The Role Of MiRNA In Innate Immunity In Human Hepatoma Cells

ObjectiveMicroRNAs (miRNA) are a class of highly conserved short noncoding RNAs origined from genome, have emerged as a major class of post-transcriptional gene-expression regulators and are involved in a wide variety of biological processes. They regulate target gene expression mainly through imperfectly base pairing with the 3'-untranslated regions (3'-UTRs) of target mRNAs in animal, preventing protein accumulation by inducing mRNA degradation or suppressing translation.To date, increasing evidence reveals that miR-155 is involved in numerous physiological and pathological processes including innate and adaptive immunity, inflammation and tumorigenesis. Moreover, miR-155 was found to be induced by poly (I:C) and other TLR ligands, and by IFN-βand several cytokines such as IL-1β, implicating that miR-155 is broadly involved in the regulation of both bacterial and viral innate immune responses. Besides, One research pointed out that the EBV-encoded latent membrane protein-1 (LMP1) substantially up-regulated miR-155, which led to a reduction of PU.1 and IKKε, suggesting that miR-155 contributed to viral-mediated infections through the regulation of different transcription factors and the NF-κB signaling. While another recent research indicated that miR-155 expression was up-regulated by VSV infection in murine macrophages, then feedback attenuated the viral replication through enhancing typeⅠIFN signaling against viral infection by targeting SOCS1.HBV is a hepatotropic DNA virus, which causes acute and always chronic HBV infection and leads to hepatoma fibrosis, cirrhosis and eventually hepatocellular carcinoma. Compared with HCV, the role of miRNAs in HBV infection has not been investigated until recently. A few studies reveal that miRNA is involved in the complicated interaction between HBV and hepatic cells. Based on these results, in the present study, we over-expressed miR-155 in human hepatoma cells (HepG2), examined the changes in innate immunity and further revealed the possible mechanism. Additionally, we found that miR-155 has mild anti-HBV effect in human hepatoma cells.Methods1. Construction of miR-155 overexpression vector:The miRNA expression cassette containing the human miRNA hairpin sequence and flanking regions was amplified from the genomic DNA using the pri-miRNA primers (Table 1). The cassette was then inserted into an expression vector (pcDNA3). The resulting construct was termed pcDNA3-hsa-miRNA. The construct was confirmed by DNA sequencing.2. Transfection:pcDNA3-hsa-miR155 plasmid or miR-155 mimics and control RNA or PAAV/HBV1.2 plasmid were transfected into HepG2 cells using Lipofectamine 2000.3. MTT asssy:MTT assay was used to determined cell proliferation changes after miR-155 over-expression.4. RT-PCR assay:RT-PCR was used to analysis the expression of the IFN inducible antiviral genes, SOCS1 and HBx.5. Western Blot assay:Western Blot was used to analysis the expression of SOCS1, 1SG15, p-STAT1 and p-STAT3.Results1. pcDNA3-hsa-miR-155,pcDNA3-mmu-miR-155,pcDNA3-hsa-miR-122,pcDNA3-mmu-miR-122,pcDNA3-hsa-miR-223,pcDNA3-mmu-miR-223 plasmid construction. All the constructs were confirmed by DNA sequencing.2. miR-155 over-expressed cell line was successfully established.3. miR-155 don't show obvious impact on the growth of human hepatoma cells.4. Over-expression of miR-155 upregulates IFN-inducible antiviral genes in human hepatoma cells.5. miR-155 post-transcriptionally regulates SOCS1.6. miR-155 promotes JAK/STAT signaling pathway in human hepatoma cells.7. Over-expression of miR-155 inhibits HBV in human hepatoma cells. ConclusionmiR-155, as a positive regulator of JAK/STAT signaling by targeting SOCS1, subsequently enhances the phosphorylation of Signal Transducers and Activators of Transcription 1 (STAT1) and Signal Transducers and Activators of Transcription 3 (STAT3) and the expression of IFN-inducible antiviral genes in human hepatoma cells but don't impact hepatoma cell proliferation. Further more over-expression of miR-155 exhibited mild anti-HBV effect in human hepatoma cells, which reveals a new miRNA and HBV interaction, and might benefit to anti-HBV when combine with other anti-virus therapeutics. The detail mechanisms will be further investigated in the future.