Entification And Functional Study Of MicroRNA In Innate Immune Response

MicroRNAs (miRNA), a class of single-stranded non-coding RNA by the length of18-26nucleotide encoded by genome, are widely present in eukaryotic cells, which are highly conserved in evolution. MicroRNA are generated from genome in the form of primary transcripts (pri-miRNAs) produced by RNA polymerase Ⅱ or Ⅲ. The process of its maturation involves a series of proteins editing and transporting on the primary transcripts, and finally assembly into RNA-induced silencing complex (RISC) in the cytoplasm together with other associated proteins, suppressing gene expression by binding to the3-untranslational region (UTR) of target mRNAs in the manners of decreasing target mRNA levels or directly inhibiting translation. Thousands of microRNAs are found in vivo so far, and they form a complex network regulating differentiation, development, immune response, tumorigenesis and other aspects.In response to the infection, the body’s innate immune response will be activated immediately and remove the microbial pathogens through the inflammation and antiviral response it mediate. NF-κB signaling plays a key role in innate immune response, a series of downstream cytokines can be transcript when it activated, which promote the inflammation. And the type I interferon induced by innate immune response at the same time has equal importance in the antivirus response. The innate immune response to infection must be robust enough to eradicate microbial but resolved in a timely manner to avoid excessive damage to the host. There is a lot of positive and negative factor in this signaling to ensure moderate activation of the response. However, more and more microRNAs have been shown to be involved in the regulation of this process. So, identification of the microRNAs with its molecular mechanism which can regulate innate immune response would deepen the understanding of the regulation in innate immune response, and further providing new targets for anti-infection drugs.In this subject, we have been preliminarily identified38microRNAs that maybe involved in innate immune response signaling regulation, and then we further functional studied miR-526a, miR-617and miR-623of them.1. High-throughput screening of microRNAs involved in innate immune response: Using IFNβ-Luc reporter gene and IL-1β (regulated by NF-κB) ELISA as high-throughput screening model,38microRNAs (from140) have been identified with involvement in innate immune response. Of these functional microRNAs,21ones up-regulate IFNp expression level, and22ones up-regulate IL-1β expression level.2. Functional study of miR-526a involved in innate immune response: The expression of IFNp and IL-1β has been shown to be significantly up-regulated by mir-526a through IFNp-Luc reporter gene and IL-1β ELISA. Thus, we further investigate its function in innate immune response. Using quantitative RT-PCR method, we found that miR-526a expression level gradually increased as time went on in response to VSV and LPS, which indicated that miR-526a may participate in the host immune response to pathogen infection. Next, predicted by bioinformatics methods, we identified CYLD may be the target gene of miR-526a. Overexpression of miR-526a in vivo led to a sharp reduction of CYLD in mRNA and protein levels detected by RT-PCR. CYLD is a deubiquitinating enzyme, which negative regulates RIG-I signaling by deubiquitination of RIG-I, and suppresses TL4signaling by interacting with MYD88inhibiting the phosphorylation of downstream proteins as well. Taken together, miR-526a may exert its innate immunity function via suppression of CYLD expression to promote the generation of IFNp and IL-1β.3. Functional study of miR-617and miR-623involved in tumorigenesis mediated by inflammatory response: The expression of IL-ip which is one of the most important cytokines regulated by NF-κB has been shown to be significantly up-regulated by mir-617and miR-623through IL-1β ELISA. Recent studies have revealed an’NF-κB signature’ in the process of tumorigenesis in which the inflammation associated molecules participate and play a decisive role in different stages. IL-1β, one of the proinflammatory cytokines, the inflammatory microenvironment which mediated could induce more genetic mutations and promote the cell proliferation. Other related subject has been shown the overexpression level of miR-617and miR-623in the breast cancer cell. Thus, we explored their roles in breast cancer. Using cell count and wound healing assay, we found overexpression of miR-617and miR-623led to a significant increase in proliferation and migration of breast cancer cell ZR-75-1. These results suggest that miR-617and miR-623may involved in tumorigenesis mediated by inflammatory response, which laid the foundation for us to further study the function of miR-617and miR-623.In summary, we preliminarily identified38microRNAs that may regulate innate immune response. Further studies suggest that miR-526a up-regulate the expression level of IFNβ and IL-1β through suppressing the negative regulator CYLD, in addition, miR-617and miR-623acting as activators in inflammation could promote breast cancer growth and metastasis, both of which provide important clues on our further research on their underlying molecular mechanism in innate immune response and tumorigenesis mediated by inflammation.