Noninvasive Prenatal Diagnosis Of Down Syndrome And Function Analysis Of Down Syndrome Related Genes

Part One Hypomethylated DSCR4 is placental derived epigenetic marker for Trisomy 21 and preliminary function analysis of DSCR4 geneDown syndrome (DS), or trisomy 21, is the most common genetic cause of developmental disabilities and mental retardation. At present there is no available cure for the disease. Prenatal diagnosis is therefore very important. Current methods for prenatal diagnosis of DS involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Hence, it would be very desirable to develop noninvasive prenatal diagnostic methods.Noninvasive screening methods, including nuchal translucency sonography and maternal serum biochemical screening, typically detect the associated epiphenomena instead of the core genetic abnormalities of chromosome dosage imbalance and are not diagnostic in nature. It has been a long-sought goal of noninvasive prenatal diagnosis to develop methods of obtaining fetal genetic materials for analysis. The discovery of intact fetal cells and cell-free fetal nucleic acids in maternal blood has opened up new possibility of noninvasive prenatal diagnosis. The low absolute number of fetal cells in maternal blood hampered the use of fetal cells in noninvasive prenatal diagnosis of DS, while the cell-free fetal DNA has the advantage of relatively abundant and remarkably stable and is becoming increasingly important.Fetal DNA circulates in maternal blood among a high background of maternal DNA sequences.The main obstacle is to discriminate the fetal DNA molecules from the maternal ones.Since the observation that cell-free fetal DNA in maternal blood originates from placenta tissue, whereas the maternal DNA is derived from haemopoietic tissue, an epigenetic approach has been explored to distinguish the fetal DNA molecules from the maternal ones in maternal plasma, which is regardless of fetal gender and genotype. For example, the SERPINB5 gene on chromosome 18 is different methylated in the placenta and maternal blood cells and the allelic ratio for placenta derived hypomethylated SERPINB5 in maternal plasma is useful for noninvasive detection of fetal trisomy 18.To develop a similar method for the noninvasive detection of trisomy 21, we chosed six genes highly or specifically expressed in placenta, they are CGI137, LRRC3, POTE, COL6A1, AIRE and DSCR4. Given the negative connection between gene expression and promoter methylation, we supposed the promoter regions of these genes were hypomethylated in placental tissues and hypermethylated in maternal blood cells. We screened the methylation status of genes by combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes assay, three of six genes (AIRE, POTE and DSCR4) showed the differential methylation patterns between placental tissues and maternal blood cells at least in 1st trimester paired samples. By using bisulfite DNA sequencing, the DSCR4 gene promoter was found to be hypomethylated in the placenta and densely methylated in maternal blood cells at each CpG site. To eliminate cloning bias, we performed combined bisulfite restriction analysis and confirmed the results.Based on the results of bisulfite sequencing, we developed a methylation specific PCR assays for DSCR4 and evaluated the specificity of the primers for methylated or unmethylated templateds by semi-quantification PCR. we subsequentially determined the relative level of unmethylated DSCR4 (U-DSCR4) in 1st trimester maternal blood cells and placental tissues with the SYBR Green I-based real-time PCR and confirmed that U-DSCR4 was only present in placental tissues. The placental derived fetal specific U-DSCR4 can be detected in 1st trimester maternal plasma by using real-time quantitative methylation-specific PCR.The gene DSCR4 locates in the band q22.2 of human chromosome 21 and encodes a protein of 118 amino acids. Expression of DSCR4 was restricted to human placenta and placental choriocarcinoma cell lines BeWo and JEG3. Down syndrome progresses during the development of fetus attached to the placenta and some serum markers for trisomy 21 are origin from the placenta. Functionally abnormal placenta and deficiency of fetal growth are seen in Down syndrome. Thus, the function of DSCR4 protein apparently related to placenta function, and the study of the mechanism of DSCR4 in trophoblast cells will contibute to the etiology of Down syndrome.We found the promoter methylation of DSCR4 gene correlates with reduced gene expression. In order to study the function of DSCR4 gene, we expressed the full length of DSCR4 protein as antigens to immunize rabbits and generated rabbit anti-human DSCR4 anti-serums. Then we purified anti-serums with protein A/G affinity columns. The results of western blot and cell immunity staining showed we obtain DSCR4 specific antibodies. We next studied the subcellular localization of DSCR4 and found DSCR4 protein localized to the nucleus of BeWo and JEG3 cells. Down-regulation of DSCR4 leaded to inhibition of migration and invasion of choriocarcinoma cells but have no effect on cell growth. The mechanism of this effect is still under investigating.Part Two Down syndrome related microRNA——study on microRNA-125b functionMicroRNAs (miRNAs) are a class of 17-to 25-nt, small, noncoding RNAs in animal. miRNA genes are transcribed by RNA polymeraseⅡto form primary miRNAs (pri-miRNAs) and then digest by ribonulease Drosha to generate precursor miRNAs (pre-miRNAs). The pre-miRNAs are then exported to the cytoplasm for processing by Dicer to generate mature miRNAs which are incorporated into a ribonulceoprotein effector complex, known as the RNA-induced silencing complex (RISC). RISC identifies target messages based on complementarity between the miRNA and the mRNA and results in either endonucleolytic cleavage of target mRNA or translational repression.It is well known that the Down syndrome increases the risks of developing leukemia. Hence we wanted to investigate the function of Hsa21-derived miRNAs in cancer. Considered the development and metastasis of breast cancer has been one of our research interests, we chose decided to study the function of Hsa21-derived miRNAs in breast cancer. By poly A-PT PCR, we characterized the expression of the Hsa21-derived miRNAs in breast cancer cells and found elevated expression of mir-125b in metastatic breast cancer cell lines. This result indicated that mir-125b may play a role in breast cancer metastasis, thus we further explored the role of mir-125b in breast cancer.Down-regulation of mir-125b leaded to inhibition of cell proliferation, migration and invasion in breast cancer cell lines. In contrast, up-regulation of mir-125b leaded to promotion of cell proliferation, migration and invasion. Inhibition of mir-125b reduces lung metastasis in nude mice. Luciferase reporter assays revealed that mir-125b binded to the the 3'-UTRs of BDP1, P53, BAP1, BAK1 and ST18 gene. mir-125b significantly repressed the transcription of BDP1 gene, slightly repressed the transcription of BAK1 gene, but showed no effect on the transcription of P53, BAP1 and ST18 gene. mir-125b also target 3'-UTR of P53, BAP1, BAK1, ST18 and MTUS1.The level of mir-125b expression was elavted in invasive breast carcinomas compared with benign ones. Taken together, these findings supported its role as an oncomir in breast cancer.