Study On The New Techniques For The Detection Of Methylation And Gene Expression That Are Based On Near Infrared Fluorescence

Epigenetics involved in the altered heritable changes of the gene expression and regulation which is not concerned with the changes of DNA sequence.Among them, methylation is the most common DNA modification. DNA methylation can control gene expression by changing chromatin structure, DNA conformation, DNA stability and protein-DNA interactions. Therefore, methylation is closely related to many diseases such as cancer, aging and senile dementia. To study the role of DNA methylation in regulation of gene transcription, methylation of genome DNA should be accurately detected.Currently, there are many methods for detection of DNA methylation, but most of them have cumbersome procedures; in addition, the link between methylation detection results and gene expression regulation effect can not be build, which is difficult to be applied in the study of mechanism of epigenetic regulation of gene expression. Therefore, establishing a simple and efficient method for the synchronous detection of DNA methylation and its effect on the regulation of gene expression is necessary. Studies have shown that a variety of gene promoter-related epigenetic hypermethylations of CpG islands resulting in silence, were usually associated with the disease. For this purpose, combined with the studies of double-stranded DNA microarray (dsDNA microarray) technology, we are aiming to establish a synchronous detection method of gene methylation, in particular detecting methylation on transcription factor binding sites (TFBS) and regulatory effects of gene expression. This method can be extended by selecting different type of solid support materials for the innovation of this new technology in methylation and gene expression detection.The main contents and achievements of this study are summarized as follows:1. Nylon membrane is a long chain synthetic polyamide film, which has a strong binding ability to nucleic acid and protein. The near-infrared fluorescence (NIRF) has the characteristic of high sensitivity and high signal-noise (S/N) ratio. Based on these advantages, we established a membrane-based near-infrared fluorescence assay (M-NIFA) for detecting DNA methylation and its effect on gene transcription. The method based on double-stranded DNA (dsDNA) microarray technology including the methylation and transcript detection system. The former system can detect DNA methylation by using anti-5-methylcytidine antibody (5-MC antibodies) and the latter can detect the gene transcript with biotinylated cDNA. The signals of 5-MC antibodies and biotinylated cDNAs can be reported by near-infrared fluorescent-labeled secondary antibody and streptavidin. The feasibility of the two systems was fully verified with the synthesized methylated and biotinylated oligonucleotides. Finally, the reliability of the two systems was demonstrated by successfully detecting the methylation and transcription of a reported hypermethylated gene, pI4ARF, in colon cancer cells (LOVO cells). The study provides a new method for the simultaneous detection of DNA methylation and gene expression regulation, avoiding of bisulfite treatment and PCR amplification and has a wide dynamic range and high sensitivity.2. In the aspect of DNA methylation detection, nylon membrane-related method can detect more genes in one sample, but the microwell-based method (modified with specific groups, e.g., active groups NOS) can detect more genes in multi-sample. Therefore, we have established a new methylation detection technology, a microwell-based near-infrared fluorescence assay (W-NIFA) for the detection of DNA methylation. The W-NIFA procedure mainly had two steps:capturing the ultrasonic interrupted genomic DNA fragments with the coupled capture probes and the simple immunological process in conjunction with the near-infrared fluorescence imaging technique. In this study, feasibility of this new method was explored by detection of synthetic oligonucleotides and the reliability of the method was successfully verified by detection of nine loci of methylation in the promoter region of KIR3DL1 gene in cancer cell line K562 (chronic myelogenous leukemia cells), p14ARF gene in cancer cell line LOVO (colon cancer cells) and TP53BP2 gene in cancer cell line HepG2 (hepatoma cells).3. Microplate combined with near-infrared fluorescent dsDNA microarray technology not only can detect methylation of promoter region, but also can explore the effect of gene expression regulation, that is the detection of mRNA molecule abundance. High sensitivity of near-infrared fluorescence, flexibility of microplate and precision of the dsDNA microarray technology make the establishment of this method possible. In this study we created a near-infrared fluorescence-based gene expression detection technology named NIRF-GED. The NIRF-GED procedure mainly has