Research On Sceening For And Determining Mechanisms Of Psoriasis HLA-Cw6miRNAs

Background:Psoriasis (Psoriasis, PS) is a kind of chronic recurrent disease which is characterized by excessive skin cell proliferation and inflammation. Using genome-wide association analysis of HLA-Cw6susceptibility genes for psoriasis research shows correlation with psoriasis immune response is the strongest MHC susceptibility genes.IL12B and IL23R are two closely related with the inflammatory response of MHC susceptibility genes.LCE3A and LCE3D coding abnormal keratinization envelope protein. Our researching group found large differences in psoriasis skin and blood miRNAs expression, but the progress of the specific mechanism is unclear. Nowadays most studies are limited in the study of miRNAs and susceptibility genes. With the development of systems biology, study the relationship between specific function and the genetic susceptibility on miRNAs becomes a new trend.Purposes:This project is an extension and on the basis of clinical and experimental research of professor Zhibo Yang for many years in psoriasis on erythema scales sex skin disease. It aimed at prophase research, on the basis of using bioinformatics methods, the preliminary construction of patients with psoriasis vulgaris differentially expressed miRNAs and HLA-Cw6, IL12B, IL23R, LCE3A and LCE3D regulation network, using real-time fluorescent quantitative PCR (rt-pcr) method and in situ hybridization technology of HLA-Cw6miRNAs related validation. Finally do some reserach through experiments in vitro cell and HLA-Cw6miRNAs related downstream cytokines and cell proliferation. From the miRNA and its downstream susceptibility genes and cytokines and cell proliferation studies the relationship between the level of miRNAs in the pathogenesis of psoriasis, so as to provide the basis for the early diagnosis and prognosis of patients with psoriasis analysis, and provide exact drug targets in the treatment of level and therapeutic targets. Methods:First of all, we chose five genes which are closely associated with psoriasis susceptibility according to previous research, differentially expressed miRNAs for my previous experiments and the results obtained using bioinformatics methods, established the control network. Secondly, we built the miRNA and gene3’UTR sequences primer design, amplification by PCR amplification purpose gene sequences, a good product for agarose gel electrophoresis separation and plastic recycling fragments, with Xhol and BamH I double enzyme PCR fragment, with Xhol and BamH I double enzyme carrier plasmid pLMP purpose, connection pieces and carrier, cell culture, cell transfection, luciferase activity test. Finally, we cultivated HacaT cells will the miRNAs associated with HLA-Cw6transfection or silence, observed by the method of passing cell meter downstream TNF alpha, the expression of IL8and IL22and record HacaT proliferation.Results:Bioinformatics analysis showed that there are42miRNAs which may be involved in regulation in five susceptibility genes.11miRNAs are associated with HLA-Cw6. We drew the map of the genes cytoscape software-micrornas network diagram (micrornas-Gene-Ne). We knew that miR-222, miR-3188, miR-3162, miR-18b luciferase activity are the most significant after many experiments. Then the mutant carrier in common transfection luciferase report, confirmed that miR-149, miR-18b can directly control the HLA-CW6genes. Finally, We transfected miR-149, miR-18b in HacaT cells and found that both have inhibitory effect on the HacaT cells, including inhibition of miR-149effect is stronger. And with the augmentation of the duration, the cell inhibition rate increased. At the same time, miR-149for cytokine TNF alpha, the secretion level of IL8and IL22all have certain inhibition, whereas miR-18b inhibitions is weak. It only has obvious restraining effect for TNF alpha.Conclusions:1. Mapped the gene regulation-micrornas network diagram through bioinformatics analysis, established a preliminary forecast miRNAs which may regulate the onset and progression in patients with psoriasis byregulating susceptibility genes expression.2. Confirmed that miR-149, miR-18b luciferase activity decline, the most significanthat miRNAs can be targeted regulation of HLA-CW06through the dual luciferase detection. And speculated that miRNAs may regulate the pathogenesis and disease progression through the regulation of HLA-Cw6psoriasis.3. Found that miR-149can make human immortalized cells to produce obvious inhibition of cell vitality through HaCaT cell culture, miR-149and miR-18b transfection. At the same time, the cytokine TNF alpha, the expressions of IL8and IL22all have certain inhibition. Confirmed that the miR-149for psoriasis pathogenesis and progression of mechanism may be associated with cytokines. While miR-18b restrain cell vitality is weak, and levels of IL8and IL22significantly cut effect, its mechanism of action needs to be studied furtherly.4. The results showed that there is some network between certain miRNAs and their susceptibility genes regulatory mechanism of the sample.Thus we speculated that miRNAs-susceptibility genes and cytokines in patients with psoriasis may exist in a large control network. We regulate the network to the pathogenesis of psoriasis and controls of disease have evolved in order to provide new thought and clue for the next step for the treatment of psoriasis gene level.