ABSTRACT
【Objective】 To investigate the role of non-coding microRNA miR-16-5p in ZIKV replication and the underlying mechanism. 【Methods】 1×105/mL HeLa cells were seeded in 24-well plate and infected with ZIKV(MOI=5). RNAs were harvested, and miR-16-5p expression levels were measured by qRT-PCR at 24, 48 and 72 hour post infection, respectively. HeLa cells were transfected with 20nM miR-16-5p mimic and infected with ZIKV(MOI=5) at 24h post transfection. RNAs were extracted and ZIKV RNA and several inflammation factors expression were tested using qRT-PCR at 48h post infection. HeLa cells were co-transfected with 1μg NFκB-luc and 10ng pRL-TK with 20nM miR-16-5p mimic, and then infected with ZIKV(MOI=5) for 24h before the luciferase expression was tested at 48h post infection. HeLa cells were transfected with 20nM miR-16-5p mimic and infected with ZIKV(MOI=5) at 24h post transfection, and cell apoptosis was assayed through flow cytometry. 【Results】 Compared with uninfected control, miR-16-5p expression was significantly decreased at 24h, 48h and 72h following ZIKV infection. MiR-16-5p over-expression inhibited ZIKV replication, while upregulated NFκB activity and inflammation factors expression compared with the negative mimic-transfected cells. MiR-16-5p overexpression also promoted HeLa cell apoptosis. 【Conclusion】 ZIKV infection downregulated intracellular miR-16-5p expression. Overexpression of miR-16-5p suppressed ZIKV infection. MiR-16-5p inhibited ZIKV replication and promoted cell apoptosis probably by activating NFκB pathway and stimulating inflammation factors expression.
ABSTRACT
【Objective】 To investigate the effect of transfusion-transmitted Zika virus (ZIKV) on the expression of non-coding circular RNA (hsa_circ_0001613) and the role of hsa_circ_0001613 in Zika virus replication. 【Methods】 Human adenocarcinomic alveolar basal epithelial cells (A549) were seeded on a 12-well plate at 1.8×105/ well and infected with ZIKV at 0.05 MOI. The Total RNAs were isolated every day for 5 days after infection, and the relative expression level of hsa_circ_0001613 was detected by qRT-PCR. In addition, 10nM siRNA-hsa_circ_0001613 was transfected into 2×105/ well A549 cells to specifically knock down the expression level of hsa_circ_0001613. 24h later, the cells were infected with ZIKV (MOI=0.05). Total RNAs were isolated at day 1-5 post-infection, proteins were extracted 96h post-infection. ZIKV replication, relative host antiviral gene expression, and interferon stimulated response element (ISRE) activity were tested using qRT-PCR, western blot and dual luciferase assay, respectively. 【Results】 The relative expression of hsa_circ_0001613 decreased significantly after 1-5 days of ZIKV infection. Knockdown of hsa_circ_0001613 inhibited ZIKV replication. Meanwhile, hsa_circ_0001613 knockdown significantly upregulated IFN-α/β and its downstream interferon-stimulated genes (ISGs) expression, also increased ISRE activity. 【Conclusion】 ZIKV infection significantly suppressed hsa_circ_0001613 expression in A4549 cells. Preliminary study indicated that hsa_circ_0001613 knockdown inhibited ZIKV replication possibly through activating type-Ⅰ IFN signaling pathway as showed by increased ISGs expression and ISRE activity.