three steps:(1) isolating total RNA from the detected samples and then reverse transcription of RNA into cDNA with a biotin-labeled oligo dT; (2) hybridizing the cDNA to oligonucleotide probes coupled to a 96-well microplate; and (3) detecting biotin with NIRF-labeled streptavidin. The transcription factor NF-κB target genes Ccl20, Cxcl2 and housekeeping gene GAPDH were studied,standard curve of NIRF-GED detection technology was established by using the oligonucleotide probes. By hybridizing the total cDNAs of HeLa cells in the concentration gradient to capture probes fixed on the 96-well microplate, number of cDNA molecules be obtained according to the standard curve, and NIRF-GED results were validated by real-time quantitative PCR. The results showed that NIRF-GED can accurately detect the gene transcription of the detected samples and can be used to achieve absolute quantification of gene expression analysis. Therefore, this study provides a new method for the detection of gene expression.4. Slides modified with special aldehyde groups are the important type of microarrays. The DNA microarray can achieve the higher throughput detection by fixing a plurality of capture probes arranged on the slide. The near infrared-aldehyde slides assay (S-NIFA) was created by using the MeDIP and ChIP techniques to enrich the methylated DNA fragments as well as the TFBS fragments specific to the E2F proteins;the enriched DNA fragments can be detected by S-NIFA technology. At last, we investigated the impact of DNA methylation, gene expression and the interactions of transcription factor with its TFBS by the quantitative PCR analysis. There are two advantages using aldehyde slides as follows:first, consumption of the experimental material, including amino-modified oligonucleotides, second antibodies and fluorescent-labeled antibodies will be greatly reduced, which make this method more cost-effective. Second, uniformity and compatibility between each site are improved by detecting a plurality of target sites in a very small area. The method can be improved and expected to play an important role in the areas such as high sensitive detection of nucleic acid molecules, for example the detection of pathogenic microorganisms.5. Embryonic development is a process in which selective gene expressing takes place in a certain temporal order. In embryonic development, epigenetic modifications play an irreplaceable role in the maintenance of normal life and activities of mammalian. To further investigate the methylation and its effect on regulation of gene expression, the research of methylation on promoter-related CpG islands of TP53BP2 and Apaf-1 genes in human embryonic lung cells (HFL-I cells) was investigated, the analysis of methylation-prone region and its relationship with transcription factor binding sites was done, the methylation regulation pattern with transcription factor binding was predicted. On the basis of bioinformatic prediction, the bisulfite sequencing was conducted aiming to the target areas. The methylation in promoter area and its impact on transcription factor binding as well as gene expression regulation effect were investigated by methylation inhibitor treatment and real-time PCR detection.The results showed that there existed a certain degree of methylation in promoter regions of both genes. The CpG methylation predicted by bioinformatic prediction were in part agree with the bisulphite sequencing results, some CpG methylation were also appeared in transcription factor binding sites.The results suggest that expression of two genes may be influenced by methylated CpG, particular methylated CpG in the transcription factor binding sites. The results of RT-PCR with 5-aza-deoxycytidine treatment of HFL-I cell also proved this conclusion. These results will help to further understand the epigenetic regulation and its impact on the embryonic development.Compared with the conventional detection methods of methylation, methods established in our study have the merits of avoiding tedious process such as bisulfite treatment, immunoprecipitation and PCR amplification. In addition, these methods can simultaneous detection of sense and antisense strands of DNA and achieve high-throughput detection of more genes and loci. In gene expression detection, our methods have the merits of high sensitivity and free of PCR amplification.These new methods have enriched the detection methods of DNA methylation and gene expression, adding new tools to the mechanism exploration of epigenetic regulation of gene expression. Besides the near-infrared fluorescence has the high sensitivity and signal-noise ratio compared with the traditional fluorescent signal,which become to an advantageous labeled molecule for the fluorescence imaging.The subject has made a good attempt on the detection of biological molecules with near-infrared fluorescence,which may promote the applications of these biological molecules.