miR-21 promotes dengue virus serotype 2 replication in HepG2 cells Sitthichai Kanokudom a , Tirayut Vilaivan b , Nitwara Wikan c , Chutima Thepparit c , Duncan R. Smith c , Wanchai Assavalapsakul a, * a Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand b Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330 Thailand c Institute of Molecular Biosciences, Mahidol University, Nakornpathom, 73170, Thailand article info Article history: Received 7 September 2016 Received in revised form 9 February 2017 Accepted 27 March 2017 Available online 30 March 2017 Keywords: Dengue virus serotype 2 (DENV 2) HepG2 MicroRNA (miRNA) miRNA-21 (miR-21) Anti-miRNA-21 oligonucleotide (AMO-21) Peptide nucleic acid-21 (PNA-21) abstract Infection with the mosquito transmitted dengue virus (DENV) remains a significant worldwide public health problem. While the majority of infections are asymptomatic, infection can result in a range of symptoms. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through repression or degradation of mRNAs. To understand the contribution of miRNAs to DENV 2 replication, we screened a number of candidate miRNAs for variations in expression levels during DENV 2 infection of HepG2 (liver) cells. Seven miRNAs were identified as differentially expressed, and one, miR-21, was differentially expressed at all time points examined. Interestingly, miR-21 was also differentially regu- lated in DENV 2 infection under conditions of antibody dependent enhancement of infection, and in direct Zika virus infection, but not in DENV 4 infection. The role of miR-21 during DENV infection was further examined by treating HepG2 cells with an anti-miR-21 (AMO-21) before DENV infection. The results showed a significant reduction in DENV 2 production, clearly suggesting that miR-21 plays a key role in DENV 2 replication. To further confirm the role of miR-21 in DENV infection, a peptide nucleic acid-21 (PNA-21) construct with a nucleotide sequence complementary to AMO-21, was co-administered with AMO-21 as an AMO-21/PNA-21 complex followed by DENV 2 infection. The results showed that AMO-21 significantly reduced DENV 2 titer, PNA-21 significantly increased DENV 2 titer and the com- bined AMO-21/PNA-21 showed no difference from non-treated infection controls. Taken together, the results show that miR-21 promotes DENV 2 replication, and this mechanism could serve as a possible therapeutic intervention point. © 2017 Elsevier B.V. All rights reserved. 1. Introduction Despite the recent introduction of a vaccine to protect against dengue virus (DENV) infection in selected countries (WHO, 2016), there remains no specific treatment for DENV infection. While the majority of DENV infections are asymptomatic (Runge-Ranzinger et al., 2014), the infection can lead to a range of symptoms from a mild fever to the more severe forms of the disease, dengue hem- orrhagic fever and dengue shock syndrome (WHO, 2009). During infection, DENV manipulates the host cell machinery to facilitate its own replication (Walsh and Mohr, 2011) and as such targeting these processes offer the potential for the development of thera- peutic agents. RNA interference (RNAi) is a process by which small, non coding RNA molecules regulate gene expression through either attenua- tion of mRNA transcription or by targeting mRNAs for degradation (Felekkis and Deltas, 2006). Currently, three classes of small RNAs associated with silencing pathways have been described in mam- mals, namely endogenous small interfering RNAs (endo-siRNAs), piwi-associated RNAs (piRNAs) and microRNAs (miRNAs). The first two classes of small RNAs are primarily involved in the repression of transposons as well as the nucleic acid of viruses (Carmell et al., 2007; Farazi et al., 2008), while the last class of small RNAs in particular regulates cellular gene expression (Felekkis et al., 2010). miRNAs are generated through transcription to generate a primary miRNAs (pri-miRNA) that is composed of exonic and intronic re- gions (Borchert et al., 2006; Lee et al., 2004). The stem loop region of the pri-miRNA is processed by a complex of proteins to yield 70e80 nucleotide stem loop precursor miRNAs (pre-miRNA) (Han et al., 2004; Landthaler et al., 2004; Lee et al., 2003), which are * Corresponding author. Department of Microbiology, Faculty of Science, Chula- longkorn University, 254 Phayathai, Pathumwan, Bangkok, 10330, Thailand. E-mail address: wanchai.a@chula.ac.th (W. Assavalapsakul). Contents lists available at ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral http://dx.doi.org/10.1016/j.antiviral.2017.03.020 0166-3542/© 2017 Elsevier B.V. All rights reserved. Antiviral Research 142 (2017) 169e177