at the mid-secretory phase HOXA11 in glandular epithelium expressed weaken or even disappeared but negatively regulated by progesterone. Such negative regulation has a close relation with endometrial differentiation, maturation and receptive state. However, the mechanism of the negative regulation is still not clear. The existence of progesterone response element on HOXA11 gene promoter region has not been reported. Therefore, this study is designed to determine the location of the HOXA11 promoter and prove theexistence of progesterone response element on HOXA11 gene promoter region through constructing HOXA11 pEGFP-promoter recombinant plasmid. In a word, the study laid experimental basis to further study the mechanism of the negative regulation.Method: Search for HOXA11 gene sequence from Genebank, forecast two promoter regions (promoter 1and promoter 2) from sense and antisense strands and respectively design PCR primers. Genomic DNA was isolated from decidua. Target gene fragments were amplified by PCR. PCR products were cloned to T-vector (including connecting, transforming, blue-white selection, Plasmid extraction, identification and sequencing). TA cloning products were digested by two restriction enzymes. The digested products were cloned to pEGFP-1 (including connecting, transforming, and kanamycin selection, plasmid extraction, identification and sequencing). The pEGFP-promoter recombinant plasmids were transfected into Ishikawa cells, which were treated with progesterone, and then observed by fluorescent microscope.Results:1. Compared with the gene sequences in Genebank, the correct rates of target gene sequences were over 99%.2. The green fluorescence was observed in Ishikawa cells transfected with pEGFP-promoter1 recombinant plasmid.3. Ishikawa cells that had been transfected with pEGFP-promoter1 recombinant plasmid were treated with progesterone, and the brighter green fluorescence was observed in cytoplasm.4. The green fluorescence was not detected in Ishikawa cells which had been transfected with pEGFP-promoter2 recombinant plasmid and then treated with and without progesterone.5. Two similar progesterone-response elements were found in promote- r1 region (1048-1062bp, 1492-1506bp), and the homology of these two elements were over 50%, compared with classic PRE (progesterone response element).Conclusion:1. Promoter1 located between 993 and 2016bp of HOXA11 sense strand (AF039307) had promoter function.2. The brighter green fluorescence was observed in Ishikawa cells transfected with pEGFP-promoter1 recombinant plasmid and treated with progesterone. It was suggested that promoter1 region contained progesterone response element.3. There were two similar progesterone response elements in HOXA11 promoter1 region. They respectively located at 1048-1062bp and 1492-1506- bp (AF039307).4. Promoter2 located between 19-1049bp of HOXA11 antisense could not contain progesterone response elements and had not promoter function.