Major Changes Of MicroRNA Profile In HSK Cornea And The Regulated Effect Of MiRNA-155 In Inflammation

MicroRNAs(miRNAs) are a set of non-coding RNA molecules with the length of 18-23nt. its discovery is known as the most meaningful one of world’s top ten scientific and technological breakthroughs in 2002. miRNAs were processed from short stem-loop precursors that call be encoded in genomes of plants, animals and viruses. According to the current understanding, miRNA is firstly transcribed as long primary micro RNA, which processed into 60-70 nt miRNA precursor by nuclear RNaseⅢ Drosha. Thus far over 3,500 microRNAs have been identified.miRNAs exist in the genome take the form of single copy, multiple copies and gene clusters and most of them located in the intergenic region. miRNAs transcripts independently without protein translation. MiRNA can produce its effect by gene silencing, which occurs either via mRNA degradation or preventing mRNA from being translated. Some miRNAs can regulate gene expression by changing the half-life period of mRNAs. A computational analysis indicated that more than one-third of protein-encoding genes are regulated by miRNAs. They are involved in the regulation of variety of biological processes including developmental timing, signal transduction, apoptosis, cell proliferation and tumorigenesis. In one word, miRNAs play an impotent role in physiological and pathological process.Herpetic simplex keratitis (HSK) is a potentially blinding corneal inflammation that accompanies herpes simplex virus (HSV) infection of the eye with high rate of recurrent rate. The inflammation in the eye could result in serious keratopathy such as corneal dissolution, corneal revascularization, corneal ulcer and panophthalmitis. Although, some developments have been made on the pathogenesis of HSK till now, there are still many blanks should be filled in these areas. More and more evidences indicated that:miRNA involved in the innate immune response and the adaptive immune response as a key mediator to various physiological and pathological processes of the body. In terms of the host and viruses, miRNAs regulates the protection mechanism and virus-inhibition of the host as well as the confrontation to host immune process of pathogens. Whether HSV-1 infection regulate the expression of miRNAs of corneal tissue in the early stage of inflammation? Whether miRNAs involved in cornea inflammation? What is the mechanism? These are the aims of the present research. Currently, very few study focused on these problems abroad and there is no systematic research in china.Therefore, we built up mice HSK inflammation model by HSV-1 cornea infection. Then, take the HSK cornea tissue as research object, First of all miRNAs expression profiles of HSK cornea and normal cornea were analyzed using microRNA microarray. Moreover, results of miRNA microarray were verified by qRT-PCR in the following research. The targets genes of miRNAs were forecast by bioinformatics and the functions of them were analyzed. According to the results of QRT-PCR and microRNA microarray, miRNA-155 is the most significantly changed one in HSK cornea tissue. The proinflammatory role and molecular mechanism of miRNA-155 to HSK inflammatory response were also analyzed in the present research. Based on the results of the problems above, we hope to clarify noncoding RNA mechanism of inflammation during HSK to some level in order to provide theoretical and experimental basis for treatment and drug development to the disease.Part 1 Analysis of expression changes of microRNA in HSK cornea after HSV-1 infectionObjective:(1) Preliminary screen the differences miRNAs in HSK cornea after HSV-1 infection by miRNA microarray.(2) Determine the miRNAs differently expressed in HSK cornea.(3) Predict target genes and the main biological functions and its role in the HSK by bioinformatics technology.Method:(1) Built up HSK mouse model by scratching corneal epithelium(2) Analyze difference of miRNA expression between HSK and normal cornea by Affymetrix miRNA3.0.(3) Verify the results of microarray by quantitative real-time PCR to determine the miRNAs differently expressed in HSK cornea.(4) Predict target genes of differentially expressed miRNAs and analyze the function of them. Determine the main biological functions of the target genes and its role in the HSK onset process by bioinformatics technology.Result(1) At 7 days post infection, HSK were built up with corneal epithelial damage, corneal opacity, corneal revascularization. The method of cornea scratch to establish the model of the HSK has good maneuverability and repeatability with the success rate of 90%. The mice model can be used in researches on the change of the miRNA expression profile.