Preliminary Study On MRNA And MiRNA Profiles Of Exosomes Secreted From IFN-α Induced Liver Non-parenchymal Cells

Hepatitis B virus (HBV) is a critical pathogen with world-wide threat to human health. Interferon α (IFN-α) is an important cytokine performing as the host natural immune defense against pathogen invasion, and also as the current first-line drugs for clinical treatment of chronic hepatitis B (CHB) patients with response rates of30-40%. However, the specific mechanism for IFN-α against HBV is still not completely known, and HBV can utilize a variety of mechanisms to inhibit the production of interferon, and impair the interferon effect pathway. Both in vitro and in HBV chimeric mice experiments confirmed that IFN-α’s capacity to directly inhibit HBV replication in liver parenchymal cells is weak, suggesting that in vivo IFN-a may control virus through some other ways.Exosome is a kind of microvessicle secreted by cells, sized from30-100nm, containing proteins, RNAs and lipids. It is widely found in various body fluids. Exosome has been reported to mediate communication between the cells by horizontally transfer functional miRNAs and mRNAs cargo to nearby or faraway acceptance cells, and regulate a variety of important cellular activity. Dr. Jianhua Li in our lab found that IFN-α can act on the liver non-parenchymal cells and transfer antiviral effects through their secreted exosomes to help HBV infected hepatocytes remodel antiviral state, suggesting a noval mechanism of IFN-α’s antiviral effects. So many questions, such as what the specific profile difference of mRNAs and miRNAs in exosomes secreted by non-parenchymal cells before and after IFN-α treatment is, and what thebiological functions of these differentially expressed molecules are, what the correlation of these molecules with clinical therapeutic efficacy of IFN-αis remain to be further studied.Microarray technology was used to detect the genome-wide miRNA and mRNA expression profile of exosomes secreted by liver sinusoidal endothelial cells (LSEC) treated with and without IFN-α.1548kinds of mRNAs and22miRNAs were found differentially expressed with over1.5fold change (FC≥1.5,3independent repeats, t-test, p<0.05), including618kinds of mRNAs and3miRNAs upregulated over1.5fold.11kinds of up-regulated mRNAs and3miRNAs were randomly selected to verify the reliability of the microarray results using qRT-PCR, and these14molecules were representatives for further investigation. Besides, we measured expression levels of these14molecules in LSEC donor cells before and after IFN-a treatment, and found that the change in the donor cells was not consistent with the up-regulated level within the exosomes, suggesting that there are specific selection mechanisms for molecules sorting into exosomes. Furthermore, we tested monocyte-macrophage derived THP-1cell line, another important liver non-parenchymal cell for the expression of these14molecules both in donor cells and their secreted exosomes, with and without IFN-a treatment. The results showed the same characterstic with LSEC, and further indicated that it may be a general phenomenon that specific RNA molecules exist in exosomes derived from IFN-a treatment non-parenchymal cells.With bioinformatics tools, we analyzed the function of up-regulated618mRNAs and3miRNAs in exosomes after IFN-a treatment. And49kinds of interferon regulatory related genes (IRGs) was enriched, as well as many other molecule were found be likely to participate in a variety of cellular function in the micro-enrivonment of the liver.. We selected14molecules to validate their anti-HBV activity. CALHM1, FABP5, DDIT3, miR-638, miR4284could significantly decrease HBsAg expression (p<0.01), and CALHM1, miR-4284on HBeAg expression decline (p<0.01) after cotransfected with HBV replicating dimer plasmids.In clinical, we examined74CHB patients, including34patients with early rapid virological response (EVR) and40patients with on response after pegylated interferon-a (PEG-IFN-a) therapy. Exosomes were purified from serum samples and14differential expressed mRNAs and miRNAs were examined by qRT-PCR. In CHB patients with EVR response, IFITM mRNA, miR-638, miR-4284, and miRNA-1260were found significantly increased (p<0.01) after3months of treatment compared with non-EVR CHB patients. Meanwhile, the raised level of IFITM have correlation with the decline degree of HBV DNA copies in EVR patient (correlation coefficient of0.4045, p<0.001). On the one hand, the result of clinical patients confirms the clinical correlation with the cell experiment results. On the other hand, it also explores the possibility of applying these mRNAs and miRNAs molecules in exosomes as biomarkers for the indication of IFN therapy response.In summary, we analyzed the differential expression profiles and functions of mRNAs and miRNAs containing in exosomes secreted from liver non-parenchymal cells (LSEC and macrophages) before and after IFN-a treatment. The results contribute to the understanding of specific molecular mechanism of IFN-a utilizing exosomes to influence in the micro-environment. Furthermore, the correlation between clinical CHB patients in PEG-IFN-a treatment with and without EVR response and the differential expression of partial molecules, provides possibility for further development of clinical IFN-a efficacy predictive biomarkers based on plasma exosomes containing mRNAs and miRNAs.