Analysis On Promoter CpG Methylation And Expression Of HOXB4Gene In Various Tissues Of Mice

Hematopoietic stem cell transplantation has become an important treatment inclinical currently, which is related with cancer, the disease of hematopoieticsystem,autoimmune diseases and a part of hereditary diseases. HOX gene encodes aclass transcript factors, playing an important role in regulating the development ofhematopoietic stem cells or progenitor cells. hoxb4was the first factor proved that itplayed the role in proliferation and self-renewal of hematopoietic stem cells, withoutaffecting the differentiation of the myeloid and lymphoid lineage. hoxb4gene wasstudied widely, it is mainly about the expression of hoxb4in CD34+cells or theabnormal expression in tumor cells, but few people studied about the expression ofhoxb4in normal tissues, and the transcriptional regulation mechanism of hoxb4wasknown a little. In addition, CD34+cells was increased in bone marrow after thepatients with bronchial asthma inhaled allergen, there is a need to further studywhether related with hoxb4gene.Purpose:To explore the expression of hoxb4gene in different tissues of normal mice, theexpression of protein and its status of promoter CpG island methylation and itsrelationship with bronchial asthma disease.Methods:(1) RNA extraction was performed from different the tissues of normal mice andasthma mice, followed synthsised of cDNA, semi-quantitative reverse transcriptionpolymerase chain reaction(RT-PCR) was used to examine the expression of hoxb4gene in different tissues.(2)To detect the expression of hoxb4gene protein usingwestern blot in different tissues of normal mouse.(3) Genome DNA was extractedfrom the different tissues of normal mice and asthmatic mice, to detect themethylation of promoter CpG islands of hoxb4gene using Bisulfite Sequencing PCR(BS-PCR).Results:(1) hoxb4, a362bp gene, was amplified using RT-PCR from fetal fibroblasts,bone marrow cells, the tissues of normal mice, containing kidney, lung, liver, brain,fetuses, and the lung, blood from asthmatic mice.(2)The hoxb4protein was detected by Western Blot from various tissues of mice.(3)Demethylation of hoxb4promoter CpG island occurred in different tissues ofnormal mice, lung tissue of asthmatic mice and blood.Conclusion:(1) The expression level of mRNA and protein of hoxb4showed consistency inthe tissues of lung and blood from and asthmatic mice and different tissues of normalmice Based on the results, we can study the function of hoxb4gene further.(2)The status of demethylation of hoxb4promoter CpG island occurred indifferent tissues of normal mice, and lung tissue, blood of asthmatic mice. Thepreliminary identification of the correlation of the expression of hoxb4andmethylation of hoxb4gene, and the correlation between hoxb4gene and asthma wereperformed.