(2) miRNA microarray showed that, there are 34 miRNAs with differently expression: mmu-miRs-5100,-21-3p,-133a-3p,-18a-5p,-150-5p,-155-5p,-133b-3p,-139-5p,-146a-5p,-206-3p,-124-3p,-300-3p,-5115,-5097,-1224-5p,-720,-712-5p,-342-3p,-223-3p,-714,-17-3p,-1934-3p,-204-5p,-3968,-21-5p,-3096b-5p,-200a-3p,-204-3p,-148a-3p,-181c-5p,-346-3p,-434-3p,-711,-762. Fifteen miRNAs’expression change more than 2 folds:mmu-miRs-223-3p,-155-5p,-21-3p,-150-5p,-18a-5p,-3096b-5p,-146a-5p, and -342-3p, mmu-miRs-434-3p,-124-3p,-204-5p,-133b-3p,-206-3p, and -133a-3p.(3) The differently of expression of miRs-204-5p,-206-3p,-155-5p,-133a-3p, and-150-5p in HSK cornea were verified by qRT-PCR.(4) There are 125 potential target genes of the 5 miRNAs. Nanty-two were up-regulated and 33 were down-regulated.(5) The main function shared with the targets genes of the were miRNAs are:negative regulation of cell proliferation and cell adhesion.Conclusion:(1) The method of cornea scratch to establish the model of the HSK has good maneuverability and repeatability. The model can be used in researches on the change of the miRNA expression profile.(2) There is significant changes in the miRNAs profile in HSK cornea compared with normal ones. The expression of 34 miRNAs changes detected by microRNA microarray.(3) The differently expression of 5 miRNAs in HSK cornea were verified by qRT-PCR with the same trend as microarray.(4) The 5 miRNAs has 125 potential target genes, which form a complex network. The main function shared with the targets genes of the were miRNAs are:negative regulation of cell proliferation and cell adhesion, which closely correlation to inflammation.Part 2 Preliminarily explore to the function of miRNA-155 in regulating the inflammation during HSKObjective:(1) Built up cornea miRNA-155 down-expression mice model in vivo.(2) Research the molecular mechanism of miRNA-155 in the regulation of inflammation during early stage of HSK.(3) To explore the function of miRNA-155 down regulation to the HSK inflammationary performance in mice.Method:(1) Built up HSK mice model as the method mentioned above. Transfect lentivirus with miRNA-155 down-regulation plasmid and negative control plasmid by scratch corneal epithelium of C57BL/6 mice and infiltrate with virus. Observe the fluorescence of GFP in corneal at day3, day5, and day7 by slit lamp. Observe fluorescence of GFP by fluorescence microscope at day 7.(2) Built up HSK model, HSK model with miRNA-155 down regulation as well as HSK model transfect with negative control plasmid by the methods mentioned above. Choose HSK cornea with the fluorescence expression of GFP take more than 20% area of the cornea at day 7 post infection. Detect the expression of IL-1β, IL-6, IL-8, IL-10, IL-17, IFN-γ, TNF-α, CXCL1 by qRT-PCR.(3) Built up HSK model, HSK model with miRNA-155 down regulation as well as HSK model transfect with negative control plasmid by the methods mentioned above. Choose HSK cornea with the fluorescence expression of GFP take more than 20% area of the cornea at day 7 post infection. Double-blinded scored the manifestation by the standard introduced by Heiligenhaus [1] and FosterResult(1) Fluorescence expression of GFP can observed by Slit lamp at day 3 post infection. The area of fluorescence become bigger at day 5 post infection and the area and fluorescence intensity and area peak at day 7 post infection. The main position of lentivirus infection is epithelium and shallow matrix of cornea.(2) There is significant differences between HSK, miRNA-155 down HSK and negative control plasmid HSK groups in the expression of IL-17 and CXCL1 detected by qRT-PCR.(3) There is no significant differences between HSK, miRNA-155 down HSK and negative control plasmid HSK groups in the score of corneal epithelium lesion, corneal opacity and corneal nevascularization at day 3, day 5, and day 7 post infection.Conclusion:(1) Fluorescence expression of GFP can be detected in epithelium and shallow matrix of cornea by transfect lentivirus with miRNA-155 down-regulation plasmid after scratch corneal epithelium of C57BL/6 mice and infiltrate with virus. The model can be used for the miRNA function research(2) miRNA-155 down-regulation can decrease the expression of IL-17 and CXCL1 in HSK cornea. MiRNA-155 may influence inflammatory response by regulate the expression of inflammatory factors.(3) Based on the sample size in the present research, miRNA-155 down regulation had no obviously effect on the clinical manifestation of HSK.