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OBJECTIVE. miR-122 is highly abundant in liver and a hepato-specific microRNA. There is evidence to show that miR-122 expression is down-regulated in human hepatocellular carcinoma (HCC). It is not known whether miR-122 affects the cellular behavior of hepatoma cells. The aim of this study was to investigate the effects of miR-122 on the viability and apoptosis of hepatoma cells. MATERIAL AND METHODS. The viability and apoptosis of Huh-7 and HepG2 cells treated with miR-122 or miR-122 antisense RNA (anti-miR-122) were analyzed by adenosine triphosphate (ATP)-based luminescent assay, annexin V-based flow cytometry, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) detection. The miR-122 coding genes in both cell lines were sequenced. RESULTS. Although two putative promoter sequences for the miR-122 gene at 18q21.31 were detected, the miR-122 coding sequence was missing in HepG2 cells, which might be the reason for the absence of miR-122 expression. There was no significant difference between the viabilities of HepG2 cells transfected with miR-122 and mock HepG2 cells (p >0.05). However, the viability of Huh-7 transfected with anti-miR-122 was significantly elevated at 24, 36, and 48 h posttransfection compared with that of mock cells (p <0.01). Both the flow cytometry and TUNEL assay showed that the apoptotic level of Huh-7 transfected with anti-miR-122 was significantly decreased at 48 h posttransfection (p <0.01). CONCLUSIONS. miR-122 down-regulated the viability but up-regulated the apoptosis of hepatoma cell Huh-7. The absence of miR-122 expression in HepG2 cells was due to the loss of the miR-122 coding sequence in chromosome 18. These results imply that aberrant expression of miR-122 may contribute to hepatocarcinogenesis.

Human enterovirus 71 (EV71) is the main causative pathogen of hand, foot, and mouth disease (HFMD) in children. The epidemic of HFMD has been a public health problem in Asia-Pacific region for decades, and no vaccine and effective antiviral medicine are available. Curcumin has been used as a traditional medicine for centuries to treat a diversity of disorders including viral infections. In this study, we demonstrated that curcumin showed potent antiviral effect again EV71. In Vero cells infected with EV71, the addition of curcumin significantly suppressed the synthesis of viral RNA, the expression of viral protein, and the overall production of viral progeny. Similar with the previous reports, curcumin reduced the production of ROS induced by viral infection. However, the antioxidant property of curcumin did not contribute to its antiviral activity, since N-acetyl-l-cysteine, the potent antioxidant failed to suppress viral replication. This study also showed that extracellular signal-regulated kinase (ERK) was activated by either viral infection or curcumin treatment, but the activated ERK did not interfere with the antiviral effect of curcumin, indicating ERK is not involved in the antiviral mechanism of curcumin. Unlike the previous reports that curcumin inhibited protein degradation through ubiquitin–proteasome system (UPS), we found that curcumin had no impact on UPS in control cells. However, curcumin did reduce the activity of proteasomes which was increased by viral infection. In addition, the accumulation of the short-lived proteins, p53 and p21, was increased by the treatment of curcumin in EV71-infected cells. We further probed the antiviral mechanism of curcumin by examining the expression of GBF1 and PI4KB, both of which are required for the formation of viral replication complex. We found that curcumin significantly reduced the level of both proteins. Moreover, the decreased expression of either GBF1 or PI4KB by the application of siRNAs was sufficient to suppress viral replication. We also demonstrated that curcumin showed anti-apoptotic activity at the early stage of viral infection. The results of this study provide solid evidence that curcumin has potent anti-EV71 activity. Whether or not the down-regulated GBF1 and PI4KB by curcumin contribute to its antiviral effect needs further studies.

MicroRNAs (miRNAs) are small non-coding RNAs that can posttranscriptionally regulate gene expression by targeting messenger RNAs. During miRNA biogenesis, the star strand (miRNA*) is generally degraded to a low level in the cells. However, certain miRNA* express abundantly and can be recruited into the silencing complex to regulate gene expression. Most miRNAs function as suppressive regulators on gene expression. Group B coxsackieviruses (CVB) are the major pathogens of human viral myocarditis and dilated cardiomyopathy. CVB genome is a positive-sense, single-stranded RNA. Our previous study shows that miR-342-5p can suppress CVB biogenesis by targeting its 2C-coding sequence. In this study, we found that the miR-10a duplex could significantly up-regulate the biosynthesis of CVB type 3 (CVB3). Further study showed that it was the miR-10a star strand (miR-10a*) that augmented CVB3 biosynthesis. Site-directed mutagenesis showed that the miR-10a* target was located in the nt6818–nt6941 sequence of the viral 3D-coding region. MiR-10a* was detectable in the cardiac tissues of suckling Balb/c mice, suggesting that miR-10a* may impact CVB3 replication during its cardiac infection. Taken together, these data for the first time show that miRNA* can positively modulate gene expression. MiR-10a* might be involved in the CVB3 cardiac pathogenesis.

BackgroundStress granules (SGs) are granular aggregates in the cytoplasm that are formed under a variety of stress situations including viral infection. Previous studies indicate that poliovirus, a member of Picornaviridae, can induce SG formation. However, the exact mechanism by which the picornaviruses induce SG formation is unknown.MethodThe localization of SG markers in cells infected with coxsackievirus B3 (CVB3) or enterovirus 71 (EV71) and in cells expressing each viral protein was determined via immunofluorescence assays or plasmid transfection. Eight plasmids expressing mutants of the 2A protease (2Apro) of CVB3 were generated using a site-directed mutagenesis strategy. The cleavage efficiencies of eIF4G by CVB3 2Apro and its mutants were determined via western blotting assays.ResultsIn this study, we found that CVB3 infection induced SG formation, as evidenced by the co-localization of some accepted SG markers in viral infection-induced granules. Furthermore, we identified that 2Apro of CVB3 was the key viral component that triggered SG formation. A 2Apro mutant with the G122E mutation, which exhibited very low cleavage efficiency toward eIF4G, significantly attenuated its capacity for SG induction, indicating that the protease activity was required for 2Apro to initiate SG formation. Finally, we observed that SGs also formed in EV71-infected cells. Expression of EV71 2Apro alone was also sufficient to cause SG formation.ConclusionBoth CVB3 and EV71 infections can induce SG formation, and 2Apro plays a crucial role in the induction of SG formation during these infections. This finding may help us to better understand how picornaviruses initiate the SG response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-014-0192-1) contains supplementary material, which is available to authorized users.