Study On The Regulation And Function Of MiR-15b During Japanese Encephalitis Virus Infection

Japanese Encephalitis virus(JEV) belongs to the genus Flavivirus of the Flaviviridae. It is a serious zoonosis pathogen and causes Japanese encephalitis(JE) in human and animal. Approximately, 35,000-50,000 cases of JE are reported annually in Asia with 10,000 deaths and nearly half of the survivors suffer from permanent neuropsychiatric sequelae. JE is also a major threat to pig industry because of abortion, stillbirth and mummy fetus in pregnant sows and orchitis in boar. JEV leads to high mortality by targeting central nervous system and causing excessive neuroinflammation. However, the mechanism by which JEV induces neuroinflam-mation remains unclear.MicroRNAs(miRNAs) are non-coding, tiny(approximately 22 nucleotides) RNAs that play a crucial role in post-transcriptional gene regulation, by targeting 3? untranslated regions(3? UTR), which results in translational tuning, repression or degradation. The role of miRNAs has been widely studied in regulating a broad spectrum of cellular processes, including proliferation and differentiation, cancer and apoptosis. Recently, these small RNAs also have been found to be involved in viral infections and regulation of TLR- and RIG-I-mediated inflammation. Although the function of mi RNAs has been widely studied, it is less clear whether microRNAs participate in JEV infection. In this study, we investigated the mechanism of regulation of miR-15 b by JEV, and revealed the roles of miR-15 b played in JEV infected glial cells. The main contents are as follows:1. Upregulation of mi R-15 b upon JEV infectionMiR-15 b expression patterns upon viral infection were analyzed by using qRT-PCR in JEV-infected human astrocytes(U251 cells) and mouse microglia(BV-2 cells). The results revealed that miR-15 b levels were significantly elevated in a time- and dose-dependent manner. We also investigated the expression of mi R-15 b in JEV-infected mouse brain samples. The results showed that miR-15 b expression was upregulated. These results strongly demonstrate that miR-15 b expression is upregulated after JEV infection in vitro and in vivo.2. Transcriptional regulation of miR-15 b by c-Rel and CREBOur data demonstrated that primary precursor form of miR-15b(pri-miR-15b) showed a similar change as the mature form(miR-15b) in JEV infection, suggesting that the upregulation of mi R-15 b may occur at the transcriptional level. Bioinformatics analysis of miR-15 b promoter region, we found potential binding sites of transcription factor c-Rel and CREB in the region. To determine if the c-Rel and CREB were responsive to mi R-15 b transcription, a panel of promoter reporters with mutations in binding site was generated. The promoter activity results showed that c-Rel and CREB can bind to mi R-15 b promoter, which regulates JEV-induced miR-15 b transcription. Furthermore, chromatin immunoprecipitation(ChIP) assay suggested that c-Rel and CREB can physically binding to the promoter element of miR-15 b. To explore the signaling pathway that involved in regulation of mi R-15 b expression, cells were treated with specific inhibitors of signaling pathways and siRNA. The results indicated that ERK and NF-κB signaling pathways play important roles in the transcriptional regulation of JEV-triggered miR-15 b, and miR-15 b induction depends on RIG-I.3. mi R-15 b modulates JEV-mediated inflammation by targeting RNF125To investigate the function of miR-15 b in JEV infection, gain of function and loss of function assays were employed. The results demonstrated that miR-15 b positively regulates the expression of inflammatory cytokines and IFN-β induced by JEV. Mechanistically, ring finger protein 125(RNF125), a negative regulator of RIG-I signaling, is identified as a direct target for miR-15 b in the context of JEV infection. mi R-15 b inhibits endogenous RNF125 expression by binding to 3? UTR of RNF125 mRNA. Furthermore, inhibition of RNF125 by miR-15 b resulted in elevation of RIG-I level, which in turn leads to RIG-I activation. Overexpression of RNF125 assays suggested that virally induced miR-15 b regulates JEV-triggered cytokine production through suppression of endogenous RNF125.4. The function of miR-15 b was studied in vivoTo confirm the proinflammatory role of miR-15 b in JEV infection mouse model, a chemically modified inhibitor for miR-15b(antagomir-15b) was delivered into mice via tail vein injections and mice tolerated antagomir-15 b well, without signs of illness or discomfort. In JEV-infected mice, antagomir-15 b treatment revealed specific inhibition of miR-15 b expression, reversed changes in RNF125 expression and reduced the levels of inflammatory cytokines. Analysis of brain sections by H&E staining, Immunohistochemistry(IHC) staining, and TUNEL assays, we found that pathological alterations were alleviated, astrocytosis and microgliosis were decreased, and neuronal death were reduced by antagomir-15 b treatment. In addition, antagomir-15 b treatment improves survival in mice challenged with lethal dose of JEV, and decreases viral burden in brains.This study firstly proved that miR-15 b plays important roles in JEV-host interaction. It preliminarily explores the molecular mechanism of regulation of miR-15 b expression by JEV, and provides evidences for the association of miR-15 b with JEV-induced inflammation. Moreover, this study may also provide insight into the investigation of deep mechanism of JEV-triggered neuroinflammation and the therapeutics against encephalitis-related diseases.